Modulation of Paracellular Permeability in SARS-CoV-2 Blood-to-Brain Transcytosis.

SARS-CoV-2 primarily infects the lungs via the ACE2 receptor but also other organs including the kidneys, the gastrointestinal tract, the heart, and the skin. SARS-CoV-2 also infects the brain, but the hematogenous route of viral entry to the brain is still not fully characterized. Understanding how SARS-CoV-2 traverses the blood-brain barrier (BBB) as well as how it affects the molecular functions of the BBB are unclear. In this study, we investigated the roles of the receptors ACE2 and DPP4 in the SARS-CoV-2 infection of the discrete cellular components of a transwell BBB model comprising HUVECs, astrocytes, and pericytes. Our results demonstrate that direct infection on the BBB model does not modulate paracellular permeability. Also, our results show that SARS-CoV-2 utilizes clathrin and caveolin-mediated endocytosis to traverse the BBB, resulting in the direct infection of the brain side of the BBB model with a minimal endothelial infection. In conclusion, the BBB is susceptible to SARS-CoV-2 infection in multiple ways, including the direct infection of endothelium, astrocytes, and pericytes involving ACE2 and/or DPP4 and the blood-to-brain transcytosis, which is an event that does not require the presence of host receptors.

[Phosphoproteomic analysis of human umbilical venous endothelial cells with DENV-2 infection].

OBJECTIVE: To analyze the differentially phosphorylated proteins in DENV-2-infected human umbilical venous endothelial cells (HUVECs) and explore the possible pathogenic mechanism of DENV-2 infection. METHODS: The total proteins were extracted from DENV-2-infected HUVECs and blank control HUVEC using SDT lysis method. The phosphorylated proteins were qualitatively and quantitatively analyzed using tandem mass spectrometry (TMT). The identified differentially phosphorylated proteins were analyzed by bioinformatics analyses such as subcellular localization analysis, GO enrichment analysis, KEGG pathway analysis and protein-protein interaction (PPI) analysis. Western blotting was used to detect the expressions of phosphorylated Jun, map2k2 and AKT1 proteins in DENV-2-infected HUVECs. RESULTS: A total of 2918 modified peptides on 1385 different proteins were detected, and among them 1346 were significantly upregulated (FC > 1.2, P < 0.05) and 1572 were significantly downregulated (FC < 0.83, P < 0.05). A total of 49 phosphorylated conserved motifs were obtained by amino acid conservative motif analysis. The most abundant differentially phosphorylated peptides in protein domain analysis included RNA recognition motif, protein kinase domain and PH domain. Subcellular localization analysis showed that the differentially modified peptides were mainly localized in the nucleus and cytoplasm. GO enrichment and KEGG pathway analysis showed that the differential peptides were mainly enriched in the regulation of stimulation response, biosynthesis of small molecules containing nuclear bases, and migration of phagosomes and leukocytes across the endothelium. PPI and KEGG joint analysis showed that the up-regulated and down-regulated differentially phosphorylated proteins were enriched in 15 pathways. In DENV-2-infected HUVECs, Western blotting detected differential expressions of phosphorylated proteins related with the autophagy pathway, namely JUN, MAP2K2 and AKT1, and among them p-JUN was significantly down-regulated and p-AKT1 and p-MAP2K2 were significantly upregulated (P < 0.01). CONCLUSION: DENV-2 infected HUVECs show numerous differentially expressed proteins. The downregulation of p-JUN and upregulation of p-MAP2K2 and p-AKT1 suggest their potential roles in regulating autophagy, which is probably involved in the mechanism of DENV-2 infection.

Transcriptional induction of TGF-ß1 and endothelial-to-mesenchymal transition cell markers in human umbilical vein endothelial cells by Ebola virus infection.

BACKGROUND: Ebola virus (EBOV) causes a serious hemorrhagic disease in humans, with a mortality rate of up to 80%. Despite significant achievements in the past decades elucidating the pathogenesis of EBOV, there is still much to be elucidated about the cell type-specific host response and their functional roles during infection. OBJECTIVE: This study aimed to gain insight into cell type-specific host responses to EBOV infection. METHODS: Real-time RT-qPCR analysis was used to identify host transcriptional changes in epithelial Caco-2 cells and endothelial HUVECs by EBOV infection. RESULTS: EBOV efficiently infected to both Caco-2 cells and HUVECs, depending on the time of infection. However, changes in the transcriptional levels of several host cellular genes following viral infection showed significant differences between Caco-2 cells and HUVECs. EBOV infection increases the transcription of TGF-ß1, a key factor in epithelium-to-mesenchyme transition (EMT), only in HUVECs, but not in Caco-2 cells. This upregulation in turn induces the transcription of other EMT signaling molecules such as snail, slug and MMP9, ultimately leading to endothelial-to-mesenchymal transition (EndMT). Furthermore, this EndMT process appears to be associated with increased transcription of stem-cell markers such as Klf4, Sox2 and Oct4. However, most of these transcriptional changes due to EBOV infection did not occur in Caco-2 cells, suggesting that EMT or EndMT by EBOV infection is cell type-specific. CONCLUSION: We propose that EBOV infection induces the expression of TGF-ß1-mediated signals in endothelial HUVECs, resulting in EndMT. This could provide broader information to elucidate the pathogenesis of Ebola virus disease.

Generation of Spike-Extracellular Vesicles (S-EVs) as a Tool to Mimic SARS-CoV-2 Interaction with Host Cells.

Extracellular vesicles (EVs) and viruses share common features: size, structure, biogenesis and uptake. In order to generate EVs expressing the SARS-CoV-2 spike protein on their surface (S-EVs), we collected EVs from SARS-CoV-2 spike expressing human embryonic kidney (HEK-293T) cells by stable transfection with a vector coding for the S1 and S2 subunits. S-EVs were characterized using nanoparticle tracking analysis, ExoView and super-resolution microscopy. We obtained a population of EVs of 50 to 200 nm in size. Spike expressing EVs represented around 40% of the total EV population and co-expressed spike protein with tetraspanins on the surfaces of EVs. We subsequently used ACE2-positive endothelial and bronchial epithelial cells for assessing the internalization of labeled S-EVs using a cytofluorimetric analysis. Internalization of S-EVs was higher than that of control EVs from non-transfected cells. Moreover, S-EV uptake was significantly decreased by anti-ACE2 antibody pre-treatment. Furthermore, colchicine, a drug currently used in clinical trials, significantly reduced S-EV entry into the cells. S-EVs represent a simple, safe, and scalable model to study host-virus interactions and the mechanisms of novel therapeutic drugs.

[Establishment of an immortalized human umbilical vein endothelial cell line used for the study of the innate immune response to Hantaan virus].

Objective To establish the immortalized human umbilical vein vascular endothelial cells (HUVECs-hTERT) by introducing hTERT gene into primary HUVECs. In order to evaluate the potential of HUVECs-hTERT as a research model of HTNV infection, we explored the infection efficiency of Hantaan virus (HTNV) in HUVECs-hTERT and the influence of celluar innate immune regulation. Methods hTERT gene was cloned into lentivirus vector pCDH-CMV-MCS-EF1-puro, resulting in pCDH-CMV-hTERT-EF1-puro plasmid which was packaged into lentivirus. Then it was infected with HUVECs, and the HUVECs which stably express hTERT gene was selected by using puromycin and named HUVECs-hTERT. The morphology of HUVECs-hTERT and endothelial cell marker molecules, such as human von Willebrand factor (vWF), CD31 and vascular endothelial cell cadherin (VE-cadherin) were identified by microscopic observation and immunofluorescence assay. The percentage of nucleocapsid protein (NP)-positive cells after HTNV infection was detected by immunofluorescence assay to identify the difference of infection efficiency in HTNV between HUVECs and HUVECs-hTERT. Subsequently, real-time quantitative PCR (RT-qPCR) and Western blot analysis were used to detect the expression of HTNV S mRNA and NP after HTNV infection to verify amplification efficiency of HTNV in HUVECs and HUVECs-hTERT. RT-qPCR were used to detect the mRNA expression level of interferon ß (IFN-ß), interferon stimulating gene (ISG), including myxovirus resistance protein A (MxA), myxovirus resistance protein B (MxB), interferon inducing protein 2 (IFIT2), interferon-induced transmembrane protein 3 (IFITM3) and inflammatory factors, such as cyclooxygenase -2 (COX2), intercellular adhesion molecule (ICAM), C-C motif chemokine ligand 5 (CCL5) and the protein expression level of IFIT2, IFITM3 and MxA in the two types of cells after HTNV infection to determine whether the cellular innate immune response between HUVECs and HUVECs-hTERT are consistent. Results The immortalized cell line HUVECs-hTERT was screened successfully and the identification results showed that HUVECs-hTERT and HUVECs are with the same phenotype and express endothelial cell marker molecules, such as vWF, CD31 and VE-cadherin. HTNV can infect HUVECs-hTERT and HUVECs with approximately the same efficiency. In HTNV infection, the expression of innate immune molecules, such as IFN-ß, MxA, MxB, IFIT2, IFITM3, COX2, ICAM, CCL5 are similar between HUVECs and HUVECs-hTERT, indicating that the innate immune regulation of HUVECs-hTERT has not changed. Conclusion HUVECs-hTERT can replace primary HUVECs for the study of innate immune response regulation during HTNV infection under certain conditions.

Interferon-Induced Transmembrane Proteins Inhibit Infection by the Kaposi's Sarcoma-Associated Herpesvirus and the Related Rhesus Monkey Rhadinovirus in a Cell-Specific Manner.

The interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral proteins that inhibit the entry of enveloped viruses. We analyzed the effect of IFITMs on the gamma-2 herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related rhesus monkey rhadinovirus (RRV). We used CRISPR/Cas9-mediated gene knockout to generate A549 cells, human foreskin fibroblasts (HFF), and human umbilical vein endothelial cells (HUVEC) with combined IFITM1/2/3 knockout and identified IFITMs as cell-dependent inhibitors of KSHV and RRV infection in A549 cells and HFF but not HUVEC. IFITM overexpression revealed IFITM1 as the relevant IFITM that inhibits KSHV and RRV infection. Fluorescent KSHV particles did not pronouncedly colocalize with IFITM-positive compartments. However, we found that KSHV and RRV glycoprotein-mediated cell-cell fusion is enhanced upon IFITM1/2/3 knockout. Taken together, we identified IFITM1 as a cell-dependent restriction factor of KSHV and RRV that acts at the level of membrane fusion. Of note, our results indicate that recombinant IFITM overexpression may lead to results that are not representative for the situation at endogenous levels. Strikingly, we observed that the endotheliotropic KSHV circumvents IFITM-mediated restriction in HUVEC despite high IFITM expression, while influenza A virus (IAV) glycoprotein-driven entry into HUVEC is potently restricted by IFITMs even in the absence of interferon. Mechanistically, we found that KSHV colocalizes less with IFITM1 and IFITM2 in HUVEC than in A549 cells immediately after attachment, potentially contributing to the observed difference in restriction. IMPORTANCE IFITM proteins are the first line of defense against infection by many pathogens and may also have therapeutic importance, as they, among other effectors, mediate the antiviral effect of interferons. Neither their function against herpesviruses nor their mechanism of action is well understood. We report here that in some cells but not in, for example, primary umbilical vein endothelial cells, IFITM1 restricts KSHV and RRV and that, mechanistically, this is likely effected by reducing the fusogenicity of the cell membrane. Further, we demonstrate potent inhibition of IAV glycoprotein-driven infection of cells of extrapulmonary origin by high constitutive IFITM expression.

The zinc finger transcription factor, KLF2, protects against COVID-19 associated endothelial dysfunction.

Coronavirus disease 2019 (COVID-19) is regarded as an endothelial disease (endothelialitis) with its patho-mechanism being incompletely understood. Emerging evidence has demonstrated that endothelial dysfunction precipitates COVID-19 and its accompanying multi-organ injuries. Thus, pharmacotherapies targeting endothelial dysfunction have potential to ameliorate COVID-19 and its cardiovascular complications. The objective of the present study is to evaluate whether kruppel-like factor 2 (KLF2), a master regulator of vascular homeostasis, represents a therapeutic target for COVID-19-induced endothelial dysfunction. Here, we demonstrate that the expression of KLF2 was reduced and monocyte adhesion was increased in endothelial cells treated with COVID-19 patient serum due to elevated levels of pro-adhesive molecules, ICAM1 and VCAM1. IL-1ß and TNF-α, two cytokines elevated in cytokine release syndrome in COVID-19 patients, decreased KLF2 gene expression. Pharmacologic (atorvastatin and tannic acid) and genetic (adenoviral overexpression) approaches to augment KLF2 levels attenuated COVID-19-serum-induced increase in endothelial inflammation and monocyte adhesion. Next-generation RNA-sequencing data showed that atorvastatin treatment leads to a cardiovascular protective transcriptome associated with improved endothelial function (vasodilation, anti-inflammation, antioxidant status, anti-thrombosis/-coagulation, anti-fibrosis, and reduced angiogenesis). Finally, knockdown of KLF2 partially reversed the ameliorative effect of atorvastatin on COVID-19-serum-induced endothelial inflammation and monocyte adhesion. Collectively, the present study implicates loss of KLF2 as an important molecular event in the development of COVID-19-induced vascular disease and suggests that efforts to augment KLF2 levels may be therapeutically beneficial.

Human rhinovirus 16 induces antiviral and inflammatory response in the human vascular endothelium.

The effect of rhinovirus on airway epithelium is very well described. However, its influence on the vascular endothelium is unknown. The current study assesses the effect of rhinovirus HRV16 on the antiviral and inflammatory response in the human vascular endothelial cells (ECs). HRV16 increased IFN-ß, RANTES, and IP-10 mRNA expression and protein release. HRV16 copy number in ECs reached maximal value 10 h after incubation. Increase in virus copies was accompanied by the enhancement of Toll- and RIG-I-like receptors: TLR3, RIG-I, and MDA5. Additionally, HRV16 increased OAS-1 and PKR mRNA expression, enzymes responsible for virus degradation and inhibition of replication. ICAM-1 blockade decreased HRV16 copy number in ECs and inhibited IFN-ß, RANTES, IP-10, OAS1, PKR, TLR3, RIG-I, and MDA5 mRNA expression increase upon subsequent induction with HRV16. The vascular endothelium may be infected by human rhinovirus and generate antiviral and inflammatory innate response. Results of the study indicate the possible involvement of the vascular endothelium in the immunopathology of rhinoviral airway infections.

Lassa hemorrhagic shock syndrome-on-a-chip.

Lack of experimental human models hinders research on Lassa hemorrhagic fever and the development of treatment strategies. Here, we report the first chip-based model for Lassa hemorrhagic syndrome. The chip features a microvessel interfacing collagen network as a simple mimic for extracellular matrix, allowing for quantitative and real-time vascular integrity assessment. Luminal infusion of Lassa virus-like particles led to a dramatic increase in vascular permeability in a viral load-dependent manner. Using this platform, we showed that Fibrin-derived peptide FX06 can be used to suppress the vascular integrity loss. This simple chip-based model proved promising in the assessment of disease severity and provides an easy-to-use platform for future investigation of Lassa pathogenesis and drug development in a human-like setting.

The Signature Amino Acid Residue Serine 31 of HIV-1C Tat Potentiates an Activated Phenotype in Endothelial Cells.

The natural cysteine to serine variation at position 31 of Tat in HIV-1C disrupts the dicysteine motif attenuating the chemokine function of Tat. We ask if there exists a trade-off in terms of a gain of function for HIV-1C Tat due to this natural variation. We constructed two Tat-expression vectors encoding Tat proteins discordant for the serine 31 residue (CS-Tat vs. CC-Tat), expressed the proteins in Jurkat cells under doxycycline control, and performed the whole transcriptome analysis to compare the early events of Tat-induced host gene expression. Our analysis delineated a significant enrichment of pathways and gene ontologies associated with the angiogenic signaling events in CS-Tat stable cells. Subsequently, we validated and compared angiogenic signaling events induced by CS- vs. CC-Tat using human umbilical vein endothelial cells (HUVEC) and the human cerebral microvascular endothelial cell line (hCMEC/D3). CS-Tat significantly enhanced the production of CCL2 from HUVEC and induced an activated phenotype in endothelial cells conferring on them enhanced migration, invasion, and in vitro morphogenesis potential. The ability of CS-Tat to induce the activated phenotype in endothelial cells could be of significance, especially in the context of HIV-associated cardiovascular and neuronal disorders. The findings from the present study are likely to help appreciate the functional significance of the SAR (signature amino acid residues) influencing the unique biological properties.

Exosomal miR-145-5p derived from orthohantavirus-infected endothelial cells inhibits HTNV infection.

Human infection of orthohantavirus can cause potentially fatal diseases, such as hemorrhagic fever with renal syndrome (HFRS) caused by Hantaan virus (HTNV) in Eurasia. Exosomes are new carriers for information exchange between cells. Cumulative findings suggest that exosomes released from parental infected cells can block or promote viral infection in recipient cells, but the role of exosomes in hantavirus infection is poorly understood. In our study, we identified the exosomes derived from HTNV-infected human vascular endothelial cells (HUVECs) (Exo-HV) and found the antiviral properties of Exo-HV in the uninfected recipient cells. High-throughput sequencing revealed the distinctly expressed miRNAs transcriptomes in Exo-HV. MiR-145-5p, one of the abundant miRNAs packaged into Exo-HV, was found to be able to transferred to recipient cells and functioned by directly targeting M RNA of HTNV 76-118 and inducing type I interferon (IFN-I) response, thus, blocking the viral replication. Concluding, this study indicated that exosomes released by HTNV-infected HUVECs were able to transfer active molecules, miR-145-5p as a proving sample, to mediate novel anti-HTNV activity in the neighboring uninfected cells, which will help us to explore new strategies for the treatment of infectious disease utilizing exosomes with miRNA.

Herpesvirus-infected Hofbauer cells activate endothelial cells through an IL-1ß-dependent mechanism.

INTRODUCTION: Placental viral infections are associated with fetal inflammation and adverse pregnancy outcomes. However, there have been limited studies on how placental macrophages in the villous and adjacent fetal umbilical endothelial cells respond to a viral insult. This study aimed to evaluate the communication between Hofbauer cells (HBCs) and human umbilical vein endothelial cells (HUVECs) during a viral infection. METHODS: HBCs were either uninfected or infected with the γ-herpesvirus, MHV-68, and the conditioned medium (CM) collected. HUVECs were exposed to HBC CM and the levels of the pro-neutrophilic response markers: IL-8; E-selectin; intercellular adhesion molecule 1 (ICAM-1); and vascular adhesion molecule 1 (VCAM-1) measured by ELISA and qPCR. The role of HBC-derived IL-1ß was investigated using an IL-1ß blocking antibody (Ab) or IL-1 receptor antagonist (IL-1Ra). RESULTS: MHV-68 infection of HBCs induced a significant increase in IL-1ß secretion. CM from infected HBCs induced HUVEC expression of IL-8, E-selectin, VCAM-1, ICAM-1 mRNA, and secretion of IL-8. The HUVEC response to the CM of MHV-infected HBCs was inhibited by a neutralizing IL-1ß Ab and by IL-1Ra. DISCUSSION: Virally-induced HBC IL-1ß activates HUVECs to generate a pro-neutrophilic response. This novel cell-cell communication pathway may play an important role in the genesis of fetal inflammation associated with placental viral infection.

Human cytomegalovirus promoting endothelial cell proliferation by targeting regulator of G-protein signaling 5 hypermethylation and downregulation.

Interactions between human cytomegalovirus (HCMV) infection and environmental factors can increase susceptibility to essential hypertension (EH). Although endothelial dysfunction is the initial factor of EH, the epigenetic mechanisms through which HCMV infection induces endothelial cell dysfunction are poorly understood. Here, we evaluated whether HCMV regulated endothelial cell function and assessed the underlying mechanisms. Microarray analysis in human umbilical vein endothelial cells (HUVECs) treated with HCMV AD169 strain in the presence of hyperglycemia and hyperlipidemia revealed differential expression of genes involved in hypertension. Further analyses validated that the regulator of G-protein signaling 5 (RGS5) gene was downregulated in infected HUVECs and showed that HCMV infection promoted HUVEC proliferation, whereas hyperglycemia and hyperlipidemia inhibited HUVEC proliferation. Additionally, treatment with decitabine (DAC) and RGS5 reversed the effects of HCMV infection on HUVEC proliferation, but not triggered by hyperglycemia and hyperlipidemia. In summary, upregulation of RGS5 may be a promising treatment for preventing HCMV-induced hypertension.

Mechanisms of mTOR and Autophagy in Human Endothelial Cell Infected with Dengue Virus-2.

The mechanistic mammalian target of rapamycin (mTOR) plays a crucial role in response to many major cellular processes, including cellular metabolism, proliferation, and autophagy. Both mTOR and autophagy are suggested to be involved in the viral infection. However, little is known about the role of mTOR and autophagy in human endothelial cell infected with dengue virus-2 (DENV-2), this study is to investigate the role of mTOR and autophagy in human umbilical vein endothelial cells (HUVECs) infected with DENV-2 and related regulatory mechanisms. HUVECs were cultured in epithelial cell medium. A series of experiments involving immunohistochemistry, TCID50 method, real-time PCR, western blot, and laser confocal were performed in this study. The cell line was identified as HUVEC by the expression of cell factor VIII. The expression level of DENV-2 mRNA increased and showed an upward trend. Compared with the control group, the fluorescence of autophagy-labeled protein LC3B and lysosome-labeled protein lysosome-associated membrane protein 1 (LAMP1) in the cytoplasm of HUVEC induced by rapamycin was observed, and intensity was significantly enhanced under confocal laser scanning microscope, after fluorescence synthesis, the fluorescence of autophagy-labeled protein LC3B and lysosome-labeled protein LAMP1 overlaps were reduced. The intensity of fluorescence of autophagy-labeled protein LC3B and lysosome-labeled protein LAMP1 increased in 1 × 104 TCID50 DENV-2 infection group, after fluorescence synthesis, fluorescence of autophagy-labeled protein LC3B, lysosome-labeled protein LAMP1, and DEN2 NS1 overlapped. Compared with the control group, the phosphorylation level of mTOR, Atg13, and p-ULK1 in DENV-2-infected group or Rapa treatment group decreased significantly (p < 0.05), and the level of LC3-II increased significantly (p < 0.05). These results suggest that DENV-2 induces autophagy in HUVECs through mTOR signaling molecule.

The role of sphingosine-1-phosphate signaling in HSV-1-infected human umbilical vein endothelial cells.

Infections with the herpes simplex virus type 1 (HSV-1) are common and widespread. Most infections remain undetected but severe forms may develop in newborns and in immunocompromised patients. Moreover, HSV-1 might be involved in the pathogenesis of atherosclerosis, which may include viral infection of the endothelium. Antiviral therapy is efficient to treat symptomatic patients. However, an increasing accumulation of resistance-associated mutations has been observed in the viral genome. Thus, new antiviral strategies are focused on host factors. Among others, signaling of bioactive sphingolipids seems to be important in mediating HSV-1 replication. With the present study, regulation and function of sphingosine-1-phosphate (S1P)-based signaling were analyzed in HSV-1-infected human umbilical vein endothelial cells (HUVEC). Our data indicate that viral replication in endothelial cells relies on sphingosine kinase (SK) activity and S1P receptor (S1PR)1,3-5 signaling, which involves the activation of phosphatidylinositol-3-kinase (PI3K) and the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac-1). Inhibitor- or siRNA-meditated reduction of Rac-1 activity decreased HSV-1 replication. In general, targeting S1P-related signaling may be a successful strategy to establish new anti-HSV-1 therapies.

Hantavirus Inhibits TRAIL-Mediated Killing of Infected Cells by Downregulating Death Receptor 5.

Cytotoxic lymphocytes normally kill virus-infected cells by apoptosis induction. Cytotoxic granule-dependent apoptosis induction engages the intrinsic apoptosis pathway, whereas death receptor (DR)-dependent apoptosis triggers the extrinsic apoptosis pathway. Hantaviruses, single-stranded RNA viruses of the order Bunyavirales, induce strong cytotoxic lymphocyte responses in infected humans. Cytotoxic lymphocytes, however, are largely incapable of eradicating hantavirus-infected cells. Here, we show that the prototypic hantavirus, Hantaan virus (HTNV), induces TRAIL production but strongly inhibits TRAIL-mediated extrinsic apoptosis induction in infected cells by downregulating DR5 cell surface expression. Mechanistic analyses revealed that HTNV triggers both 26S proteasome-dependent degradation of DR5 through direct ubiquitination of DR5 and hampers DR5 transport to the cell surface. These results corroborate earlier findings, demonstrating that hantavirus also inhibits cytotoxic cell granule-dependent apoptosis induction. Together, these findings show that HTNV counteracts intrinsic and extrinsic apoptosis induction pathways, providing a defense mechanism utilized by hantaviruses to inhibit cytotoxic cell-mediated eradication of infected cells.

Dentin matrix protein 1 correlates with the severity of hemorrhagic fever with renal syndrome and promotes hyper-permeability of endothelial cells infected by Hantaan virus.

Hantaviruses are the major causative agents of hemorrhagic fever with renal syndrome (HFRS) in humans, which is characterized by increased capillary permeability. Dentin matrix protein 1 (DMP1) has been shown to degrade components of the basal membrane and interendothelial junctions via matrix metalloproteinase-9. To study the changes of serum DMP1 in HFRS, we determined the concentration of DMP1 using sandwich enzyme-linked immunosorbent assay. We found that serum DMP1 concentrations increased significantly, and reached peak value during the oliguric phase and in the critical group in HFRS patients. Moreover, serum DMP1 concentrations were closely related to blood urea nitrogen, creatinine, cystatin C, and vascular endothelial growth factor (VEGF). We further explored the role of DMP1 in HTNV-infected human umbilical vein endothelial cells (HUVECs) model. Data from immunocytochemistry showed that VEGF and tumor necrosis factor-α (TNF-α) promoted the expression of DMP1 on HTNV-infected HUVECs. Results from transwell assays demonstrated that the permeability of HUVECs increased significantly after HTNV infection with the addition of DMP1, VEGF, and TNF-α. This study suggests that elevated DMP1 concentrations may be associated with disease stage, severity, and the degree of acute kidney injury. DMP1 is involved in the regulation of capillary permeability in HFRS caused by hantavirus infection.

Epstein-Barr virus noncoding RNAs from the extracellular vesicles of nasopharyngeal carcinoma (NPC) cells promote angiogenesis via TLR3/RIG-I-mediated VCAM-1 expression.

Viral noncoding RNAs (Epstein-Barr virus-encoded RNAs, EBERs) are believed to play a critical role in the progression of lymphoma and nasopharyngeal carcinoma (NPC). However, the accurate mechanisms accounting for their oncogenic function have not been elucidated, especially in terms of interaction between tumor cells and mesenchymal cells. Here, we report that, in addition to NPC cells, EBERs are also found in endothelial cells in Epstein-Barr virus (EBV)-infected NPC parenchymal tissues, which implicates NPC-derived extracellular vesicles (EVs) in transmitting EBERs to endothelial cells. In support of this hypothesis, we first ascertained if EBERs could be transferred to endothelial cells via EVs isolated from NPC culture supernatant. Then, we clarified that EVs-derived EBERs could promote angiogenesis through stimulation of VCAM-1 expression. Finally, we explored the involvement of EBER recognition by TLR3 and RIG-I in NPC angiogenesis. Our observations collectively illustrate the significance and mechanism of EVs-derived EBERs in angiogenesis and underlie the interaction mechanisms between EBV-infected NPC cells and the tumor microenvironment.

IL-8 as a potential in-vitro severity biomarker for dengue disease.

Dengue is a common infection, caused by dengue virus. There are four different dengue serotypes, with different capacity to cause severe dengue infections. Besides, secondary infections with heterologous serotypes, concurrent infections of multiple dengue serotypes may alter the severity of dengue infection. This study aims to compare the severity of single infection and concurrent infections of different combinations of dengue serotypes in-vitro. Human mast cells (HMC)-1.1 were infected with single and concurrent infections of multiple dengue serotypes. The infected HMC-1.1 supernatant was then added to human umbilical cord vascular endothelial cells (HUVEC) and severity of dengue infections was measured by the percentage of transendothelial electrical resistance (TEER). Levels of IL10, CXCL10 and sTRAIL in HMC-1.1 and IL-8, IL-10 and CXCL10 in HUVEC culture supernatants were measured by the ELISA assays. The result showed that the percentage of TEER values were significantly lower in single infections (p< 0.05), compared to concurrent infections on day 2 and 3, indicating that single infection increase endothelial permeability greater than concurrent infections. IL-8 showed moderate correlation with endothelial permeability (r > 0.4), indicating that IL-8 may be suitable as an in-vitro severity biomarker. In conclusion, this in-vitro model presented few similarities with regards to the conditions in dengue patients, suggesting that it could serve as a severity model to test for severity and levels of severity biomarkers upon different dengue virus infections.

Human Herpesvirus 6A and 6B inhibit in vitro angiogenesis by induction of Human Leukocyte Antigen G.

We have previously reported that human herpesvirus 6 (HHV-6) infection of endothelial cells (ECs) induces the loss of angiogenic properties, through the expression of HHV-6 U94, possibly associated to the release of a soluble mediator. It is also known that the soluble isoform of HLA-G exhibits an anti-angiogenic function, important in implantation, transplantation and neoplastic development. In this study, we analyzed the expression of HLA-G in HHV-6 infected ECs, showing that both HHV-6A and HHV-6B infection induce a potent up-modulation of HLA-G, including both membrane and soluble isoforms. Interestingly, HHV-6A and HHV-6B induced different isoforms of HLA-G. The virus-induced increase of HLA-G was likely due to the expression of the U94 viral gene, that by itself was able to reproduce the effect of whole virus. The effect of U94 was mediated by human transcription factor ATF3, that induced HLA-G activation by recognizing a consensus sequence on its promoter. Virus-induced inhibition of ECs angiogenic ability directly correlated to HLA-G expression and release, and the addition of anti-HLA-G antibody restored the angiogenic properties of HHV6-infected ECs. The induction of HLA-G expression in ECs might represent an important mediator of HHV-6 induced effects.