An In Vitro Model of Blood-Brain Barrier for Studies on HIV Neuroinflammation and CNS Antibody Penetration.

The blood-brain barrier (BBB) is one of several barriers between the brain and the peripheral blood system to maintain homeostasis. Understanding the interactions between infectious agents such as human immunodeficiency virus type 1 (HIV-1), which are capable of traversing the BBB and causing neuroinflammation requires modeling an authentic BBB in vitro. Such an in vitro BBB model also helps develop means of targeting viruses that reside in the brain via natural immune effectors such as antibodies. The BBB consists of human brain microvascular endothelial cells (HBMECs), astrocytes, and pericytes. Here we report in vitro methods to establish a dual-cell BBB model consisting of primary HBMECs and primary astrocytes to measure the integrity of the BBB and antibody penetration of the BBB, as well as a method to establish a single cell BBB model to study the impact of HIV-1 infected medium on the integrity of such a BBB.

SDF-1/CXCR4 axis participants in the pathophysiology of adult patients with moyamoya disease.

BACKGROUND: Moyamoya disease (MMD) is characterized by an abundance of moyamoya vessels; however, the precise mechanism driving the spontaneous angiogenesis of these compensatory vessels remains unclear. Previous research has established a link between the stromal cell-derived factor-1 (SDF-1)/ CXC receptor 4 (CXCR4) axis and angiogenesis under hypoxic conditions. Nevertheless, the alterations in this axis within the cerebrospinal fluid, arachnoid membranes and vascular tissue of MMD patients have not been fully investigated. METHODS: Our study enrolled 66 adult MMD patients and 61 patients with atherosclerotic vascular disease (ACVD). We investigated the SDF-1 concentration in cerebrospinal fluid (CSF) and CXCR4 expression level on the arachnoid membranes and vascular tissue. We utilized enzyme-linked immunosorbent assay and immunohistochemistr. Additionally, we cultured and stimulated human brain microvascular endothelial cells (HBMECs) and smooth muscle cells (SMCs) under oxygen and glucose deprivation (OGD) conditions followed by reoxygenation, to examine any changes in the SDF-1/CXCR4 axis. RESULTS: The results demonstrated an elevation in the level of SDF-1 in CSF among MMD patients compared to those with ACVD. Moreover, the expression of CXCR4 in arachnoid membranes and vascular tissue showed a similar trend. Furthermore, the content of CXCR4 in HBMECs and SMCs increased with the duration of ischemia and hypoxia. However, it was observed that the expression of CXCR4 decreased at OGD/R 24h compared to OGD 24h. The temporal pattern of SDF-1 expression in HBMECs and SMCs mirrored that of CXCR4 expression. CONCLUSION: These findings indicate a critical role for the SDF-1/CXCR4 axis in the angiogenesis of moyamoya disease.

Sox17 protects human brain microvascular endothelial cells from AngII-induced injury by regulating autophagy and apoptosis.

Intracranial aneurysm (IA), is a localized dilation of the intracranial arteries, the rupture of which is catastrophic. Hypertension is major IA risk factor that mediates endothelial cell damage. Sox17 is highly expressed in intracranial vascular endothelial cells, and GWAS studies indicate that its genetic alteration is one of the major genetic risk factors for IA. Vascular endothelial cell injury plays a vital role in the pathogenesis of IA. The genetic ablation of Sox17 plus hypertension induced by AngII can lead to an increased incidence of intracranial aneurysms had tested in the previous animal experiments. In order to study the underlying molecular mechanisms, we established stable Sox17-overexpressing and knockdown cell lines in human brain microvascular endothelial cells (HBMECs) first. Then flow cytometry, western blotting, and immunofluorescence were employed. We found that the knockdown of Sox17 could worsen the apoptosis and autophagy of HBMECs caused by AngII, while overexpression of Sox17 had the opposite effect. Transmission electron microscopy displayed increased autophagosomes after the knockdown of Sox17 in HBMECs. The RNA-sequencing analysis shown that dysregulation of the Sox17 gene was closely associated with the autophagy-related pathways. Our study suggests that Sox17 could protect HBMECs from AngII-induced injury by regulating autophagy and apoptosis.

Type I/type III IFN and related factors regulate JEV infection and BBB endothelial integrity.

BACKGROUND: Japanese encephalitis virus (JEV) remains a predominant cause of Japanese encephalitis (JE) globally. Its infection is usually accompanied by disrupted blood‒brain barrier (BBB) integrity and central nervous system (CNS) inflammation in a poorly understood pathogenesis. Productive JEV infection in brain microvascular endothelial cells (BMECs) is considered the initial event of the virus in penetrating the BBB. Type I/III IFN and related factors have been described as negative regulators in CNS inflammation, whereas their role in JE remains ambiguous. METHODS: RNA-sequencing profiling (RNA-seq), real-time quantitative PCR, enzyme-linked immunosorbent assay, and Western blotting analysis were performed to analyze the gene and protein expression changes between mock- and JEV-infected hBMECs. Bioinformatic tools were used to cluster altered signaling pathway members during JEV infection. The shRNA-mediated immune factor-knockdown hBMECs and the in vitro transwell BBB model were utilized to explore the interrelation between immune factors, as well as between immune factors and BBB endothelial integrity. RESULTS: RNA-Seq data of JEV-infected hBMECs identified 417, 1256, and 2748 differentially expressed genes (DEGs) at 12, 36, and 72 h post-infection (hpi), respectively. The altered genes clustered into distinct pathways in gene ontology (GO) terms and KEGG pathway enrichment analysis, including host antiviral immune defense and endothelial cell leakage. Further investigation revealed that pattern-recognition receptors (PRRs, including TLR3, RIG-I, and MDA5) sensed JEV and initiated IRF/IFN signaling. IFNs triggered the expression of interferon-induced proteins with tetratricopeptide repeats (IFITs) via the JAK/STAT pathway. Distinct PRRs exert different functions in barrier homeostasis, while treatment with IFN (IFN-ß and IFN-λ1) in hBMECs stabilizes the endothelial barrier by alleviating exogenous destruction. Despite the complex interrelationship, IFITs are considered nonessential in the IFN-mediated maintenance of hBMEC barrier integrity. CONCLUSIONS: This research provided the first comprehensive description of the molecular mechanisms of host‒pathogen interplay in hBMECs responding to JEV invasion, in which type I/III IFN and related factors strongly correlated with regulating the hBMEC barrier and restricting JEV infection. This might help with developing an attractive therapeutic strategy in JE.

Upregulation of girdin delays endothelial cell apoptosis via promoting engulfment of platelets.

BACKGROUND: Endothelial cells are crucial in maintaining the homeostasis of the blood-brain barrier. Girders of actin filament (Girdin) and phosphor (p)-Girdin are essential for the engulfment of human brain microvascular endothelial cells (HBMECs) into platelets (PLTs), but the potential mechanism remains unclear and requires further study. METHODS: Following PLT and cytochalasin D treatment, Hoechst 33,342 detected apoptosis. The transfection efficiency of the short hairpin RNA targeting Girdin (sh-Girdin) or overexpressing Girdin (OE-Girdin) was determined using western blotting. Sh-Girdin, OE-Girdin, mutated Girdin (m-Girdin), and microfilament binding region deleted Girdin (Del-Girdin) were transfected into HBMECs under PLT conditions. Subsequently, the engulfment of HBMECs by PLTs was detected by flow cytometry and transmission electron microscopy. Girdin and phosphorylated (p)-Girdin levels were quantified by western blot. The positive expression of Girdin was measured by immunohistochemistry (IHC). The localization of PLT, Girdin, and p-Girdin and the engulfment of HBMECs in PLTs were analyzed by confocal microscopy. RESULT: Cytochalasin D overturned the inhibitory effect of PLT on cell apoptosis. OE-Girdin enhanced the fluorescent intensity of PLT-labelling and the engulfment of HBMECs by PLTs, while sh-Girdin, m-Girdin, and Del-Girdin ran reversely. OE-Girdin elevated the Girdin and p-Girdin levels, while sh-Girdin and Del-Girdin were the opposite, but m-Girdin did not affect the p-Girdin and Girdin levels. CONCLUSION: Girdin and p-Girdin were co-located with PLTs in HBMECs. The over-expression of Girdin was identified as being associated with the increasing engulfment of PTLs. Girdin may be an effective target to alleviate endothelial cell apoptosis.

Enterovirus 71 enters human brain microvascular endothelial cells through an ARF6-mediated endocytic pathway.

Infection of the central nervous system caused by enterovirus 71 (EV71) remains the main cause of death in hand-foot-and-mouth disease. However, the mechanism responsible for how EV71 breaks through the blood-brain barrier to infect brain cells has yet to be elucidated. By performing a high-throughput small interfering RNA (siRNA) screening and validation, we found that the infection of human brain microvascular endothelial cells (HBMECs) by EV71 was independent of the endocytosis pathways mediated by caveolin, clathrin, and macropinocytosis but dependent on ADP-ribosylation factor 6 (ARF6), a small guanosinetriphosphate (GTP)-binding protein of the Ras superfamily. The specific siRNA targeting ARF6 markedly inhibited HBMECs susceptibility to EV71. EV71 infectivity was inhibited by NAV-2729, a specific inhibitor of ARF6, in a dose-dependent manner. The subcellular analysis demonstrated the co-localization of the endocytosed EV71 and ARF6, while knockdown of ARF6 with siRNA remarkably influenced EV71 endocytosis. By immunoprecipitation assays, we found a direct interaction of ARF6 with EV71 viral protein. Furthermore, ARF1, another small GTP-binding protein, was also found to participate in ARF6-mediated EV71 endocytosis. Murine experiments demonstrated that NAV-2729 significantly alleviated mortality caused by EV71 infection. Our study revealed a new pathway by which EV71 enters the HBMECs and provides new targets for drug development.

Open source board based acoustofluidic transwells for reversible disruption of the blood-brain barrier for therapeutic delivery.

BACKGROUND: Blood-brain barrier (BBB) is a crucial but dynamic structure that functions as a gatekeeper for the central nervous system (CNS). Managing sufficient substances across the BBB is a major challenge, especially in the development of therapeutics for CNS disorders. METHODS: To achieve an efficient, fast and safe strategy for BBB opening, an acoustofluidic transwell (AFT) was developed for reversible disruption of the BBB. The proposed AFT was consisted of a transwell insert where the BBB model was established, and a surface acoustic wave (SAW) transducer realized using open-source electronics based on printed circuit board techniques. RESULTS: In the AFT device, the SAW produced acousto-mechanical stimulations to the BBB model resulting in decreased transendothelial electrical resistance in a dose dependent manner, indicating the disruption of the BBB. Moreover, SAW stimulation enhanced transendothelial permeability to sodium fluorescein and FITC-dextran with various molecular weight in the AFT device. Further study indicated BBB opening was mainly attributed to the apparent stretching of intercellular spaces. An in vivo study using a zebrafish model demonstrated SAW exposure promoted penetration of sodium fluorescein to the CNS. CONCLUSIONS: In summary, AFT effectively disrupts the BBB under the SAW stimulation, which is promising as a new drug delivery methodology for neurodegenerative diseases.

Usefulness of a humanized tricellular static transwell blood-brain barrier model as a microphysiological system for drug development applications. - A case study based on the benchmark evaluations of blood-brain barrier microphysiological system.

Microphysiological system (MPS), a new technology for in vitro testing platforms, have been acknowledged as a strong tool for drug development. In the central nervous system (CNS), the blood‒brain barrier (BBB) limits the permeation of circulating substances from the blood vessels to the brain, thereby protecting the CNS from circulating xenobiotic compounds. At the same time, the BBB hinders drug development by introducing challenges at various stages, such as pharmacokinetics/pharmacodynamics (PK/PD), safety assessment, and efficacy assessment. To solve these problems, efforts are being made to develop a BBB MPS, particularly of a humanized type. In this study, we suggested minimal essential benchmark items to establish the BBB-likeness of a BBB MPS; these criteria support end users in determining the appropriate range of applications for a candidate BBB MPS. Furthermore, we examined these benchmark items in a two-dimensional (2D) humanized tricellular static transwell BBB MPS, the most conventional design of BBB MPS with human cell lines. Among the benchmark items, the efflux ratios of P-gp and BCRP showed high reproducibility in two independent facilities, while the directional transports meditated through Glut1 or TfR were not confirmed. We have organized the protocols of the experiments described above as standard operating procedures (SOPs). We here provide the SOPs with the flow chart including entire procedure and how to apply each SOP. Our study is important developmental step of BBB MPS towards the social acceptance, which enable end users to check and compare the performance the BBB MPSs.

Tumor Treating Fields (TTFields) Induce Cell Junction Alterations in a Human 3D In Vitro Model of the Blood-Brain Barrier.

In a recent study, we showed in an in vitro murine cerebellar microvascular endothelial cell (cerebEND) model as well as in vivo in rats that Tumor-Treating Fields (TTFields) reversibly open the blood-brain barrier (BBB). This process is facilitated by delocalizing tight junction proteins such as claudin-5 from the membrane to the cytoplasm. In investigating the possibility that the same effects could be observed in human-derived cells, a 3D co-culture model of the BBB was established consisting of primary microvascular brain endothelial cells (HBMVEC) and immortalized pericytes, both of human origin. The TTFields at a frequency of 100 kHz administered for 72 h increased the permeability of our human-derived BBB model. The integrity of the BBB had already recovered 48 h post-TTFields, which is earlier than that observed in cerebEND. The data presented herein validate the previously observed effects of TTFields in murine models. Moreover, due to the fact that human cell-based in vitro models more closely resemble patient-derived entities, our findings are highly relevant for pre-clinical studies.

High and low permeability of human pluripotent stem cell-derived blood-brain barrier models depend on epithelial or endothelial features.

The search for reliable human blood-brain barrier (BBB) models represents a challenge for the development/testing of strategies aiming to enhance brain delivery of drugs. Human-induced pluripotent stem cells (hiPSCs) have raised hopes in the development of predictive BBB models. Differentiating strategies are thus required to generate endothelial cells (ECs), a major component of the BBB. Several hiPSC-based protocols have reported the generation of in vitro models with significant differences in barrier properties. We studied in depth the properties of iPSCs byproducts from two protocols that have been established to yield these in vitro barrier models. Our analysis/study reveals that iPSCs derivatives endowed with EC features yield high permeability models while the cells that exhibit outstanding barrier properties show principally epithelial cell-like (EpC) features. We found that models containing EpC-like cells express tight junction proteins, transporters/efflux pumps and display a high functional tightness with very low permeability, which are features commonly shared between BBB and epithelial barriers. Our study demonstrates that hiPSC-based BBB models need extensive characterization beforehand and that a reliable human BBB model containing EC-like cells and displaying low permeability is still needed.

ß-Estradiol 17-acetate enhances the in vitro vitality of endothelial cells isolated from the brain of patients subjected to neurosurgery.

In the current landscape of endothelial cell isolation for building in vitro models of the blood-brain barrier, our work moves towards reproducing the features of the neurovascular unit to achieve glial compliance through an innovative biomimetic coating technology for brain chronic implants. We hypothesized that the autologous origin of human brain microvascular endothelial cells (hBMECs) is the first requirement for the suitable coating to prevent the glial inflammatory response triggered by foreign neuroprosthetics. Therefore, this study established a new procedure to preserve the in vitro viability of hBMECs isolated from gray and white matter specimens taken from neurosurgery patients. Culturing adult hBMECs is generally considered a challenging task due to the difficult survival ex vivo and progressive reduction in proliferation of these cells. The addition of 10 nM ß-estradiol 17-acetate to the hBMEC culture medium was found to be an essential and discriminating factor promoting adhesion and proliferation both after isolation and thawing, supporting the well-known protective role played by estrogens on microvessels. In particular, ß-estradiol 17-acetate was critical for both freshly isolated and thawed female-derived hBMECs, while it was not necessary for freshly isolated male-derived hBMECs; however, it did counteract the decay in the viability of the latter after thawing. The tumor-free hBMECs were thus cultured for up to 2 months and their growth efficiency was assessed before and after two periods of cryopreservation. Despite the thermal stress, the hBMECs remained viable and suitable for re-freezing and storage for several months. This approach increasing in vitro viability of hBMECs opens new perspectives for the use of cryopreserved autologous hBMECs as biomimetic therapeutic tools, offering the potential to avoid additional surgical sampling for each patient.

Ruxolitinib enhances cytotoxic and apoptotic effects of temozolomide on glioblastoma cells by regulating WNT signaling pathway-related genes.

Although temozolomide is the primary chemotherapeutic agent in glioblastoma, current studies have focused on its combinational applications to overcome resistance by targeting multiple pathways. JAK/STAT and WNT are among the essential cancer-related signaling pathways. Ruxolitinib, the first approved JAK1/2 inhibitor, has promise in glioblastoma with its blood-brain barrier (BBB) crossing ability. The mentioned study aims to evaluate the anti-cancer potential of ruxolitinib individually and in combination with temozolomide on glioblastoma cells, brain cancer stem cells (BCSCs), and BBB-forming healthy cells. It also intends to determine the effects of JAK inhibitor treatment in combination with temozolomide on WNT signaling, which is known to cross-talk with the JAK/STAT pathway. The U87MG, BCSC, and HBMEC cell lines were the in vitro models. The cytotoxic and apoptotic effects of ruxolitinib and the combination were determined by the WST-1 test and Annexin V assay, respectively. The expression level changes of WNT signaling pathway genes caused by ruxolitinib and the combination treatments were defined by the qRT-PCR method. Network analysis of significantly upregulated and downregulated genes was performed via the GO KEGG pathway enrichment module of the String V11.5 database. The IC50 value of the ruxolitinib on U87MG glioblastoma cells was determined as 94.07 µM at 24th h. The combination of temozolomide and ruxolitinib had a synergistic effect on U87MG cells at 24th h. The combination index (CI) was determined as 0.796, and ED60 values of ruxolitinib and temozolomide were determined as 89.75 and 391.48 µM, respectively. Ruxolitinib improves the apoptotic effect of temozolomide on glioblastoma cells and brain cancer stem cells. Ruxolitinib regulates the WNT signaling pathway both individually and in combination with temozolomide. Our study indicates the potential of ruxolitinib to increase the cytotoxic and apoptotic activity of temozolomide in glioblastoma cells, also considering CSCs and healthy BBB-forming cells. As supported by gene expression and network analyses, the BBB-crossing agent ruxolitinib promises the potential to increase the efficacy of temozolomide in glioblastoma by affecting multiple signaling pathways in both cancer cells and CSCs.

Cholesterol efflux regulator ABCA1 exerts protective role against high shear stress-induced injury of HBMECs via regulating PI3K/Akt/eNOS signaling.

BACKGROUND: In brain, microvascular endothelial cells are exposed to various forces, including shear stress (SS). However, little is known about the effects of high shear stress (HSS) on human brain microvascular endothelial cells (HBMECs) and the underlying mechanism. The cholesterol efflux regulator ATP-binding cassette subfamily A member 1 (ABCA1) has been demonstrated to exert protective effect on HBMECs. However, whether ABCA1 is involved in the mechanism underneath the effect of HSS on HBMECs remains obscure. In the present study, a series of experiments were performed to better understand the effect of HSS on cellular processes of HBMECs and the possible involvement of ABCA1 and PI3K/Akt/eNOS in the underlying mechanisms. RESULTS: HBMECs were subjected to physiological SS (PSS) or high SS (HSS). Cell migration was evaluated using Transwell assay. Apoptotic HBMECs were detected by flow cytometry or caspase3/7 activity. IL-1ß, IL-6, MCP-1 and TNF-α levels were measured by ELISA. RT-qPCR and western blotting were used for mRNA and protein expression detection, respectively. ROS and NO levels were detected using specific detection kits. Compared to PSS, HBMECs exhibited decreased cell viability and migration and increased cell apoptosis, increased levels of inflammatory cytokines, and improved ROS and NO productions after HSS treatment. Moreover, HSS downregulated ABCA1 but upregulated the cholesterol efflux-related proteins MMP9, AQP4, and CYP46 and activated PI3K/Akt/eNOS pathway. Overexpression of ABCA1 in HBMECS inhibited PI3K/Akt/eNOS pathway and counteracted the deleterious effects of HSS. Contrary effects were observed by ABCA1 silencing. Inhibiting PI3K/Akt/eNOS pathway mimicked ABCA1 effects, suggesting that ABCA1 protects HBMECs from HSS via PI3K/Akt/eNOS signaling. CONCLUSION: These results advanced our understanding on the mechanisms of HSS on HBMECs and potentiated ABCA1/PI3K/Akt/eNOS pathway as therapeutic target for cerebrovascular diseases.

The role of glutathione conjugation on the transcellular transport process of PEGylated liposomes across the blood brain barrier.

Notwithstanding the growing evidence of improved drug delivery efficiency to the brain by ligand modification of PEGylated liposomes, the comprehensive knowledge of their transport processes and payload across the BBB is yet to be revealed. Herein, this study sought to understand the glutathione (GSH) ligand effect on transcellular transport mechanisms of liposomes through the blood-brain barrier (BBB) by comparing PEGylated liposomes (PEG-L) and GSH PEGylated liposomes (GSH-PEG-L). Endocytosis and exocytosis of liposomes including the role of secreted extracellular vesicles (EVs) of brain endothelial cells (BECs) were assessed. Furthermore, pharmacokinetics and brain distribution analysis of gemcitabine loaded liposomes were carried out in healthy rats to ascertain the in vivo applicability. Our findings suggested that the presence of GSH increased the cellular uptake of liposomes by up to 3-fold in human brain microvascular endothelial cells depending on the dose but not in astrocytes. The cell exposure to liposomes particularly GSH-PEG-L dramatically increased the cell secretion of small and microvesicles with liposomal components, though different liposomes preferred different vesicles for exocytosis. This correlated with GSH-PEG-L transport efficiency of 4 % across the in vitro BBB model in 24 h, 1.7-fold higher than that of PEG-L (p < 0.05). In rats, while PEG-L and GSH-PEG-L showed similar pharmacokinetic profiles and prolonged circulation properties, 3.8 % of the total injected dose (ID) of gemcitabine was found in the brain of the GSH-PEG-L group at 8 h post-injection, compared with 2.8 % ID in the PEG-L group. A brain: blood concentration ratio of 1.27 ± 0.12 indicated that an active transport mechanism to cross the BBB for GSH-PEG-L. Overall, this study revealed that GSH augmented the transcellular transport efficiency of liposomes through BBB to improve targeted brain delivery by enhancing cellular uptake and vesicular exocytosis route of BECs.

LncRNA-Profile-Based Screening of Extracellular Vesicles Released from Brain Endothelial Cells after Oxygen-Glucose Deprivation.

Brain microvascular endothelial cells (BMECs) linked by tight junctions play important roles in cerebral ischemia. Intercellular signaling via extracellular vesicles (EVs) is an underappreciated mode of cell-cell crosstalk. This study aims to explore the potential function of long noncoding RNAs (lncRNAs) in BMECs' secreted EVs. We subjected primary human and rat BMECs to oxygen and glucose deprivation (OGD). EVs were enriched for RNA sequencing. A comparison of the sequencing results revealed 146 upregulated lncRNAs and 331 downregulated lncRNAs in human cells and 1215 upregulated lncRNAs and 1200 downregulated lncRNAs in rat cells. Next, we analyzed the genes that were coexpressed with the differentially expressed (DE) lncRNAs on chromosomes and performed Gene Ontology (GO) and signaling pathway enrichment analyses. The results showed that the lncRNAs may play roles in apoptosis, the TNF signaling pathway, and leukocyte transendothelial migration. Next, three conserved lncRNAs between humans and rats were analyzed and confirmed using PCR. The binding proteins of these three lncRNAs in human astrocytes were identified via RNA pulldown and mass spectrometry. These proteins could regulate mRNA stability and translation. Additionally, the lentivirus was used to upregulate them in human microglial HMC3 cells. The results showed NR_002323.2 induced microglial M1 activation. Therefore, these results suggest that BMECs' EVs carry the lncRNAs, which may regulate gliocyte function after cerebral ischemia.

Disturbance of Key Cellular Subproteomes upon Propofol Treatment Is Associated with Increased Permeability of the Blood-Brain Barrier.

BACKGROUND: Propofol is a short-acting anesthetic, which is often used for induction and maintenance of general anesthesia, sedation for mechanically ventilated adults and procedural sedation. Several side effects of propofol are known and a substantial number of patients suffer from post-operative delirium after propofol application. In this study, we analyzed the effect of propofol on the function and protein expression profile on a proteome-wide scale. METHODS: We cultured human brain microvascular endothelial cells in absence and presence of propofol and analyzed the permeability of the blood-brain barrier (BBB) by fluorescein passage and protein abundance on a proteome-wide scale by mass spectrometry. RESULTS: Propofol interfered with the function of the blood-brain barrier. This was not due to decreased adhesion of propofol-treated human brain microvascular endothelial cells. The proteomic analysis revealed that some key pathways in these cells were disturbed, such as oxygen metabolism, DNA damage recognition and response to stress. CONCLUSIONS: Propofol has strong effects on protein expression which could explain several side effects of propofol.

miRNA expression in COVID-19.

Coronavirus disease 2019 (COVID-19) is regarded as a challenge in health system. Several studies have assessed the immune-related aspect of this disorder to identify the host-related factors that affect the course of COVID-19. microRNAs (miRNAs) as potent regulators of immune responses have gained much attention in this regard. Recent studies have shown aberrant expression of miRNAs in COVID-19 in association with disease course. Differentially expressed miRNAs have been enriched in pathways related with inflammation and antiviral immune response. miRNAs have also been regarded as potential therapeutic targets in COVID-19, particularly for management of pathological consequences of COVID-19. In the current review, we summarize the data about dysregulation of miRNAs in COVID-19.

Calcium and the Ca-ATPase SPCA1 modulate plasma membrane abundance of ZIP8 and ZIP14 to regulate Mn(II) uptake in brain microvascular endothelial cells.

Manganese (II) accumulation in human brain microvascular endothelial cells is mediated by the metal-ion transporters ZRT IRT-like protein 8 (ZIP8) and ZRT IRT-like protein 14 (ZIP14). The plasma membrane occupancy of ZIP14, in particular, is increased in cells treated with Mn2+, lipopolysaccharide, or IL-6, but the mechanism of this regulation has not been elucidated. The calcium-transporting type 2C member 1 ATPase, SPCA1, is a Golgi-localized Ca2+-uptake transporter thought to support Golgi uptake of Mn2+ also. Here, we show using surface protein biotinylation, indirect immunofluorescence, and GFP-tagged proteins that cytoplasmic Ca2+ regulates ZIP8- and ZIP14-mediated manganese accumulation in human brain microvascular endothelial cells by increasing the plasma membrane localization of these transporters. We demonstrate that RNAi knockdown of SPCA1 expression results in an increase in cytoplasmic Ca2+ levels. In turn, we found increased cytoplasmic Ca2+ enhances membrane-localized ZIP8 and ZIP14 and a subsequent increase in 54Mn2+ uptake. Furthermore, overexpression of WT SPCA1 or a gain-of-function mutant resulted in a decrease in cytoplasmic Ca2+ and 54Mn2+ accumulation. While addition of Ca2+ positively regulated ZIP-mediated 54Mn2+ uptake, we show chelation of Ca2+ diminished manganese transport. In conclusion, the modulation of ZIP8 and ZIP14 membrane cycling by cytoplasmic calcium is a novel finding and provides new insight into the regulation of the uptake of Mn2+ and other divalent metal ions-mediated ZIP metal transporters.

EGFR Activation Impairs Antiviral Activity of Interferon Signaling in Brain Microvascular Endothelial Cells During Japanese Encephalitis Virus Infection.

The establishment of Japanese encephalitis virus (JEV) infection in brain microvascular endothelial cells (BMECs) is thought to be a critical step to induce viral encephalitis with compromised blood-brain barrier (BBB), and the mechanisms involved in this process are not completely understood. In this study, we found that epidermal growth factor receptor (EGFR) is related to JEV escape from interferon-related host innate immunity based on a STRING analysis of JEV-infected primary human brain microvascular endothelial cells (hBMECs) and mouse brain. At the early phase of the infection processes, JEV induced the phosphorylation of EGFR. In JEV-infected hBMECs, a rapid internalization of EGFR that co-localizes with the endosomal marker EEA1 occurred. Using specific inhibitors to block EGFR, reduced production of viral particles was observed. Similar results were also found in an EGFR-KO hBMEC cell line. Even though the process of viral infection in attachment and entry was not noticeably influenced, the induction of IFNs in EGFR-KO hBMECs was significantly increased, which may account for the decreased viral production. Further investigation demonstrated that EGFR downstream cascade ERK, but not STAT3, was involved in the antiviral effect of IFNs, and a lowered viral yield was observed by utilizing the specific inhibitor of ERK. Taken together, the results revealed that JEV induces EGFR activation, leading to a suppression of interferon signaling and promotion of viral replication, which could provide a potential target for future therapies for the JEV infection.

Early biomolecular changes in brain microvascular endothelial cells under Epstein-Barr virus influence: a Raman microspectroscopic investigation.

The brain microvascular endothelial cells (ECs) play an important role in protecting the brain from hazardous pathogens. However, some viral pathogens can smartly modulate the endothelial pathways to gain entry inside the brain. Further, these viruses can cause endothelial dysfunction which could develop serious neurological ailments. Epstein-Barr virus (EBV), an oncogenic virus, has also been linked to various neurological disorders. The virus primarily infects epithelial and B cells, however, it also has a tendency to infect ECs and cause endothelial activation. However, the impact of EBV influence on ECs is still underexplored. Studying the early events of virus-mediated cellular modulation could help in understanding the virus' infection strategy or aftermath. Raman microspectroscopy has been widely utilized in biomedical sciences to decipher cellular changes. To understand the EBV-influenced EC modulation by studying intracellular biomolecular changes at early time points, we utilized the Raman microspectroscopy tool. We treated the ECs with EBV and acquired the Raman spectra at different time points (2, 4, 6, 12, 24 and 36 h) and different sites (nucleus and periphery) to check changes in Raman intensities associated with specific biomolecules. In the EBV-treated cells, the status of various biomolecules in terms of Raman intensities was observed to be altered compared with uninfected cells. Specifically, the cholesterol, polysaccharide, nucleotides, nucleic acid and proline moieties were altered at different time points. We also investigated the possible correlation between these molecules using molecular network analysis and observed various associated factors. These factors could be influenced by EBV to alter the associated biomolecular levels. Our study paves the pathway to study EBV infection in human brain microvascular ECs and highlights specific biomolecular alterations, which can be focused for further mechanistic investigations.