Human Cerebral Microvascular Endothelial Cells Research Articles

TGF-β induces oncofetal fibronectin that, in turn, modulates TGF-β superfamily signaling in endothelial cells.

Gene splicing profiles are frequently altered in cancer, and the splice variants of fibronectin (FN) that contain the extra-domains A (EDA) or B (EDB), referred to as EDA+FN or EDB+FN, are highly upregulated in tumor vasculature. Transforming growth factor β (TGF-β) signaling has been attributed a pivotal role in glioblastoma, with TGF-β promoting angiogenesis and vessel remodeling. By using immunohistochemistry staining, we observed that the oncofetal FN isoforms EDA+FN and EDB+FN are expressed in glioblastoma vasculature. Ex vivo single-cell gene expression profiling of tumors by using CD31 and α-smooth muscle actin (αSMA) as markers for endothelial cells, and pericytes and vascular smooth muscle cells (VSMCs), respectively, confirmed the predominant expression of FN, EDA+FN and EDB+FN in the vascular compartment of glioblastoma. Specifically, within the CD31-positive cell population, we identified a positive correlation between the expression of EDA+FN and EDB+FN, and of molecules associated with TGF-β signaling. Further, TGF-β induced EDA+FN and EDB+FN in human cerebral microvascular endothelial cells and glioblastoma-derived endothelial cells in a SMAD3- and SMAD4-dependent manner. In turn, we found that FN modulated TGF-β superfamily signaling in endothelial cells via the EDA and EDB, pointing towards a bidirectional influence of oncofetal FN and TGF-β superfamily signaling.

Regulation of Reduced Folate Carrier (RFC) by Vitamin D Receptor at the Blood-Brain Barrier.

Folates are essential for brain development and function. Folate transport in mammalian tissues is mediated by three major folate transport systems, i.e., reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), and folate receptor alpha (FRα), known to be regulated by ligand-activated nuclear receptors, such as vitamin D receptor (VDR). Folate uptake at the choroid plexus, which requires the actions of both FRα and PCFT, is critical to cerebral folate delivery. Inactivating FRα or PCFT mutations cause severe cerebral folate deficiency resulting in early childhood neurodegeneration. The objective of this study was to investigate the role of RFC in folate uptake at the level of the blood-brain barrier (BBB) and its potential regulation by VDR. We detected robust expression of RFC in different in vitro BBB model systems, particularly in immortalized cultures of human cerebral microvascular endothelial cells (hCMEC/D3) and isolated mouse brain capillaries. [3H]-methotrexate uptake by hCMEC/D3 cells at pH 7.4 was inhibited by PT523 and pemetrexed, antifolates with high affinity for RFC. We also showed that activation of VDR through calcitriol (1,25-dihydroxyvitamin D3) exposure up-regulates RFC mRNA and protein expression as well as function in hCMEC/D3 cells and isolated mouse brain capillaries. We further demonstrated that RFC expression could be down-regulated by VDR-targeting siRNA, further confirming the role of VDR in the direct regulation of this folate transporter. Together, these data suggest that augmenting RFC functional expression could constitute a novel strategy for enhancing brain folate delivery for the treatment of neurometabolic disorders caused by loss of FRα or PCFT function.

Long-term exposure to ethanol downregulates tight junction proteins through the protein kinase Cα signaling pathway in human cerebral microvascular endothelial cells.

Brain microvascular endothelial cells (BMECs) are the primary component of the blood-brain barrier (BBB). Tight junction (TJ) proteins, including claudin, occludin and zonula occludens (ZO)-1, ZO-2 and ZO-3, maintain the structural integrity of BMECs. Ethanol activates the assembly and disassembly of TJs, which is a process that is regulated by protein kinase C (PKC). In addition, ethanol treatment leads to the loss of structural integrity, which damages the permeability of the BBB and subsequently affects central nervous system homeostasis, thus allowing additional substances to enter the brain. However, the mechanisms underlying ethanol-induced loss of BBB structure remain unknown. It has been hypothesized that long-term exposure to ethanol reduces the expression of claudin-5, occludin and ZO-1 via the PKC signaling pathway, thereby affecting BBB structural integrity. In the current study, the human cerebral microvascular endothelial cell line, HCMEC/D3, was treated with 50, 100, 200 and 400 mM ethanol for 24, 48 and 72 h. Cell viability was determined using an MTS assay. The expression of claudin-5, occludin and ZO-1 protein and mRNA was measured using western blot analysis and reverse transcription-quantitative polymerase chain reaction, respectively. Following the pretreatment of HCMEC/D3 cells with the PKCα-specific inhibitor, safingol (10 µmol/l), the expression of claudin-5, occludin, ZO-1 and phosphorylated (p)-PKCα was measured using western blot analysis, and PKCα localization was determined by immunofluorescence. With increasing concentrations of ethanol, the expression of claudin-5, occludin and ZO-1 protein decreased, while the expression of claudin-5, occludin and ZO-1 mRNA increased. Exposure to ethanol significantly increased the expression of p-PKCα, whereas no significant effect on the expression of PKCα was observed. Following 48 h treatment with 200 mM ethanol, the expression of claudin-5, occludin and ZO-1 protein was significantly decreased when compared with the control. By contrast, the expression of p-PKCα was increased, and increased translocation of PKCα from the cytoplasm to the nuclear membrane and nucleus was observed. In addition, the results demonstrated that safingol significantly reversed these effects of ethanol. In conclusion, long-term exposure to ethanol downregulates the expression of claudin-5, occludin and ZO-1 protein in HCMEC/D3 s, and this effect may be mediated via activation of PKCα.

Effect of low-frequency but high-intensity noise exposure on swine brain blood barrier permeability and its mechanism of injury.

Vibroacousitic disease (VAD) is caused by excessive exposure to low-frequency but high-intensity noise. The integrity of the brain blood barrier (BBB) is essential for the brain. The study aimed to investigate the effect of noise exposure on the BBB. Healthy male Bama swine were exposed to 50, 70, 100, and 120Hz, 140dB noise for 30min. After exposure, CT brain imaging and ex vivo fluorescent imaging of parenchymal EB leakage were performed (each group consisted of N=3 swine). The human cerebral microvascular endothelial cells were exposed to 70Hz, 140dB noise for 5min. The BBB permeability assay showed that 50, 70, and 100Hz with 140dB noise exposure accelerated BBB permeability, and the BBB opening at 70Hz was most serious and reversible. Additionally, CT images demonstrated that the noise-induced opening of the BBB caused no intracerebral hemorrhage. This noise-induced BBB opening was related to the downregulation of zo-1 and occludin. Finally, cysteinyl leukotriene receptor 1 (CysLT1 receptor) was found to regulate noise-induced tight junction defects in vitro. In conclusion, noise exposure accelerates the formation of a high-permeability BBB with leaky tight junctions through a CysLT1-mediated mechanism, which warrants additional research.

SNHG15 affects the growth of glioma microvascular endothelial cells by negatively regulating miR-153

Malignant glioma, the most common intracranial primary tumor, is characterized by increased angiogenesis. Accumulating evidence has shown that long non-coding RNAs (lncRNAs) play an important role in a variety of biological behaviors of tumors. However, the role of lncRNAs in the regulation of glioma vascular endothelial cell function remains to be investigated. To simulate the glioma microenvironment, we applied glioma conditioned medium (GCM) to human cerebral microvascular endothelial cells (hCMECs). In the present study, the lncRNA SNHG15 was found to be highly expressed in glioma vascular endothelial cells. Cell Counting Kit-8 (CCK-8), migration and tube formation assays demonstrated that knockdown of SNHG15 inhibited glioma vascular endothelial cell proliferation, migration and tube formation invitro. Furthermore, knockdown of SNHG15 downregulated the expression of VEGFA and Cdc42, which are known to promote angiogenesis. Bioinformatics software and dual-luciferase system analysis confirmed that SNHG15 affected endothelial cell function by targeting miR-153. Additionally, the present study showed that miR-153 targeted the 3'‑untranslated region of VEGFA and Cdc42 and downregulated their expression. In conclusion, knockdown of SNHG15 downregulated the expression of VEGFA and Cdc42 by targeting miR-153, consequently suppressing glioma vascular endothelial cell proliferation, migration and tube formation. Therefore, SNHG15 and miR-153 are new potential therapeutic targets for anti-angiogenesis treatment of glioma.

OP.8A.06] PULSATILE STRETCH ALTERS AMYLOID PRECURSOR PROTEIN EXPRESSION AND NITRIC OXIDE SIGNALLING IN HUMAN CEREBRAL ENDOTHELIAL CELLS PRE-EXPOSED TO GLYCOSPHINGOLIPID INHIBITION

Objective: Amyloid β (Aβ) deposits that arise from amyloid precursor protein (APP) are a hallmark of Alzheimer's disease (AD). Elevated pulsatility of arterial pressure and deregulation of ceramide and nitric oxide (NO) metabolism are associated with AD. Glycosphingolipid inhibitors that inhibit the production of the enzyme glucosylceramide synthase by blocking ceramide glycosylation are known to increase gamma secretase-mediated-Aβ release. This study investigates whether pulsatile stretch of human cerebral microvascular endothelial cells (hCMEC) pre-treated with the glycosphingolipid inhibitor, D-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), alters expression and/or phosphorylation of APP and endothelial NO synthase (eNOS). Design and method: hCMECs were pre-treated with 20 μM D-PDMP for 2 hours prior to 15% cyclic stretch for 18 hours at 1 Hz (n = 7–8). Protein expression and phosphorylation (phospho-eNOS, Ser1177) were quantified using western blots. Results were analyzed using one way ANOVA (mean ± SEM, % control). Results: APP expression increased significantly in response to stretch in ECs pre-treated with D-PDMP (170.2 ± 27%, p < 0.01) compared to cells stretched without pre-treatment (114.5 ± 23%). eNOS phosphorylation was down-regulated in ECs subjected to stretch (9.9 ± 4%) compared to vehicle control (100.0 ± 0%, p < 0.0001) and D-PDMP-treated-static control (107.0 ± 17%, p < 0.01). In ECs pre-treated with D-PDMP prior to stretch, phospho-eNOS levels decreased significantly (40.3 ± 15%) compared to vehicle control (100.0 ± 0%, p < 0.05) and PDMP-treated-static control (107.0 ± 17%, p < 0.01). Conclusions: APP expression increased in cerebral ECs pre-treated with D-PDMP after being exposed to cyclic stretch. eNOS phosphorylation was down-regulated by cyclic stretch. D-PDMP pre-treatment appeared to have a restoring effect on phospho-eNOS, although it did not statistically significance. Glycosphingolipid inhibition with D-PDMP and cyclic stretch may lead to ceramide accumulation thereby increasing the Aβ load. This can affect NO production and modulation of Aβ deposits.

The interaction of clozapine loaded nanocapsules with the hCMEC/D3 cells – In vitro model of blood brain barrier

Despite progress in the development of novel pharmacological compounds, their efficacy in the treatment of neuropathologies is not satisfactory. One strategy to achieve safe and efficient brain targeting therapy is to design nanocarriers capable of transporting antipsychotic drugs through the BBB (without affecting the normal functions of the barrier) in a defined part of the brain. Here we investigate the interaction of clozapine-loaded polymeric Nano capsules (CLO-NCs) with hCMEC/D3 (human cerebral microvascular endothelial cells, D3 clone) cells that constitutes an in vitro model of the blood brain barrier (BBB). CLO-NCs (average size of 100nm) were constructed by the technique of sequential adsorption of polyelectrolytes (LbL), using biocompatible polyanion PGA (Poly-l-glutamic acid sodium salt) and polycation PLL (poly L-lysine) on clozapine-loaded nano-emulsion cores. Pegylated external layers were prepared using PGA-g(39)-PEG (PGA grafted by PEG poly(ethylene glycol)). The influence of the physicochemical properties of the CLO-NCs (charge, size, surface modification) on cell viability was determined. Advanced studies of CLO-NCs internalization (including endocytosis and transcytosis experiments) using confocal microscopy, flow cytometry and fluorescence spectroscopy are presented. Our results indicate that among the studied NCs, the pegylated clozapine-loaded NCs were the most protected from their uptake by macrophages, and they were the least toxic to hCMEC/D3 cells. They were also the most efficient in the transcytosis experiment, which serves as an indicator of their ability to cross a model BBB.

CYCLIC STRETCH MEDIATES CHANGES IN AMYLOID PRECURSOR PROTEIN EXPRESSION AND NITRIC OXIDE SIGNALLING IN HUMAN CEREBRAL ENDOTHELIAL CELLS PRE-EXPOSED TO A GLYCOSPHINGOLIPID INHIBITOR

Amyloid β (Aβ) deposits, a hallmark of Alzheimer's disease (AD), are aggregates of Aβ peptides that are cleaved of amyloid precursor protein (APP). Elevated pulsatility of arterial pressure and deregulation of ceramide and nitric oxide (NO) metabolism are independently associated with AD (Nation et al. 2013; Takasugi et al. 2015; Austin et al. 2013). Glycosphingolipid inhibitors that inhibit the enzyme glucosylceramide synthase by blocking ceramide glycosylation are known to increase gamma secretase-mediated-Aβ release (Takasugi et al. 2015). This study investigates whether pulsatile stretch of human cerebral microvascular endothelial cells (hCMEC) pre-treated with the glycosphingolipid inhibitor, D-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) alters expression and/or phosphorylation of APP and endothelial NO synthase (eNOS). hCMECs were pre-treated with 20 μM D-PDMP for 2 hours prior to 15% cyclic stretch for 18 hours at 1 Hz (n=7–8). Protein expression and phosphorylation (phospho-eNOS, Ser1177) were quantified using western blots. Results were analyzed using one way ANOVA (mean ± SEM, % control). APP expression significantly increased in response to stretch in ECs pre-treated with D-PDMP (170.2±27%, p<0.01) compared to those that have been stretched without pre-treatment (114.5±23%). eNOS phosphorylation was down-regulated in ECs subjected to stretch (9.9±4%) compared to vehicle control (100.0±0%, p<0.0001) and PDMP-treated-static control (107.0±17%, p<0.01). In ECs pre-treated with D-PDMP prior to stretch, phospho-eNOS levels significantly decreased (40.3±15%) compared to vehicle control (100.0±0%, p<0.05) and PDMP-treated-static control (107.0±17%, p<0.01). APP expression increased in cerebral ECs pre-exposed to D-PDMP after being exposed to cyclic stretch. eNOS phosphorylation was down-regulated by cyclic stretch. D-PDMP pre-treatment appeared to have a restoring effect on peNOS, although was not statistically significant. Glucosyl and/or lactosyl ceramide synthases may have regulatory roles in modulating APP expression and NO signalling in cerebral ECs and may have implications on Aβ load, NO production and related vascular pathologies in the context of AD.

Andrographolide-loaded nanoparticles for brain delivery: Formulation, characterisation and in vitro permeability using hCMEC/D3 cell line

Andrographolide (AG) is a major diterpenoid of the Asian medicinal plant Andrographis paniculata which has shown exciting pharmacological potential for the treatment of inflammation-related pathologies including neurodegenerative disorders. Conversely, the low bioavailability of AG still represents a limiting factor for its use. To overcome these limitations, AG was loaded into human serum albumin based nanoparticles (HSA NPs) and poly ethylcyanoacrylate nanoparticles (PECA NPs). HSA NPs were prepared by thermal (HSAT AG NPs) and chemical cross-linking (HSAC AG NPs), while PECA AG NPs were produced by emulsion-polymerization. NPs were characterized in terms of size, zeta (ζ)-potential, polydispersity, and release studies of AG. In addition, the ability of free AG and AG-loaded in PECA and HSAT NPs to cross the blood-brain barrier (BBB) was assessed using an in vitro BBB model based on human cerebral microvascular endothelial cell line (hCMEC/D3). For BBB drug permeability assays, a quantitative UPLC-MS/MS method for AG in Ringer HEPES buffer was developed and validated according to international regulatory guidelines for industry. Free AG did not permeate the BBB model, as also predicted by in silico studies. HSAT NPs improved by two-fold the permeation of AG while maintaining the integrity of the cell layer, while PECA NPs temporarily disrupted BBB integrity.

Transthyretin stability is critical in assisting beta amyloid clearance- Relevance of transthyretin stabilization in Alzheimer's disease.

The absence of transthyretin (TTR) in AD mice decreases brain Aβ clearance and reduces the low-density lipoprotein receptor-related protein 1 (LRP1). It is possible that neuroprotection by TTR is dependent on its tetramer structural stability, as studies using TTR mutants showed that unstable L55P TTR has low affinity for Aβ, and TTR tetrameric stabilizers such as iododiflunisal ameliorate AD features in vivo. We firstly investigated TTR folding status in human plasma measuring the resistance to urea denaturation. The importance of TTR stability on Aβ internalization was studied in human cerebral microvascular endothelial (hCMEC/D3) and hepatoma cells (HepG2), by flow cytometry. To investigate the fate of Aβ at the blood-brain barrier, Aβ efflux from hCMEC/D3 cells seeded on transwells was measured using ELISA. Further, to assess Aβ colocalization with lysosomes, Lysotracker was used. Moreover, levels of LRP1 were assessed in the liver and plasma of mice with different TTR backgrounds or treated with iododiflunisal. We showed that TTR stability is decreased in AD and that WT TTR and drug-stabilized L55P TTR are able to increase uptake of Aβ.Furthermore, measurement of Aβ efflux showed that stable or stabilized TTR increased Aβ efflux from the basolateral to the apical side. Moreover, HepG2 cells incubated with Aβ in the presence of WT TTR, but not L55P TTR, showed an increased number of lysosomes. Further, in the presence of WT TTR, Aβ peptide colocalized with lysosomes, indicating that only stable TTR assists Aβ internalization, leading to its degradation. Finally, we demonstrated that only stable TTR can increase LRP1 levels. TTR stabilization exerts a positive effect on Aβ clearance and LRP1 levels, suggesting that TTR protective role in AD is dependent on its stability. These results provide relevant information for the design of TTR-based therapeutic strategies for AD.

Abstract 574: High-Density Lipoproteins Suppress Amyloid Beta Induced Human Brain Microvascular Endothelial Cell Activation

Introduction: Epidemiological studies suggest a link between plasma high-density lipoprotein (HDL) cholesterol levels and Alzheimer’s disease (AD) risk through mechanisms that are not understood. We hypothesize that HDL protects against AD through actions at the blood-brain-barrier. HDL has vasoprotective functions in large peripheral arteries, however, it is unknown if these functions extend to cerebral vessels to reduce the contribution of cerebrovascular dysfunction in AD pathogenesis. We investigated in vitro interactions between HDL and amyloid beta (Aβ), the toxic peptide known to accumulate in AD, in peripheral and brain-derived endothelial cells (EC). Methods: HDL was isolated by density gradient ultracentrifugation and added to human umbilical vein endothelial cells (HUVEC) or human cerebral microvascular endothelial cells (hCMEC/D3). Cell activation was measured by counting adhered labelled peripheral blood mononuclear cells (PBMC) after stimulation with tumour necrosis factor α (TNFα) or Aβ. Aβ binding and uptake into cells was measured using ELISA and immunofluorescence. All experiments included at least 4 independent replicates. Results: We demonstrate that HDL attenuates Aβ-induced EC activation independent of nitric oxide production, miR-233 and changes in adhesion molecule expression. Rather, HDL acts through scavenger receptor BI to block Aβ uptake into ECs and, in vitro , can maintain Aβ in a soluble state. We validated our results using three dimensional engineered vessels composed of primary human endothelial and smooth muscle cells. Following Aβ addition to the abluminal (brain) side, we demonstrated that HDL circulated within the lumen attenuates EC activation, again independent of intracellular adhesion molecule changes. Conclusions: We show that the anti-inflammatory activities of HDL extend to cerebrovascular endothelial cells and work to suppress Aβ-induced activation through a novel mechanism involving the inhibition of Aβ binding and uptake into cells through SR-BI. The protective role for HDL against Aβ may explain the epidemiological evidence supporting a protective effect of high plasma HDL cholesterol levels against dementia.

Regulation of Tight-Junction Integrity by Insulin in an In Vitro Model of Human Blood–Brain Barrier

Although insulin receptor is expressed at the human blood–brain barrier (BBB), the physiological and pathologic roles of insulin signaling in biologic responses at the BBB remain unclear. Here, we investigate insulin signaling at the human BBB using human cerebral microvascular endothelial cell line (hCMEC/D3) as a well-established in vitro model. Western blot analysis showed that insulin induced phosphorylation of extracellular signal-regulated kinase and insulin receptor substrate-1 in hCMEC/D3 cells. Short-term insulin stimulation increased cell proliferation via the canonical phosphoinositide-3 kinase/protein kinase B and mitogen-activated protein kinase signaling pathways, suggesting that insulin signaling is involved in the regulation of biologic responses in the human BBB. We also found that insulin rapidly increased tight-junction integrity of hCMEC/D3 cells via the phosphoinositide-3 kinase/protein kinase B/glycogen synthase kinase-3 β signaling pathway. Inhibition of insulin/insulin-like growth factor-1 receptor kinase by AG1024 blocked the increase of tight-junction integrity. In addition, high-insulin/high-glucose treatment (as a model of hyperglycemia and hyperinsulinemia) synergistically reduced the tight-junction integrity in hCMEC/D3 cells, although either condition alone had little or no effect. Our findings suggest that, in addition to the established role of interactions of astrocytes and pericytes with brain capillary endothelial cells, insulin signaling from the blood side of the BBB contributes to maintenance of homeostasis by regulating cell proliferation and tight-junction integrity.

The blood-brain barrier fatty acid transport protein 1 (FATP1/SLC27A1) supplies docosahexaenoic acid to the brain, and insulin facilitates transport.

We purposed to clarify the contribution of fatty acid transport protein 1 (FATP1/SLC 27A1) to the supply of docosahexaenoic acid (DHA) to the brain across the blood-brain barrier in this study. Transport experiments showed that the uptake rate of [14 C]-DHA in human FATP1-expressing HEK293 cells was significantly greater than that in empty vector-transfected (mock) HEK293 cells. The steady-state intracellular DHA concentration was nearly 2-fold smaller in FATP1-expressing than in mock cells, suggesting that FATP1 works as not only an influx, but also an efflux transporter for DHA. [14 C]-DHA uptake by a human cerebral microvascular endothelial cell line (hCMEC/D3) increased in a time-dependent manner, and was inhibited by unlabeled DHA and a known FATP1 substrate, oleic acid. Knock-down of FATP1 in hCMEC/D3 cells with specific siRNA showed that FATP1-mediated uptake accounts for 59.2-73.0% of total [14 C]-DHA uptake by the cells. Insulin treatment for 30min induced translocation of FATP1 protein to the plasma membrane in hCMEC/D3 cells and enhanced [14 C]-DHA uptake. Immunohistochemical analysis of mouse brain sections showed that FATP1 protein is preferentially localized at the basal membrane of brain microvessel endothelial cells. We found that two neuroprotective substances, taurine and biotin, in addition to DHA, undergo FATP1-mediated efflux. Overall, our results suggest that FATP1 localized at the basal membrane of brain microvessels contributes to the transport of DHA, taurine and biotin into the brain, and insulin rapidly increases DHA supply to the brain by promoting translocation of FATP1 to the membrane. Read the Editorial Comment for this article on page 324.

Aryl hydrocarbon receptor upregulates IL-1β expression in hCMEC/D3 human cerebral microvascular endothelial cells after TCDD exposure

The AhR is a cytosolic ligand-dependent transcription factor activated by both endogenous and exogenous chemicals. It can regulate expression of many target genes including some inflammatory cytokines and chemokines. To date AhR implication in the regulation of inflammatory cytokines and chemokines at human cerebral endothelium has not been addressed. In the present study, we investigated whether AhR could regulate the expression of two pro-inflammatory cytokines and one chemokine i.e. IL-1β, IL-6 and IL-8 in the hCMEC/D3 human cerebral microvascular endothelial cell line. Exposure to TCDD increased IL-1β mRNA expression levels in hCMEC/D3. By using small interfering RNA against AhR we demonstrated that TCDD effects on IL-1β expression were mediated through AhR activation. Regarding IL-6 and IL-8, TCDD exposure had little or no effect on their mRNA levels in hCMEC/D3. In conclusion, our findings suggest that AhR-mediated IL-1β regulation in cerebral endothelium could induce BBB breakdown and contribute to the pathogenesis of a variety of neurologic disorders.

PVT1 affects growth of glioma microvascular endothelial cells by negatively regulating miR-186.

Vigorous angiogenesis is one of the reasons for the poor prognosis of glioma. A number of studies have shown that long non-coding RNA can affect a variety of biological behaviors of tumors. However, the influence of long non-coding RNAs on glioma vascular endothelial cells remains unclear. To simulate the glioma microenvironment, we applied glioma-conditioned medium to human cerebral microvascular endothelial cells. The long non-coding RNA PVT1 was found to be highly expressed in glioma vascular endothelial cells. Cell Counting Kit-8, migration, and tube formation assays showed that PVT1 overexpression promoted glioma vascular endothelial cells proliferation, migration, and angiogenesis. We also found that PVT1 overexpression upregulated the expression of the autophagy-related proteins Atg7 and Beclin1, which induced protective autophagy. Bioinformatics software and dual-luciferase system analysis confirmed that PVT1 acts by targeting miR-186. In addition, our study showed that miR-186 could target the 3' untranslated region of Atg7 and Beclin1 to decrease their expression levels, thereby inhibiting glioma-conditioned human cerebral microvascular endothelial cell autophagy. In conclusion, PVT1 overexpression increased the expression of Atg7 and Beclin1 by targeting miR-186, which induced protective autophagy, thus promoting glioma vascular endothelial cell proliferation, migration, and angiogenesis. Therefore, PVT1 and miR-186 can provide new therapeutic targets for future anti-angiogenic treatment of glioma.

Adenosine receptors regulate gap junction coupling of the human cerebral microvascular endothelial cells hCMEC/D3 by Ca2+ influx through cyclic nucleotide-gated channels.

Gap junction channels are essential for the formation and regulation of physiological units in tissues by allowing the lateral cell-to-cell diffusion of ions, metabolites and second messengers. Stimulation of the adenosine receptor subtype A2B increases the gap junction coupling in the human blood-brain barrier endothelial cell line hCMEC/D3. Although the increased gap junction coupling is cAMP-dependent, neither the protein kinase A nor the exchange protein directly activated by cAMP were involved in this increase. We found that cAMP activates cyclic nucleotide-gated (CNG) channels and thereby induces a Ca2+ influx, which leads to the increase in gap junction coupling. The report identifies CNG channels as a possible physiological link between adenosine receptors and the regulation of gap junction channels in endothelial cells of the blood-brain barrier. The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A2A and A2B adenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the gap junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of gap junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor l-cis-diltiazem, as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+ , suppressed the 2-PAA-related enhancement of gap junction coupling. Moreover, we observed a 2-PAA-dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+ influx by opening CNG channels in a cAMP-dependent manner. Ca2+ in turn induces the formation of new gap junction plaques and a consecutive sustained enhancement of gap junction coupling. The report identifies CNG channels as a physiological link that integrates gap junction coupling into the adenosine receptor-dependent signalling of endothelial cells of the blood-brain barrier.

HIV-1 Latency-Reversing Agents Prostratin and Bryostatin-1 Induce Blood-Brain Barrier Disruption/Inflammation and Modulate Leukocyte Adhesion/Transmigration.

A shock-and-kill approach involving the simultaneous treatment of HIV-1-infected patients with latency-reversing agents (LRAs) and combination antiretroviral therapy was proposed as a means to eradicate viral reservoirs. Currently available LRAs cannot discriminate between HIV-1-infected and uninfected cells. Therefore, the risks and benefits of using broad-spectrum LRAs need to be carefully evaluated, particularly in the CNS, where inflammation and leukocyte transmigration must be tightly regulated. We used a real-time impedance-sensing system to dynamically record the impact of different classes of LRAs on the integrity of tight monolayers of the immortalized human cerebral microvascular endothelial cell line hCMEC/D3. Results show that prostratin and bryostatin-1 can significantly damage the integrity of an endothelial monolayer. Moreover, prostratin and bryostatin-1 induce secretion of some proinflammatory cytokines and an increase of ICAM-1 expression. Additional studies demonstrated that prostratin and bryostatin-1 also affect adhesion and transmigration of CD4+ and CD8+ T cells as well as monocytes in an in vitro human blood-brain barrier (BBB) model. Prostratin and bryostatin-1 could thus be considered as potent regulators of BBB permeability and inflammation that influence leukocyte transport across the BBB. Altogether, these findings contribute to a better understanding of the potential risks and benefits of using a shock-and-kill approach with LRAs on the normal physiological functions of the BBB.

Increased Transendothelial Transport of CCL3 Is Insufficient to Drive Immune Cell Transmigration through the Blood-Brain Barrier under Inflammatory Conditions In Vitro.

Many neuroinflammatory diseases are characterized by massive immune cell infiltration into the central nervous system. Identifying the underlying mechanisms could aid in the development of therapeutic strategies specifically interfering with inflammatory cell trafficking. To achieve this, we implemented and validated a blood–brain barrier (BBB) model to study chemokine secretion, chemokine transport, and leukocyte trafficking in vitro. In a coculture model consisting of a human cerebral microvascular endothelial cell line and human astrocytes, proinflammatory stimulation downregulated the expression of tight junction proteins, while the expression of adhesion molecules and chemokines was upregulated. Moreover, chemokine transport across BBB cocultures was upregulated, as evidenced by a significantly increased concentration of the inflammatory chemokine CCL3 at the luminal side following proinflammatory stimulation. CCL3 transport occurred independently of the chemokine receptors CCR1 and CCR5, albeit that migrated cells displayed increased expression of CCR1 and CCR5. However, overall leukocyte transmigration was reduced in inflammatory conditions, although higher numbers of leukocytes adhered to activated endothelial cells. Altogether, our findings demonstrate that prominent barrier activation following proinflammatory stimulation is insufficient to drive immune cell recruitment, suggesting that additional traffic cues are crucial to mediate the increased immune cell infiltration seen in vivo during neuroinflammation.

HIV-1 Transactivator Protein Induces ZO-1 and Neprilysin Dysfunction in Brain Endothelial Cells via the Ras Signaling Pathway.

Amyloid beta (Aβ) deposition is increased in human immunodeficiency virus-1- (HIV-1-) infected brain, but the mechanisms are not fully understood. The aim of the present study was to evaluate the role of Ras signaling in HIV-1 transactivator protein- (Tat-) induced Aβ accumulation in human cerebral microvascular endothelial cells (HBEC-5i). Cell viability assay showed that 1 μg/mL Tat and 20 μmol/L of the Ras inhibitor farnesylthiosalicylic acid (FTS) had no significant effect on HBEC-5i cell viability after 24 h exposure. Exposure to Tat decreased protein and mRNA levels of zonula occludens- (ZO-) 1 and Aβ-degrading enzyme neprilysin (NEP) in HBEC-5i cells as determined by western blotting and quantitative real-time polymerase chain reaction. Exposure to Tat also increased transendothelial transfer of Aβ and intracellular reactive oxygen species (ROS) levels; however, these effects were attenuated by FTS. Collectively, these results suggest that the Ras signaling pathway is involved in HIV-1 Tat-induced changes in ZO-1 and NEP, as well as Aβ deposition in HBEC-5i cells. FTS partially protects blood-brain barrier (BBB) integrity and inhibits Aβ accumulation.

Ocimum sanctum Linn. stimulate the expression of choline acetyltransferase on the human cerebral microvascular endothelial cells.

Aim:This research was conducted to identify the expression of choline acetyltransferase (ChAT) in human cerebral microvascular endothelial cells (HCMECs) and to clarify the capability of Ocimum sanctum Linn. ethanolic extract to stimulate the presence of ChAT in the aging HCMECs.Materials and Methods:In this study, we perform an in vitro analysis some in the presence of an ethanolic extract of O. sanctum Linn. as a stimulator for the ChAT expression. HCMECs are divided become two groups, the first is in low passage cells as a model of young aged and the second is in a high passage as a model of aging. Furthermore to analysis the expression of ChAT without and with extract treatments, immunocytochemistry and flow cytometry analysis were performed. In addition, ChAT sandwich enzyme-linked immunosorbent assay is developed to detect the increasing activity of the ChAT under normal, and aging HCMECs on the condition treated and untreated cells.Results:In our in vitro models using HCMECs, we found that ChAT is expressed throughout intracytoplasmic areas. On the status of aging, the ethanolic extract from O. sanctum Linn. is capable to stimulate and restore the expression of ChAT. The increasing of ChAT expression is in line with the increasing activity of this enzyme on the aging treated HCMECs.Conclusions:Our observation indicates that HCMECs is one of the noncholinergic cells which is produced ChAT. The administrated of O. sanctum Linn. ethanolic extract may stimulate and restore the expression of ChAT on the deteriorating cells of HCMECs, thus its may give nerve protection and help the production of acetylcholine.