Untreated Human Umbilical Vein Endothelial Cells Research Articles

Pseudomonas aeruginosa LecB suppresses immune responses by inhibiting transendothelial migration.

Pseudomonas aeruginosa is a Gram-negative bacterium causing morbidity and mortality in immuno-compromised humans. It produces a lectin, LecB, that is considered a major virulence factor, however, its impact on the immune system remains incompletely understood. Here we show that LecB binds to endothelial cells in human skin and mice and disrupts the transendothelial passage of leukocytes in vitro. It impairs the migration of dendritic cells into the paracortex of lymph nodes leading to a reduced antigen-specific T cell response. Under the effect of the lectin, endothelial cells undergo profound cellular changes resulting in endocytosis and degradation of the junctional protein VE-cadherin, formation of an actin rim, and arrested cell motility. This likely negatively impacts the capacity of endothelial cells to respond to extracellular stimuli and to generate the intercellular gaps for allowing leukocyte diapedesis. A LecB inhibitor can restore dendritic cell migration and T cell activation, underlining the importance of LecB antagonism to reactivate the immune response against P.aeruginosa infection.

APOL1 variant-expressing endothelial cells exhibit autophagic dysfunction and mitochondrial stress.

Polymorphisms in the Apolipoprotein L1 (APOL1) gene are common in ancestrally African populations, and associate with kidney injury and cardiovascular disease. These risk variants (RV) provide an advantage in resisting Trypanosoma brucei, the causal agent of African trypanosomiasis, and are largely absent from non-African genomes. Clinical associations between the APOL1 high risk genotype (HRG) and disease are stronger in those with comorbid infectious or immune disease. To understand the interaction between cytokine exposure and APOL1 cytotoxicity, we established human umbilical vein endothelial cell (HUVEC) cultures representing each APOL1 genotype. Untreated HUVECs were compared to IFNɣ-exposed; and APOL1 expression, mitochondrial function, lysosome integrity, and autophagic flux were measured. IFNɣ increased median APOL1 expression across all genotypes 22.1 (8.3 to 29.8) fold (p=0.02). Compared to zero risk variant-carrying HUVECs (0RV), HUVECs carrying 2 risk variant copies (2RV) showed both depressed baseline and maximum mitochondrial oxygen consumption (p<0.01), and impaired mitochondrial networking on MitoTracker assays. These cells also demonstrated a contracted lysosomal compartment, and an accumulation of autophagosomes suggesting a defect in autophagic flux. Upon blocking autophagy with non-selective lysosome inhibitor, hydroxychloroquine, autophagosome accumulation between 0RV HUVECs and untreated 2RV HUVECs was similar, implicating lysosomal dysfunction in the HRG-associated autophagy defect. Compared to 0RV and 2RV HUVECs, HUVECs carrying 1 risk variant copy (1RV) demonstrated intermediate mitochondrial respiration and autophagic flux phenotypes, which were exacerbated with IFNɣ exposure. Taken together, our data reveal that IFNɣ induces APOL1 expression, and that each additional RV associates with mitochondrial dysfunction and autophagy inhibition. IFNɣ amplifies this phenotype even in 1RV HUVECs, representing the first description of APOL1 pathobiology in variant heterozygous cell cultures.

CircDIP2C ameliorates oxidized low-density lipoprotein-induced cell dysfunction by binding to miR-556-5p to induce TET2 in human umbilical vein endothelial cells

Circular RNAs (circRNAs) are a group of conserved noncoding RNAs. Recent reports reveal that circRNAs play vital parts in cardiovascular system, including atherosclerosis (AS). The present study is designed to reveal the role of circRNA DIP2C-disco interacting protein 2 homolog C (circDIP2C) in oxidized low-density lipoprotein (ox-LDL)-triggered damage of human umbilical vein endothelial cells (HUVECs). The expression levels of circDIP2C, microRNA-556-5p (miR-556-5p) and tet methylcytosine dioxygenase 2 (TET2) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Protein expression was determined by western blot analysis. Cell viability and angiogenesis were demonstrated by cell counting kit-8 and tube formation assays, respectively. The levels of reactive oxygen species (ROS) and malondialdehyde (MDA) were checked by ROS and MDA determination assays. Superoxide dismutase (SOD) and lactate dehydrogenase (LDH) activity assays were performed to detect the activity of SOD and LDH. The binding sites of miR-556-5p in circDIP2C or TET2 were predicted by online databases, and identified by dual-luciferase reporter, RNA immunoprecipitation and RNA pull-down assays. CircDIP2C and TET2 expression were obviously decreased, while miR-556-5p expression was increased in ox-LDL-induced HUVECs in comparison with untreated HUVECs. Ox-LDL treatment inhibited cell viability and angiogenesis, promoted oxidative stress, enhanced cytotoxicity and activated NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway. CircDIP2C upregulation protected HUVECs from ox-LDL-induced injury. Additionally, circDIP2C directly bound to miR-556-5p, which was further found to target TET2. MiR-556-5p mimics or TET2 silencing could attenuate the effect of circDIP2C overexpression on ox-LDL-induced cell disorder. Thus, we came a conclusion that circDIP2C protected against ox-LDL-induced HUVEC damage by upregulating TET2 expression through sponging miR-556-5p, which provided a strategy for the therapy of AS.

L-Threoascorbic acid treatment promotes S. aureus-infected primary human endothelial cells survival and function, as well as intracellular bacterial killing, and immunomodulates the release of IL-1β and soluble ICAM-1

BackgroundVitamin C (ascorbic acid, AscH2) has been shown to enhance immunity. Here, we studied its immunomodulatory effect on human endothelial cells (ECs) during S. aureus infection. Materials and methodsThe ex vivo effects of AscH2 were performed on primary human umbilical vein endothelial cells (HUVECs) infected or not with S. aureus. ResultsAscH2 treatment induced a marked downregulation of nitric oxide (NO) production and a moderate upregulation of arginase activity in S. aureus-infected HUVECs (respectively, p < 0.05 and p > 0.05). Although the upregulated release levels of soluble intercellular adhesion molecular 1 (sICAM-1/sCD54) and sE-selectin (sCD62E) molecules were not significantly different between treated and untreated S. aureus-infected HUVECs, AscH2 treatment induced reversing effect on sICAM-1 release when comparing to uninfected control HUVECs. Moreover, AscH2 treatment appears to have a significant effect on preventing HUVEC necrosis induced by S. aureus infection (p < 0.05). Furthermore, AscH2 treatment induced a significant upregulation of cell protective redox biomarker in S. aureus-infected, as shown by superoxide dismutase (SOD) activity (p < 0.05), but not by catalase activity (p > 0.05). Additionally, S. aureus infection markedly downregulated total bound calcium ions (bCa2+) levels as compared to control HUVECs, whereas, AscH2 treatment induced a slight upregulation of bCa2+ levels in infected HUVECs as compared to infected and untreated HUVECs (p > 0.05). On the other hand, AscH2 treatment downregulated increased total cellular cholesterol content (tccCHOL) levels in HUVECs induced by S. aureus infection (p < 0.05). In addition, AscH2 treatment markedly reversed S. aureus effect on upregulation of intracellular glucose (iGLU) levels within infected HUVECs (p < 0.05). Moreover, AscH2 treatment significantly downregulated S. aureus growth (p < 0.05), and significantly upregulated bacterial internalization and intracellular killing by HUVECs (p < 0.05), as well as their cell cycle activation (p < 0.01). Finally, AscH2 treatment has a slight effect on the production of interleukin 6 (IL-6), but induced a marked downregulation of that of IL-1β in S. aureus-infected HUVECs (respectively, p > 0.05, and p < 0.05). ConclusionsOur outcomes demonstrated that, during S. aureus infection, AscH2 treatment promotes human ECs survival and function, as well as prevents inflammatory response exacerbation, while inducing bactericidal activity.

The Effect of Acanthopanax sessiliflorum Cheonghyeol Plus on NF-κB and MAPKs Signaling and Vascular Attachment Factors

Objective: This study was performed to investigate the effect of Acanthopanax sessiliflorum Cheonghyeol plus (ASCP) on NF-κB and MAPK signaling and vascular adhesion factors associated with dyslipidemia in human umbilical vein endothelial cells (HUVECs). Methods: We measured the scavenging activity of DPPH radical and ABTS radical by ASCP in HUVECs. We measured the protein expression levels of NF-κB, IκBα, ERK, JNK, and p38 after treatment of HUVECs with TNF-α. We measured the expression levels of MCP-1, ICAM-1, and VCAM-1 mRNA and of MCP-1, ICAM-1, and VCAM-1 biomarkers after treatment of HUVECs with TNF-α. Results: The DPPH and ABTS radical scavenging activity of ASCP increased in a concentration-dependent manner. NF-κB, IκB, ERK, p38 protein expression levels decreased following ASCP treatment at all concentrations compared to untreated control HUVECs. JNK protein expression levels decreased in ASCP-treated HUVECs compared to untreated controls at concentrations of 100 μg/mL. MCP-1 mRNA expression level decreased with ASCP treatment ≥200 μg/mL compared to the control. ICAM-1 and VCAM-1 mRNA expression levels decreased at all concentrations compared to the control. MCP-1 protein expression level was reduced compared to the control at concentrations ≥200 μg/mL, ICAM-1 protein expression level was reduced compared to the control at concentrations ≥100 μg/mL, and VCAM-1 protein expression level was reduced at all concentrations. Conclusions: These results suggest that ASCP has an antioxidative and hypolipidemic effect and that ASCP could treat and prevent dyslipidemia, atherosclerosis, and cardio-cerebrovascular diseases. Keywords: Acanthopanax sessiliflorum, Cheonghyeol-Plus, dyslipidemia, HUVECs, antioxidation activity

Uptake of Schistosoma mansoni extracellular vesicles by human endothelial and monocytic cell lines and impact on vascular endothelial cell gene expression

The ability of the parasitic blood fluke Schistosoma mansoni and other parasitic helminths to manipulate host biology is well recognised, but the mechanisms that underpin these phenomena are not well understood. An emerging paradigm is that helminths transfer their biological cargo to host cells by secretion of extracellular vesicles (EVs). Herein, we show that two populations of S. mansoni secreted EVs – exosome-like vesicles (ELVs) and microvesicles (MVs) – are actively internalised in two distinct human cell lines that reflect the resident cell types encountered by the parasite in vivo: human umbilical vein endothelial cells (HUVECs) and THP-1 monocytes. RNA-sequencing of HUVECs co-cultured with S. mansoni ELVs compared with untreated HUVECs revealed differential expression of genes associated with intravascular parasitism, including vascular endothelial contraction, coagulation, arachidonic acid metabolism and immune cell trafficking and signalling. Finally, we show that antibodies raised against recombinant tetraspanin (TSP) proteins from the surface of S. mansoni EVs significantly blocked EV uptake by both HUVECs and THP-1 monocytes whereas pre-immunisation antibodies did not. To our knowledge, this is the first evidence demonstrating the internalisation of secreted EVs from any helminth into vascular endothelial cells, providing novel insight into the potential mechanisms underlying host-schistosome interactions. The ability of anti-TSP antibodies to block vesicle uptake by host target cells further supports the potential of TSPs as promising antigens for an anti-fluke vaccine. It also suggests a potential mechanism whereby the current candidate human schistosomiasis vaccine, Sm-TSP-2, exerts its protective effect in animal models.

Reparative effect of mesenchymal stromal cells on endothelial cells after ischemic and inflammatory injury

Background & Aim Renal endothelium is damaged by ischemia reperfusion injury (IRI) during transplantation as a result of the hypoxic metabolism and the resumption of oxygen supply after reperfusion. It has been shown that mesenchymal stromal cells (MSC) are effective in enhancing kidney function after IRI. However, the cellular targets and mechanisms of action behind MSC effects are still poorly understood. Therefore, our objective was to evaluate the role of MSC in repairing injured endothelium as it is the first tissue with which they interact after infusion and to determine the specific interactions involved in this process. The role of physical communication and cytokine secretion in the reparative action of MSC was studied as well as its molecular mechanisms. Methods, Results & Conclusion Human umbilical vein endothelial cells (HUVEC) were submitted to hypoxic and inflammatory insult (TNF-a). This increased the expression of activation molecules such as CD54, CD62e and HLA-II on HUVEC's cell surface. Consequently, an increase of almost 20% in the adherence of MSC to HUVEC was observed in comparison to untreated HUVEC. Moreover, an increase of around 20% in the migratory capacity of MSC towards injured HUVEC was observed. MSC showed a 15 to 20% increase in their ability to transmigrate through injured HUVEC monolayers. Once MSC were in contact with HUVEC, the oxidative stress of HUVEC decreased as observed by a decrease in reactive oxygen species levels, their proliferative status improved and MSC restored the angiogenic potential to levels close to those from healthy HUVEC. To determine whether physical interaction or soluble factors released by MSC are fundamental for MSC regenerative capacity, a Boyden chamber system experiment was performed and we observed that both were required to achieve the full reparative effect of MSC. Moreover, we observed an increase in the expression of CD44 and CD29 adhesion molecules on MSC membrane after co-culture with injured HUVEC, as well as their ligands CD62e and CD106 on HUVEC membrane. We conclude that MSC are able to repair the injury caused by ischemic and inflammatory injury on HUVEC via a mechanism that requires both physical and paracrine interaction between MSC and HUVEC. We are currently studying the specific effector molecules involved in MSC and HUVEC cell-to-cell interaction which will allow targeted modification to improve the regenerative effects of MSC therapy.

Piwi-interacting RNAs play a role in vitamin C-mediated effects on endothelial aging.

The underlying mechanisms that mediate the effects of vitamin C on endothelial cell aging are widely unknown. To investigate whether Piwi-interacting RNAs (piRNAs) are involved in this process, an endothelial aging model was induced in vitro using H2O2 in human umbilical vein endothelial cells (HUVECs) and then treated with vitamin C (VC). Untreated HUVECs without H2O2 exposure were used to serve as the negative control group. Cell cycle, cell viability, and aging-associated protein expression were assessed, and RNA sequencing was performed to reveal the piRNA profile. Functional and regulatory networks of the different piRNA target genes were predicted by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) analysis. H2O2 induced G1 phase cell arrest, decreased cell viability, and upregulated the senescence marker p16 in HUVECs. We found that VC treatment inhibited G1 phase cell arrest, increased the number of cells in the S and G2/M phases, increased cell viability, and decreased p16 expression. The piRNA expression profiles revealed that a large proportion of piRNAs that were differentially expressed in H2O2-treated HUVECs were partly normalized by VC. Furthermore, a number of piRNAs associated with the response to VC in H2O2-treated HUVECs were linked with senescence and cell cycle-related pathways and networks. These results indicate that the ability of VC to attenuate H2O2-mediated endothelial cell senescence may be associated with changes in expression of piRNAs that are linked to the cell cycle.

Tetramethylpyrazine alleviates LPS-induced inflammatory injury in HUVECs by inhibiting Rho/ROCK pathway

Endothelial dysfunction plays an important role in the pathogenesis of acute lung injury (ALI). Tetramethylpyrazine (TMP) has been reported to attenuate harmful changes in ALI rats. However, the effects of TMP on endothelial cell injury and its underlying mechanisms remain unknown. In this study, human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS) was used as an inflammatory injury model, also served as LPS group. HUVECs pretreated with TMP for 2 h before induced by LPS was served as LPS + TMP group. Untreated HUVECs was served as control group. After incubation with LPS for 12 h, cell viability and morphology, cell apoptosis rate, CD31-positive endothelial microparticles (EMPs) release, proinflammatory cytokines secretion, and ROCK II expression were evaluated. Compared with LPS group, TMP pretreatment improved cell viability and morphology. Besides, cell apoptosis rate, CD31-positive EMPs amount, TNF-α and IL-1β concentrates, and ROCK II mRNA and protein levels in LPS + TMP group were significantly decreased when compared with LPS group. To further confirm the mechanism, HUVECs in all the above groups were pretreated with Y27632 (ROCK II inhibitor) for 30 min before grouping, then treated as above. No significant differences in cell apoptosis rate, CD31-positive EMPs amount, and ROCK II expression between Y27632 + LPS group and Y27632 + LPS + TMP group were found. To sum up, our study found that TMP alleviated LPS-induced inflammatory injury in HUVECs by inhibiting Rho/ROCK pathway.

Thrombopoietin Prevents CoCl2-inducing Apoptosis of HUVEC through the PI3K/AKT Pathway

To investigate the effect of thrombopoietin (TPO) on chemical hypoxia-induced apoptosis of human umbilical vein endothelial cells (HUVEC), and to explore its potential mechanism. The experiment was divided into 4 groups. The untreated HUVECs were used as normal control group. HUVECs treated with CoCl2 was CoCl2 group, and TPO was added into the culture medium 48 h before CoCl2 treatment as TPO + CoCl2 group. The cells was treated with TPO alone as TPO group. The cell viability and apoptosis of each groups were tested by Cell Counter Kit 8 (CCK-8) assay and flow cytometry. The expression of Caspase-3 and mitochondrial membrane potential (MMP) were then determined by flow cytometry with Caspase-3-PE and JC-1. The effect of TPO in PI3K/AKT pathway was detected by using Western blot. CoCl2 significantly inhibited the growth of HUVECs. The cell viability of HUVECs decreased gradually with enhancement of CoCl2 at a gradient of chemical concentrations (r= -0.997). CoCl2 dramatically increased apoptosis of HUVECs, whereas pre-treatment with TPO rescued cell apoptosis induced by CoCl2 (P<0.001). Further investigation found that TPO decreased the expression of Caspase-3 and inhibited the reduction of MMP induced by CoCl2 (P<0.05). TPO could increased the activation of PI3K/AKT pathway in HUVECs. TPO has a protective effect against CoCl2-induced apoptosis of HUVECs through activating the PI3K/AKT pathway, thus decreasing the expression of apoptosis protease Caspase-3 and inhibiting the reduction of MMP.

Effects of interaction between dengue virus type 2-infected HUVECs and human CD4+ T cells on the expression of adhesion molecules and immunosuppressive factors

Objective To investigate the effects of interaction between human umbilical vein endothelial cells (HUVECs) which were infected with dengue virus type 2 (DENV-2) and CD4+ T cells on the expression of ICAM-1 (intercellular adhesion molecule 1), VCAM-1 (vascular cell adhesion molecule 1), IL-10 and TGF-β1 at mRNA level for further understanding the immunological mechanism of DENV infection. Methods HUVECs were treated with CYM-5442, a selective agonist for sphingosine-1-phosphate receptor 1 (S1P1), for 24 hours and then infected with 103 TCID50 (50% tissue culture infective dose) of DENV-2 before co-culturing with CD4+ T cells. Changes in the expression of NS1 (DENV-2 nonstructural protein), SPHK1 (sphingosine kinase 1, phosphorylating sphingosine to S1P), ICAM-1, VCAM-1, IL-10 and TGF-β1 at mRNA level were detected by real-time PCR after 4, 8, 12, 24, 48 and 72 hours of co-culturing. Results There was a certain timeliness in the expression of NS1 at mRNA level after infecting HUVECs with DENV-2 and the expression reached a peak at 24 h. Treating HUVECs with or without CYM-5442 had no significant influence on the expression of DENV-2 NS1 at mRNA level. The expression of SPHK1 at mRNA level was significantly increased after treating HUVECs with CYM-5442 and DENV-2 (P<0.05). Compared with DENV-2-infected or untreated HUVECs, Co-culturing DENV-2-infected HUVECs with CD4+ T cells increased the expression of ICAM-1 and VCAM-1 in HUVECs at mRNA level (P<0.01) as well as the expression of IL-10 in CD4+ T cells at mRNA level (P<0.05), but had no significant influence on the expression of TGF-β1 in CD4+ T cells at mRNA level. Conclusion This study shows that DENV-2 can replicate and proliferate in HUVECs, but CD4+ T cells inhibit the replication and proliferation. CD4+ T cells play an important role in promoting the expression of VCAM-1 and ICAM-1 in DENV-2-infected HUVECs at mRNA level, activating HUVECs and increasing inflammation, which may be associated with increased vascular permeability induced by DENV-2 infection. Co-culturing CD4+ T cells with DENV-2-infected HUVECs promotes the expression of IL-10 in CD4+ T cells at mRNA level, but has no significant effect on TGF-β1. Key words: HUVEC; CD4+ T cell; DENV-2; Cytokine; Immunoregulation

A Facile Method to Probe the Vascular Permeability of Nanoparticles in Nanomedicine Applications

The effectiveness of nanoparticles (NP) in nanomedicine depends on their ability to extravasate from vasculature towards the target tissue. This is determined by their permeability across the endothelial barrier. Unfortunately, a quantitative study of the diffusion permeability coefficients (Pd) of NPs is difficult with in vivo models. Here, we utilize a relevant model of vascular-tissue interface with tunable endothelial permeability in vitro based on microfluidics. Human umbilical vein endothelial cells (HUVECs) grown in microfluidic devices were treated with Angiopoietin 1 and cyclic adenosine monophosphate (cAMP) to vary the Pd of the HUVECs monolayer towards fluorescent polystyrene NPs (pNPs) of different sizes, which was determined from image analysis of their fluorescence intensity when diffusing across the monolayer. Using 70 kDa dextran as a probe, untreated HUVECs yielded a Pd that approximated tumor vasculature while HUVECs treated with 25 μg/mL cAMP had Pd that approximated healthy vasculature in vivo. As the size of pNPs increased, its Pd decreased in tumor vasculature, but remained largely unchanged in healthy vasculature, demonstrating a trend similar to tumor selectivity for smaller NPs. This microfluidic model of vascular-tissue interface can be used in any laboratory to perform quantitative assessment of the tumor selectivity of nanomedicine-based systems.

Disruption of Endothelial Cell Homeostasis Plays a Key Role in the Early Pathogenesis of Coronary Artery Abnormalities in Kawasaki Disease

Disruption of endothelial cell homeostasis may be associated with the pathogenesis of coronary artery abnormalities (CAA) in Kawasaki disease (KD). We sought to clarify the poorly understood pathogenic role of endothelial cell survival and death in KD vasculitis. Human umbilical vein endothelial cells (HUVECs) stimulated with sera from KD patients, compared with sera from patients with bacterial infections, exhibited significant increases in cytotoxicity, high mobility group box protein 1 (HMGB-1), and caspase-3/7 and a decrease in phosphorylated Akt/Akt (pAkt/Akt) ratios. HUVECs stimulated with sera from KD patients treated with immunoglobulin (IG) showed significantly decreased cytotoxicity, HMGB-1, and caspase-3/7 levels and increased pAkt/Akt ratios, as compared with results for untreated HUVECs (P < 0.001, P = 0.008, P = 0.040, and P < 0.001, respectively). In HUVECs stimulated with sera from KD patients, the increased cytotoxicity levels and the suppression of increased pAkt/Akt ratios after subsequent IG treatment were closely related to the development of CAA (P = 0.002 and P = 0.035). Our data reveal that shifting the balance toward cell death rather than survival appears to perturb endothelial cell homeostasis and is closely related to the development of CAA. The cytoprotective effects of IG treatment appear to ameliorate endothelial cell homeostasis.

Impact of Oxidised Very Low-Density Lipoproteins on Endothelial Cell Viability and Migration: Implications for Vascular Repair

Background: Population studies consistently demonstrate a direct relationship between triglyceride levels and cardiovascular risk. The influence of triglyceride rich lipoproteins on the artery wall have not been well characterised. Here, we investigated the ex vivo effects of very low-density lipoproteins (VLDL) on vascular endothelial cells. Methods: VLDL was extracted from plasma of healthy human subjects by ultracentrifugation and modified by copper induced oxidation or glycation. The impact of co-incubation of native, oxidised and glycated VLDL (0-100 μg/ml) with human umbilical vein endothelial cells (HUVECs) for 12 hours on cellular viability was assessed using a colorimetric WST-1 assay and cellular migration using a scratch assay (50 μg/ml). These experiments were repeated following co-incubation of HUVECs with high-density lipoproteins isolated from healthy human subjects. Results: Oxidised, but not native or glycated VLDL, inhibited HUVECs viability by 15% (p<0.05) at concentrations greater than 50 μg/ml. In a similar fashion, oxidised, but not native or glycated VLDL, reduced HUVECs migration by 28% (p<0.05) compared with untreated HUVECs. Co-incubation of HUVECs with native HDL reduced the impact of oxidised VLDL on HUVEC viability by 21% (p<0.05). Conclusions: Oxidation of VLDL results in impairment of endothelial cell viability and migration, providing an important adverse mechanistic role of triglyceride rich lipoproteins in vascular repair. The ability of co-incubation with HDL to inhibit this effect provides another potential mechanism by which HDL exerts atheroprotective effects.

Microvesicles derived from hypoxia/reoxygenation-treated human umbilical vein endothelial cells promote apoptosis and oxidative stress in H9c2 cardiomyocytes.

BackgroundVascular endothelial dysfunction is the closely related determinant of ischemic heart disease (IHD). Endothelial dysfunction and ischemia/reperfusion injury (IRI) have been associated with an increase in microvesicles (MVs) in vivo. However, the potential contribution of endothelial microvesicles (EMVs) to myocardial damage is unclear. Here we aimed to investigate the role of EMVs derived from hypoxia/reoxygenation (H/R) -treated human umbilical vein endothelial cells (HUVECs) on cultured H9c2 cardiomyocytes.ResultsH/R injury model was established to induce HUVECs to release H/R-EMVs. The H/R-EMVs from HUVECs were isolated from the conditioned culture medium and characterized. H9c2 cardiomyocytes were then incubated with 10, 30, 60 μg/mL H/R-EMVs for 6 h. We found that H9c2 cells treated by H/R-EMVs exhibited reduced cell viability, increased cell apoptosis and reactive oxygen species (ROS) production. Moreover mechanism studies demonstrated that H/R-EMVs could induce the phosphorylation of p38 and JNK1/2 in H9c2 cells in a dose-dependent manner. In addition, H/R-EMVs contained significantly higher level of ROS than EMVs generated from untreated HUVECs, which might be a direct source to trigger a cascade of myocardial damage.ConclusionWe showed that EMVs released during H/R injury are pro-apoptotic, pro-oxidative and directly pathogenic to cardiomyocytes in vitro. EMVs carry ROS and they may impair myocardium by promoting apoptosis and oxidative stress. These findings provide new insights into the pathogenesis of IRI.

Candida albicans triggers the expression of inflammatory genes in human umbilical vein endothelial cells.

The aim of the present study was to investigate the pathogenesis of Candida albicans in human umbilical vein endothelial cells (HUVECs) and to screen for aberrantly expressed genes during the process of infection. GSE7355 (accession no.) was downloaded from the National Center of Biotechnology Information Gene Expression Omnibus database and used to identify the differentially-expressed genes (DEGs) between the two groups, which included 4 samples from an untreated HUVEC control group, and 4 samples from HUVECs exposed to C. albicans. Subsequently, the gene ontology (GO) function package was used to perform GO and pathway enrichment analysis, prior to the extraction of DEG correlations in the Kyoto Encyclopedia of Genes and Genomes. A protein-protein interaction (PPI) network was constructed using the String database. In total, 77 DEGs were identified, including 69 upregulated and 8 downregulated DEGs in the C. albicans-infected HUVEC samples. DEGs were significantly enriched in response to external stimuli and chemokine activity. In addition, DEG FBJ murine osteosarcoma viral oncogene homolog (FOS) and interleukin (IL)-6 were significantly enriched in the Toll-like receptor signaling pathway. Nuclear factor κ light polypeptide gene enhancer in B cells 2 (NFKB2) was significantly enriched in the mitogen-activated protein kinase signaling pathway. In the interaction network of DEGs, according data included in the KEGG database, FOS and NFKB2 had higher connectivity degrees. Notably, FOS, IL-6 and intercellular adhesion molecule 1 were demonstrated to have higher connectivity degrees in the PPI network. FOS, IL-6 and NFKB2 may be important genes for C. albicans infection in HUVECs, and these genes may act as therapeutic targets to treat patients infected with C. albicans.

Lactobacillus acidophilus Increases the Anti-apoptotic Micro RNA-21 and Decreases the Pro-inflammatory Micro RNA-155 in the LPS-Treated Human Endothelial Cells.

Given the anti-inflammatory and protective role of probiotics in atherosclerosis and the regulatory role of micro RNA (miRNA) in endothelial cell (dys) functions, this study aimed to investigate the effect of Lactobacillus acidophilus (La) on cellular death and the expression of miRNA-21, 92a, 155, and 663 in human umbilical vein endothelial cell (HUVEC) induced by Escherichia coli lipopolysaccharide (Ec-LPS). LPS-treated and untreated HUVECs were cultured in the presence of different La conditions such as La-conditioned media (LaCM), La water extract (LaWE), La culture-filtered (LaFS) and unfiltered supernatants (LaUFS). After 24h, apoptosis, necrosis and the levels of the mentioned miRNAs were measured using flow cytometry and real-time PCR methods, respectively. LaCM decreased apoptosis, necrosis and inflammatory miR-155 and conversely increased anti-apoptotic miR-21 in Ec-LPS-treated HUVECs. Association analysis revealed negative correlations between necrosis and the levels of miR-21, miR-92a, and miR-155. The beneficial effects of L. acidophilus on the ECs death and expression of atherosclerosis related miRNAs in these cells imply a new aspect of its regulation in cardiovascular diseases rather than previously described ones and suggest this probiotic bacterium as a candidate in the preventative therapy of atherosclerosis.

The differential roles of Slit2-exon 15 splicing variants in angiogenesis and HUVEC permeability.

Slit2, a secreted glycoprotein, is down-regulated in many cancers. Slit2/Robo signaling pathway plays an important, but controversial, role in angiogenesis. We identified splicing variants of Slit2 at exon 15, Slit2-WT and Slit2-ΔE15, with differential effects on proliferation and invasive capability of lung cancer cells. The aim of this study was to elucidate the differential roles of these exon 15 splicing variants in angiogenesis. Our results revealed that both Slit2-WT and Slit2-ΔE15 inhibit motility of human umbilical vein endothelial cells (HUVECs). The conditioned medium (CM) collected from CL1-5/VC or CL1-5/Slit2-WT lung adenocarcinoma cells blocked HUVEC tube formation and angiogenesis on chorioallantoic membrane (CAM) assay when compared with untreated HUVECs and CAM, respectively. However, CM of CL1-5/Slit2-ΔE15 restored the quality of tubes and the size of vessels. Although both Slit2-WT and Slit2-ΔE15 inhibited permeability induced by CM of cancer cells, Slit2-ΔE15 exhibited stronger effect. These results suggested that Slit2-ΔE15 plays important roles in normalization of blood vessels by enhancing tube quality and tightening endothelial cells, while Slit2-WT only enhances tightening of endothelial cells. It appears that Robo4 is responsible for Slit2 isoform-mediated inhibition of permeability, while neither Robo1 nor Robo4 is required for Slit2-ΔE15-enhanced tube quality. The results of this study suggest that Slit2-ΔE15 splicing form is a promising molecule for normalizing blood vessels around a tumor, which, in turn, may increase efficacy of chemotherapy and radiotherapy.

Role of Sigma-1 Receptor in Cocaine-Induced Endothelial Cell Damage

Background :Cocaine abuse is associated with an increased risk of cardiac and cerebrovascular events, such as myocardial infarction, sudden cardiac death, and ischemic stroke. The underlying mechanisms leading to these complications are not fully understood although intravascular thrombus formation and accelerated atherosclerosis are prominent findings. We showed that chronic cocaine use is associated with markers of endothelial dysfunction (ED) (Sáez et al. Thromb Res 2011; 128:18). The hallmark of ED is a change to a prothrombotic and proadhesive phenotype associated with a decreased bioavailability of nitric oxide (NO). Several experimental studies have shown that cocaine induces ED in vitro and in vivo; however, the receptor and the signaling pathways involved in the interaction of cocaine with endothelial cells (EC) are unknown. Sigma-1 receptor (sigma-1R) belongs to the family of nonopioid sigma receptors that regulate a variety of cellular functions in neuronal, glial and peripheral cells and are involved in pathological conditions such as neurodegenerative diseases, drug addiction, smooth muscle cell contraction and ischemia. Cocaine binding to sigma-1R has been demonstrated in microglial cell lines, where is located in the lipid raft microdomains of the endoplasmic reticulum being translocated into the plasma membrane upon activation. The effect of cocaine on sigma-1R expression and function in systemic endothelial cells EC are largely unknown. We hypothesize that activation of sigma-1R in EC and its downstream signaling pathway may be involved in the pathogenesis of cocaine-associated endothelial dysfunction, a key phenomenon in the onset and progression of atherosclerosis.Objectives:The main aim of this study was to demonstrate the presence of sigma-1R in EC and its role in cocaine-induced EC damage/activation.Methods:Human umbilical vein endothelial cells (HUVECs) were cultured under standard conditions and exposed to cocaine (10μM) or vehicle in the presence or absence of BD1047 (sigma-1 receptor antagonist). Sigma-1R was demonstrated by western blotting using rabbit-anti-OPRS1 polyclonal antibody. Sub-cellular distribution of sigma-1R and von Willebrand factor (VWF) bound to the membrane were studied by indirect immunofluorescence (IF) microscopy. The production of NO was determined by fluorometric assay using DAF-2DA (5μM) and results expressed as arbitrary fluorescence units (AFU). Platelet adhesion under static conditions was assessed by co-incubation of resting platelets with cultured HUVEC; platelets and EC were identified and quantified by IF microscopy using anti-glycoprotein Ib and anti-VE-cadherin antibodies, respectively.Results:HUVEC expressed sigma-1R protein as demonstrated by Western blot analysis. By IF the receptor showed a perinuclear pattern in untreated HUVEC. Treatment with cocaine resulted in translocation to the cytoplasm and suggest co-localization with actin. These changes were abrogated completely by pre-incubation of the cells with BD1047. HUVEC treated with cocaine exhibited an increase in VWF deposited on the cell membrane with the formation of strings, phenomena that were inhibited by incubation with BD1047. With respect to platelet adhesion, cocaine induced a significant increase in the number of platelets adhered to HUVEC as compared with the controls (46±24 versus 13.3±7 platelets per cell, respectively), which was drastically reduced by BD1047 (10.5± 6.3 platelets per cell). NO production in HUVEC exposed to cocaine was significantly reduced with respect to cells treated with vehicle (10.7±0.67 vs 19.5± 6.4 nM/ml, P=0.002), the use of BD1047 only partially restored NO production (12.1±2.5 nM/ml).Conclusions: Our results show that in cultured HUVEC expressed sigma 1-R with a sub-cellular distribution and changes upon cell activation similar to those observed in other cell systems. The blockade of sigma-1R inhibited significantly the pro-adhesive and pro-thrombotic changes observed in HUVEC exposed to cocaine, which support the notion that the receptor may play an important role in cocaine-induce endothelial cell dysfunction. These findings have implications for the management of drug addiction, since sigma-1R antagonists may provide a novel therapeutic intervention to protect the endothelium from the deleterious effects associated with cocaine abuse. DisclosuresNo relevant conflicts of interest to declare.

Int6/eIF3e silenced HIF2α stabilization enhances migration and tube formation of HUVECs via IL-6 and IL-8 signaling

We previously identified the tumor suppressor INT6/eIF3e as a novel down regulator of HIF2α. Small interfering RNA targeting Int6 (siRNA-Int6) in HeLa cells led to normoxic stabilization of HIF2α, with concomitant transcription of angiogenic factors, including angiopoietin, basic fibroblast growth factor, and vascular endothelial growth factor. Here we used HIF2α normoxic up-regulation via Int6 silencing to investigate the role of HIF2α in endothelial cells. As a result Int6 silencing in human umbilical vein endothelial cells (HUVECs) and in human aortic endothelial cells (HAECs) led to robust enhanced cord formation and the medium supernatant of Int6 silenced HUVECs enhanced migration of untreated HUVECs, indicating a HIF2α triggered secretory signaling. Within the responsible genes were the cytokines interleukin-6 (IL-6) and IL-8 and not unlike in HeLa cells vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and epidermal growth factor (EGF). In addition application of IL-6 and IL-8 antibodies to the medium of Int6 silenced HUVECs could reverse the enhanced migration effect and also abrogated their tube formation. Finally, a CHIP assay analysis confirmed hypoxia-responsive elements (HREs) in the IL-6 and IL-8 promoters. Our results demonstrate that expression of both IL-6 and IL-8 is regulated by HIF2α and we suggest that IL-6 and IL-8 are HIF2α controlled cytokines for angiogenesis particularly in endothelial cells.