Vascular Endothelial Cell Line Research Articles

Repairing Effect of New Dexamethasone Nanoparticles in the Treatment of Acute Lung Injury and Cluster Nursing.

The repairing effect of new dexamethasone nanoparticles in the treatment of acute lung injury was investigated in this study, as well as cluster nursing. In this study, new dexamethasone model drugs were prepared by the aqueous solvent diffusion method, such as anti-ICAM-I monoclonal antibody-modified anionic dexamethasone NLCs and anti-ICAM-I monoclonal antibody-modified cationic dexamethasone NLCs. Besides, the physical and chemical properties and repairing effects of cationic dexamethasone on acute lung injury were compared. A mouse model of acute lung injury was established, and the anti-inflammatory effect of dexamethasone was evaluated by intravenous injection of dexamethasone nanoparticles in the intervention group and normal healthy mice in the control group. A total of 100 patients with acute lung injury in Hai'anPeople's Hospital Affiliated with Nantong University were selected, of which 50 cases were given cluster nursing intervention and the other 50 cases were taken as the control group. A human vascular endothelial cell line was applied to establish the model cells, and a model of inflammatory endothelial cells in acute lung injury was constructed using lipopolysaccharide stimulation, to verify the cytotoxicity of dexamethasone NLCs. It was found that the anion particle size was 250.12 ± 20.15 nm, the cationic particle size was 245.7 ± 2.1 nm; their Zeta potentials were -31 ± 0.5 mV and 38 ± 0.6 mV in turn; their encapsulation rates were 91% and 83%, respectively; the drug loading was 3.7% and 3.4% in sequence; the release lowest rate was 60%. The 50% lethal dose of anionic cells was higher than 600 g/mL, while that of cationic cells was lower. The respiratory function of the cluster nursing intervention group was better markedly than that of the control group, and the lung infection rate was 2.5% in the intervention group and 15% in the control group. In conclusion, dexamethasone nanoparticles had good anti-inflammatory effects. Anionic ICAM NLCs were less toxic than cationic cells and could better bind to lung vascular endothelial cells, which might reduce adverse drug reactions. Therefore, the building of bundled nursing could effectively alleviate respiratory dysfunction and reduce the infection rate of patients.

Study on the Mechanism of Platelet-Released Clusterins Inducing Restenosis after Carotid Endarterectomy by Activating TLR3/NF-κb p65 Signaling Pathway.

This study aimed to explore the role of clusterin released by platelet aggregation in restenosis after carotid endarterectomy. 35 patients who underwent carotid endarterectomy due to carotid artery stenosis were enrolled in this study. They were admitted to the Third Affiliated Hospital of Qiqihar Medical University from January 2018 to January 2019. All the patients were divided into two groups: the restenosis group and the nonrestenosis group, according to the follow-up results within 12 months. Peripheral blood was collected on the first day, 6 months, and 12 months after operation. The expression of CLU in serum of plasma and platelet culture medium was detected by an ELISA experiment. The vascular endothelial cells were cultured in vitro with 100 ng/mL of human recombinant CLU added to the medium. Cell proliferation, migration, and invasion were detected by CCK8, scratch, and Transwell invasion tests. The expression level of TLR3 and NF-κb p65 proteins in cells was detected by western blot. TLR3 knockout plasmids in vascular endothelial cell lines were transfected. Cell proliferation and migration were detected by CCK8 and the scratch assay. The CLU content in peripheral blood plasma and supernatant of platelet culture medium was significantly higher in the restenosis group than that of the control group (p=0.003) 6 months after operation (p=0.047) and 12 months after operation (p=0.011). When CLU was added to vascular endothelial cell culture medium, the proliferation and migration were significantly enhanced. The TLR3/NF-κb p65 protein expression level in cells also significantly increased. After the transfection of TLR3 knockout plasmids into vascular endothelial cell lines, CLU cannot promote the proliferation and migration of vascular endothelial cells. Platelet-released clusterin can induce vascular endothelial cell proliferation and migration by activating the TLR3/NF-kb p65 signaling pathway, leading to carotid artery restenosis after carotid endarterectomy.

Mechanisms of cardiovascular toxicity induced by PM2.5: a review.

An increasing number of studies have shown that exposure to particulate matter with a diameter ≤ 2.5 μm (PM2.5) could affect the onset and development of cardiovascular diseases. To explore the underlying mechanisms, the studies conducted in vitro investigations using different cell lines. In this review, we examined recently published reports cited by PubMed or Web of Science on the topic of cardiovascular toxicity induced by PM2.5 that carried the term in vitro. Here, we summarized the suggested mechanisms of PM2.5 leading to adverse effects and cardiovascular toxicity including oxidative stress; the increase of vascular endothelial permeability; the injury of vasomotor function and vascular reparative capacity in vascular endothelial cell lines; macrophage polarization and apoptosis in macrophage cell lines; and hypermethylation and apoptosis in the AC16 cell line and the related signaling pathways, which provided a new research direction of cardiovascular toxicity of PM2.5.

Cadmium induces plasminogen activator inhibitor-1 via Smad2/3 signaling pathway in human endothelial EA.hy926 cells.

Modulation of the blood coagulation fibrinolytic system is an essential function of vascular endothelial cells. Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) are major fibrinolytic regulatory proteins synthesized by vascular endothelial cells; fibrinolytic activity is dependent on the balance between these proteins. Previously, we have reported that cadmium, an initiator of ischemic heart disease, induces PAI-1 expression and suppresses fibrinolytic activity in cultured human vascular endothelial cells. However, the key molecules involved in cadmium-induced PAI-1 induction remain unclear. Herein, we investigated the contribution of Smad2 and Smad3, transcriptional factors involved in PAI-1 induction via transforming growth factor-β, using the human vascular endothelial cell line EA.hy926 cells in culture. Our findings indicated that cadmium induces PAI-1 expression without affecting t-PA expression up to 20 µM, a non-cytotoxic concentration, and PAI-1 induction by cadmium is partly mediated via Smad2 and Smad3. This study provides a possible mechanism underlying cadmium-induced vascular disorders.

Hepatocellular carcinoma cell-derived extracellular vesicles encapsulated microRNA-584-5p facilitates angiogenesis through PCK1-mediated nuclear factor E2-related factor 2 signaling pathway.

Hepatocellular carcinoma (HCC) is a fatal disease characterized by poor liver function with increasing morbidity and poor prognosis. Extracellular vesicles, released by different cells, have been associated with HCC development. Nevertheless, the mechanisms beyond extracellular vesicles in HCC remain uncharacterized. Therefore, the current study aimed to clarify the mechanism of pro-angiogenic microRNA-584-5p in hepatocellular carcinoma. Our results showed that miR-584-5p was highly-expressed in both cancer cells (Hep3B) and their extracellular vesicles. Hep3B and extracellular vesicles were then respectively co-cultured with human vascular endothelial cell line (Ea.hy926), and they both accelerated Ea.hy926 proliferation and migration. Ea.hy926 cells could internalize extracellular vesicles carrying microRNA-584-5p. Of note, microRNA-584-5p could bind to phosphoenolpyruvate carboxykinase 1 to promote nuclear factor E2-related factor 2. Moreover, silencing microRNA-584-5p was found to decline microvessel density, vascular endothelial growth factor A, and tumor growth in vivo and in vitro. Taken altogether, our findings demonstrated that extracellular vesicles-derived microRNA-584-5p promotes angiogenesis by inhibiting PCK1 -mediating NRF2 activation, which highlights the theoretical basis for potential treatments for HCC.

Abstract P2-20-04: Controlling tight junctions in the blood brain barrier (BBB) with artemisinin as a potential adjuvant in the treatment of metastatic disease

Abstract Introduction: Brain metastatic disease (BMD) is attributed to significant mortality and morbidity in breast cancer patients. Strict policing of molecular traffic across the blood-brain barrier (BBB) precludes brain parenchymal penetration of most small molecule drugs, chemotherapy included. Consequently, the treatment of BMD remains difficult, particularly if the BBB remains intact during disease progression. Artemisinin has been efficacious in treating multi-drug resistant cerebral malaria likely indicative of an ability to traverse the BBB. Thus, Artemisinin may be able to modulate the restrictive nature of the BBB and provide a potential adjunctive therapy to established drug therapy in the treatment of BMD. Emerging evidence suggests that Artemisinin itself may harbour potent anti-cancer properties. Aim: To investigate in vitro the effect on barrier function in particular paracellular permeability of brain vascular endothelial cells compared to non-brain vascular endothelial cells when treated with Artemisinin. Materials and Methods: Inter-individual BBB differences were modelled using the human brain vascular endothelial cell lines hCMEC-D3, TY09 and TY10. In addition to the brain cell lines, a HUVEC cell line was treated with incremental concentrations of artemisinin. Adhesion, migration and electrical resistance across cellular monolayers were quantified using Electric Cell-Substrate Impedance Sensing (ECIS). Trans- Endo/Epithelial Resistance (TEER) and Paracellular Permeability (PCP) were used to assess barrier function. Immunofluorescence was used to qualitatively assess tight junction (TJ) integrity of all cell lines. RNAseq was used to determine changes in message after treatment with Artemisinin in all 4 cell lines. Results: A significant reduction in electrical resistance across all cell lines was observed when treated with 1mM Artemisinin and may be suggestive of a cytotoxic concentration. In comparison when treated with 0.01mM Artemisinin the electrical resistance decreased which could suggest restrictive barrier properties were enhanced, in addition evidencing Artemisinin may exhibit biphasic activity. The reduction in electrical resistance across the brain endothelial cell lines was significant (p<0.05) but deemed not significant across HUVEC, perhaps mirroring Artemisinin’s pharmacological action at differing vascular interfaces in vivo. Higher Artemisinin concentrations significantly reduced TEER across the brain cell lines and increased paracellular permeability, the same trends were observed in HUVEC. IF demonstrated that Artemisinin was able to effect changes in TJ protein location which supports the hypothesis that Artemisinin reduces barrier function across the brain endothelial lines, suggested by an increased permeability in the presence of Artemisinin. Electrical resistance across all cell lines significantly reduced when treated with 1mM Artemisinin. When compared to negative control, there was a significant decrease in electrical resistance across all cell lines treated with 1mM Artemisinin. RNAseq comparisons revealed that in TY10 cells, 1167 genes were upregulated and 94 downregulated in comparison to HUVEC where 185 and 0 genes were up or downregulated respectively and that in all 3 brain endothelial cell lines that the TJ protein CLDN5 exhibited a significant reduction in expression at gene level (p=0.03). Conclusion: The results demonstrate that Artemisinin may have the capacity to transiently increase or decrease the permeability of the BBB. Therefore, Artemisinin may have a role in facilitating drug delivery to the cerebral parenchyma providing potential therapeutic benefit for the treatment of BMD or other related neurological conditions difficult to treat due to the BBB. Citation Format: Emily Appadurai, Fiona Ruge, Andrew Sanders, Wen G Jiang, Tracey A. Martin. Controlling tight junctions in the blood brain barrier (BBB) with artemisinin as a potential adjuvant in the treatment of metastatic disease [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-20-04.

Aspirin, a Potential GLUT1 Inhibitor in a Vascular Endothelial Cell Line.

Recent epidemiological and preclinical studies have revealed that aspirin possesses antitumor properties; one of the mechanisms results from inhibition of angiogenesis. However, the underlying mechanisms of such action remain to be elucidated, in particular, the effect of aspirin on glucose metabolism of vascular endothelial cells (ECs) has not yet been reported. Herein, we demonstrate that glucose transporter 1 (GLUT1), a main glucose transporter in ECs, can be down-regulated by aspirin. Exposure to 4-mM aspirin significantly decreased GLUT1 at the mRNA and protein level, resulting in impaired glucose uptake capacity in vascular ECs. In addition, we also showed that exposure to 4-mM aspirin led to an inhibition of intracellular ATP and lactate synthesis in vascular ECs, and a down-regulation of the phosphorylation level of NF-κB p65 was observed. Taken together, these findings indicate 4-mM aspirin inhibits glucose uptake and glucose metabolism of vascular ECs through down-regulating GLUT1 expression and suggest that GLUT1 has potential to be a target for aspirin in vascular ECs.

Angiotensin II-Induced Oxidative Stress in Human Endothelial Cells: Modification of Cellular Molecules through Lipid Peroxidation.

Angiotensin (Ang) II is a major bioactive peptide of the renin/angiotensin system and is involved in various cardiovascular functions and diseases. Ang II type 1 (AT1) receptor mediates most of the physiological effects of Ang II. Previous studies have revealed that the lipid peroxidation products 4-oxo-2(E)-nonenal (ONE) and 4-hydroxy-2(E)-nonenal (HNE) readily modify the N-terminus and Asp1, Arg2, and His6 residues of Ang II, and these modifications alter the biological activities of Ang II. Ang II is known to stimulate the formation of reactive oxygen species (ROS) that mediate cardiovascular remodeling. Another major consequence of ROS-derived damage is lipid peroxidation, which generates genotoxic aldehydes such as ONE and HNE. This study demonstrated that Ang II induced lipid peroxidation-derived modifications of cellular molecules in EA.hy926 cells, a human vascular endothelial cell line. Ang P (ONE- and ROS-derived N-terminal pyruvamide Ang II) and [His6(HNE)]-Ang II were detected in the medium of EA.hy926 cells incubated with Ang II, and their concentrations increased dose-dependently upon the addition of ascorbic acid (AscA) and CuSO4. Cells were then subjected to metabolic labeling using SILFAC (stable isotope labeling by fatty acids in cell culture) with [13C18]-linoleic acid. Analysis of cellular phospholipids indicated over 90% labeling. [13C9]-Thiadiazabicyclo-ONE-glutathione adduct as well as Ang P and [His6([13C9]-HNE)]-Ang II was detected in the labeled cells upon treatment with Ang II and their concentrations increased in an Ang II dose-dependent manner. Incubation of the labeled cells with losartan, an AT1 receptor blocker, inhibited the formation of modified Ang IIs in a dose-dependent manner. These results indicate that Ang II induces lipid peroxidation and modification of various cellular molecules and these reactions are mediated by the activation of AT1 receptor. Therefore, lipid peroxidation could be one mechanism by which Ang II contributes to cardiovascular dysfunction.

Activation of AMPK restricts Zika virus replication in endothelial cells by potentiating antiviral response and inhibiting viral-induced glycolysis

Abstract Viruses are known to perturb host cellular metabolism to enable their replication and spread. However, studies examining Zika virus (ZIKV) infection and metabolism are limited. Here, using primary human retinal vascular endothelial (HRvEC) cells and HUVEC cell line, we investigated the role of AMPK, a master regulator of energy metabolism, in response to ZIKV challenge. ZIKV infection caused a time-dependent reduction in the phosphorylation of AMPK and its downstream target ACC. Pharmacological activation of AMPK using AICAR or metformin attenuated ZIKV replication, and similar findings were observed using GSK621, a specific activator of AMPKα. This phenomenon was reversed by incubation of cells with an AMPK inhibitor, compound C. Lentivirus-mediated knockdown of AMPK and the use of AMPK−/− MEFs further supporting the notion that AMPK restricts ZIKV replication. Interestingly, AMPK activation potentiated the expression of genes with antiviral properties (e.g., IFNs, OAS2, ISG15, MX1) and inhibited inflammatory mediators (e.g., TNFα, CCL5). Bio-energetic analysis revealed that ZIKV infection evoked a glycolytic response, as evidenced by elevated ECAR (extracellular acidification rate) levels and increased expression of key glycolytic genes (GLUT1, HK1, TPI, MCT4); AICAR treatment reduced this response. Consistent with this, 2-deoxyglucose (2DG), an inhibitor of the glycolysis, augmented AMPK activity, and attenuated ZIKV replication. Collectively, our study demonstrates that AMPK activation restricts ZIKV replication by promoting an antiviral responses and inhibiting ZIKV-induced glycolysis in endothelial cells. AMPK signaling thus represents a new potential antiviral target for ZIKV infections.

CXC chemokine ligand 1 mediates adiponectin-induced angiogenesis in ovarian cancer.

Adiponectin is a cytokine secreted from adipose tissue that regulates energy homeostasis, inflammation, and cell proliferation. Obesity is associated with increased risk of various cancers, including ovarian cancer. Adipokines, including adiponectin, have been implicated as a factor linking obesity and carcinogenesis. The oncogenic role of adiponectin is not known with regard to various cancer types. We sought to determine the role of adiponectin in angiogenesis in ovarian cancer in vitro. We transfected SKOV3 cells with vascular endothelial growth factor small interfering RNA in order to identify the independent angiogenic role of adiponectin in ovarian cancer. The vascular endothelial growth factor knockdown SKOV3 cell lines were treated with adiponectin for 48 h. The cytokines involved in adiponectin-mediated angiogenesis were explored using the human angiogenesis cytokine array and were verified with the enzyme-linked immunosorbent assay. The angiogenic effect of adiponectin was evaluated using the human umbilical vein endothelial cell tube formation assay. We also investigated the effects of adiponectin treatment on the migration and invasion of SKOV3 cells. The number of tubes formed by human umbilical vein endothelial cell decreased significantly after knockdown of vascular endothelial growth factor (via transfection of vascular endothelial growth factor small interfering RNA into SKOV3 cells). When these vascular endothelial growth factor knockdown SKOV3 cells were treated with adiponectin, there was an increase in the number of tubes in a tube formation assay. Following adiponectin treatment, the CXC chemokine ligand 1 secretion increased in a cytokine array. This was confirmed by both enzyme-linked immunosorbent assay and Western blot. The increased secretion of CXC chemokine ligand 1 by adiponectin occurred regardless of vascular endothelial growth factor knockdown. In addition, the induction of migration and invasion of SKOV3 cells were significantly stronger with adiponectin treatment than they were without. Adiponectin treatment of ovarian cancer cells induces angiogenesis via CXC chemokine ligand 1 independently of vascular endothelial growth factor. These findings suggest that adiponectin may serve as a novel therapeutic target for ovarian cancer.

Anti-ICAM-1 antibody-modified nanostructured lipid carriers: a pulmonary vascular endothelium-targeted device for acute lung injury therapy

BackgroundAcute lung injury (ALI) is a life-threatening clinical syndrome without effective treatment. Targeting delivery of glucocorticoid to lung shows potential efficacy for ALI based on their anti-inflammatory and anti-fibrotic properties, breaking through their clinical application limitation due to systemic side effects. This work was aimed to establish lung-targeted dexamethasone (DEX) loaded nanostructured lipid carriers (NLCs) with opposite surface charge and investigate their therapeutic effects on lipopolysaccharide (LPS)-induced ALI mice.ResultsThe diameter of anionic anti-intercellular adhesion molecule 1 (anti-ICAM-1) antibody-conjugated DEX-loaded NLCs (ICAM/DEX/NLCs) and the cationic ones with octadecylamine (ODA) modification (ICAM/DEX/ODA-NLCs) was about 249.9 and 235.9 nm. The zeta potential of ICAM/DEX/NLCs and ICAM/DEX/ODA-NLCs was about − 30.3 and 37.4 mV, respectively. Relative to the non-targeted control and ICAM/DEX/ODA-NLCs, ICAM/DEX/NLCs exhibited higher in vitro cellular uptake in LPS-activated human vascular endothelial cell line EAhy926 after CAM-mediated endocytosis, and stronger in vivo pulmonary distribution in the ALI model mice. In vivo i.v. administration of ICAM/DEX/NLCs significantly attenuated pulmonary inflammatory cells infiltration, and the production of pro-inflammatory cytokine TNF-α and IL-6 in ALI mice. H&E stain also revealed positive histological improvements by ICAM/DEX/NLCs.ConclusionsICAM/DEX/NLCs may represent a potential pulmonary endothelium targeted device, which facilitate translation of DEX into clinical ALI treatment.

Diphlorethohydroxycarmalol Isolated from Ishige okamurae Represses High Glucose-Induced Angiogenesis In Vitro and In Vivo.

Diabetes mellitus causes abnormalities of angiogenesis leading to vascular dysfunction and serious pathologies. Diphlorethohydroxycarmalol (DPHC), which is isolated from Ishige okamurae, is well known for its bioactivities, including antihyperglycemic and protective functions against diabetes-related pathologies. In the present study, the inhibitory effect of DPHC on high glucose-induced angiogenesis was investigated on the human vascular endothelial cell line EA.hy926. DPHC inhibited the cell proliferation, cell migration, and tube formation in cells exposed to 30 mM of glucose to induce angiogenesis. Furthermore, the effect of DPHC against high glucose-induced angiogenesis was evaluated in zebrafish embryos. The treatment of embryos with DPHC suppressed high glucose-induced dilation in the retinal vessel diameter and vessel formation. Moreover, DPHC could inhibit high glucose-induced vascular endothelial growth factor receptor 2 (VEGFR-2) expression and its downstream signaling cascade. Overall, these findings suggest that DPHC is actively involved in the suppression of high glucose-induced angiogenesis. Hence, DPHC is a potential agent for the development of therapeutics against angiogenesis induced by diabetes.

Expression and clinical significances of miR-215 and RUNX1 in retinoblastoma

Objective To investigate the expression and clinical significances of miR-215 and runt-related protein1 (RUNX1) in retinoblastoma (RB), and to study the regulation effect of miR-215 on RUNX1 in the retinoblastoma cell line PMC-RB. Methods The expressions of miR-215 and RUNX1 in the tumor tissue, non tumor tissues adjacent to cancer, human RB cell line FMC-RB and human normal retinal vascular endothelial cell line ATCC of RB patients were detected by quantitative real-time polymerase chain reaction (qRT-PCR). miR-215 mimics (miR-215-mimic), miR-NC, si-RUNX1 and si-NC were transfected into FMC-RB cell line respectively. Cell proliferation, migration and invasion ability were measured respectively, thus detecting the regulation effect of miR-215 on RUNX1. Results The expression of miR-215 in RB tissues was significantly lower than that in non tumor tissues adjacent to cancer, while the mRNA expression of RUNX1 was higher than that in non tumor tissues adjacent to cancer (P<0.05). The expression of miR-215 in PMC-RB cells was lower than that in ATCC, while the mRNA expression of RUNX1 was higher than that in ATCC (P<0.05). The expression of miR-215 and RUNX1 in RB tumor tissues were closely related to the clinicopathological features of optic nerve infiltration, tumor tissue differentiation and lymph node metastasis (P<0.05). Cell proliferation, migration and invasion in miR-215-mimic group were significantly lower than those in miR-NC group (P<0.05). In transfected 3′ untranslated region (3′UTR)-Wt cells, the luciferase activity in miR-215-mimic group was lower than that in miR-NC group (P<0.05); the expression level of RUNX1 protein in transfected miR-215-mimic cells was lower than that in transfected miR-NC cells (P<0.05). Cell proliferation, migration and invasion in si-RUNX1 group were all lower than those in si-NC group (P<0.05). There was a negative correlation between mRNA expression level of miR-215 and RUNX1 in RB tumor tissues. Conclusions During the occurrence and development of RB, the down-regulation of miR-215 expression can promote malignant progression of tumor by targeting RUNX1. miR-215 can be used as a biological markers and therapeutic target for RB diagnosis. Key words: Retinoblastoma; MicroRNAs; Core binding factor alpha 2 subunit; Cell line, tumor

Combined delivery of angiopoietin-1 gene and simvastatin mediated by anti-intercellular adhesion molecule-1 antibody-conjugated ternary nanoparticles for acute lung injury therapy

Effective treatment for acute lung injury (ALI) is in high demand. Lung-targeted ternary nanoparticles containing anti-intercellular adhesion molecule-1 (ICAM-1) antibody-conjugated simvastatin-loaded nanostructured lipid carrier (ICAM/NLC), protamine (Pro), and angiopoietin-1 (Ang-1) gene (ICAM-NLC/Pro/Ang) were developed for ALI therapy. The ternary nanoparticles with different weight ratios of ICAM-NLC to Ang-1 gene were prepared via charge interaction. The anti-ICAM-1 antibody-conjugated ternary nanoparticles exhibited higher cellular uptake and transfection efficiency (from 26.7% to 30.9%) in human vascular endothelial cell line EAhy926 than the non-targeted control. The largest size of ICAM-NLC/Pro/Ang (357.1 nm) was employed for further study, which significantly up-regulated in vitro and in vivo Ang-1 protein expression. In vivo i.v. administration of ICAM-NLC/Pro/Ang (357.1 nm) significantly attenuated pulmonary TNF-α and IL-6 levels, inflammatory cell infiltration, and led to positive histological improvements in lipopolysaccharide-induced ALI mice. Collectively, the ICAM-NLC/Pro/Ang that co-delivered simvastatin and Ang-1 gene may represent a potential treatment modality for ALI.

Apatinib targets both tumor and endothelial cells in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed malignancies worldwide with poor prognosis and tends to be hypervascular. Aberrant expression of the vascular endothelial growth factor 2 (VEGFR‐2) has been implicated in the progression of HCC and represents a valid target for anticancer therapy. Apatinib, a small molecule inhibitor of VEGFR‐2 tyrosine kinase, shows strong antitumor activity in various tumors. This study is designed to evaluate the activity of apatinib on both human umbilical vein vascular endothelial cells (HUVECs) and HCC cell lines (in vitro and in vivo), and also to investigate the characteristics and possible mechanisms underlying these effects by molecular biology methods. Following the results in our study, apatinib inhibited phosphorylation of VEGFR‐2 in HUVECs and blocked in vitro endothelial cell migration and tube formation. Concentration‐dependent antiproliferative effects of apatinib were also observed in all 6 HCC cell lines including SK‐Hep‐1, HepG2, Hep3B, Huh‐7, PLC/PRF/5, SMMC‐7721. Moreover, response to apatinib of HCC cell lines was significantly correlated with VEGFR‐2 expression level. Additionally, apatinib significantly inhibit VEGF‐triggered VEGFR‐2 phosphorylation and activation of downstream signaling molecules such as Akt and ERK1/2 in HCCs. Apatinib can also induce a cell cycle arrest at G2/M phase and promote HCC apoptosis tested in vitro. In vivo data showed that apatinib can effectively inhibit tumor growth, decreased angiogenesis, as well as induced HCC apoptosis (in some tumors), and thus prolonged animal survival in a mouse xenograft model of human HCC. Our findings suggested that apatinib is a highly potent, oral active anti‐angiogenic, and anti‐HCC agent. The results from current study provide a clear biological rationale to evaluate apatinib as a new agent in HCC in clinical setting, especially for the VEGFR‐2 overexpression ones.

Nicotine treatment ameliorates DSS-induced colitis by suppressing MAdCAM-1 expression and leukocyte recruitment.

The enhanced recruitment of leukocytes to the inflamed colon is a key feature of ulcerative colitis (UC). The gut-specific adhesion molecules involved in leukocyte recruitment have emerged as recent therapeutic targets. Nicotine absorbed from smoking has been reported to work protectively in UC patients. Our hypothesis is that nicotine may suppress the aberrant leukocyte recruitment and colonic inflammation via the suppression of the overexpressed gut-specific adhesion molecules in the inflamed colon. To test this hypothesis, the severity of colitis and the degree of leukocyte recruitment induced by gut-specific adhesion molecules were assessed in dextran sulfate sodium (DSS) colitis mice (C57BL/6J mice treated with 3% DSS) with or without nicotine treatment. We also studied the in vitro changes in the expression of adhesion molecules by using a vascular endothelial cell line. DSS-induced colitis was accompanied by increases in disease activity index (DAI), histological score, recruitment of leukocytes, and the expression of adhesion molecules, mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) and VCAM-1. Nicotine treatment significantly attenuated MAdCAM-1 expression, leukocyte recruitment, DAI, and histological score. The expression of β7-integrin, the ligand for MAdCAM-1, on leukocytes was not affected by nicotine treatment. In vitro study, the TNF-α-enhanced mRNA expression of MAdCAM-1 was reduced by the coadministration of nicotine in a dose-dependent manner, possibly via nicotinic receptor activation. These results supported our hypothesis that nicotine treatment ameliorated colitis through the suppression of MAdCAM-1 expression on the microvessels in the inflamed colon. Further investigation is warranted on the role of nicotine in the treatment of UC.

Conventional and electronic cigarettes dysregulate the expression of iron transporters and detoxifying enzymes at the brain vascular endothelium: In vivo evidence of a gender-specific cellular response to chronic cigarette smoke exposure

It is well established that tobacco smoking is associated with vascular endothelial dysfunction in a causative and dose dependent manner primarily related to the tobacco smoke (TS) content of reactive oxygen species (ROS), nicotine, and oxidative stress (OS) –driven inflammation. Preclinical studies have also shown that nicotine (the principal e-liquid’s ingredient used in e-cigarettes (e-Cigs) can also cause OS, exacerbation of cerebral ischemia and secondary brain injury. Likewise, chronic e-Cig vaping could be prodromal to vascular endothelial dysfunctions. Herein, we provide direct evidence that similarly to TS, e-Cig promotes mitochondrial depolarization in primary brain vascular endothelial cells as well as the vascular endothelial cell line bEnd3. In addition, both TS and e-Cig exposure upregulated the transmembrane iron exporter Slc40a1 (crucial to maintain cellular iron and redox homeostasis) and that of porphyrin importer Abcb6 (linked to accelerated atherosclerosis). We then investigated in vivo whether gender plays a role in how chronic TS affect vascular endothelial functions. Our results clearly show chronic TS exposure differentially impacts the expression levels of Phase-II enzymes as well as the iron transporters previously investigated in vitro. Although the physiological implications of the gender-specific differential responses to TS are not fully clear, they do demonstrate that gender is a risk factor that needs to be investigated when assessing the potential impact of chronic smoking and perhaps e-Cig vaping.

Dimer-monomer equilibrium of human HSP27 is influenced by the in-cell macromolecular crowding environment and is controlled by fatty acids and heat

Small heat shock protein 27 (HSP27) is an essential element of the proteostasis network in human cells. The HSP27 monomer coexists with the dimer, which can bind unfolded client proteins. Here, we evaluated the in-cell dimer-monomer equilibrium and its relevance to the binding of client proteins in a normal human vascular endothelial cell line. When cells were treated with a membrane-permeable crosslinker, the protein existed primarily as a free monomer (27 kDa) with a markedly smaller percentage of dimer (54 kDa), hetero-conjugates, and minor smear-like bands. When the protein was crosslinked in a cell-free lysate, two of the hetero-conjugates that were crosslinked in live cells were also detected, but the dimer and other complexes were absent. However, when cells were pretreated with fatty acid (FA) and/or heat (42.5 °C), dissociation of the dimer was selectively prevented and two types of covalently linked dimers were increased. These changes occurred most prominently in cells treated with docosahexaenoic acid (DHA) and heat, which appeared to intensify the heat resistance of the cell. Both the formation of covalently linked dimers and heat resistance were prevented by N-acetylcysteine. By contrast, nearly all of the free monomers in the lysate converted to disulfide bond-linked dimers by a simple, long incubation at 4 °C. These results strongly suggest that the monomer-dimer equilibrium of HSP27 was inversed between the in-cell and cell-free systems. Temperature- and amphiphile-regulated dimerization was restricted probably due to the low hydration of the in-cell crowding environment.

Interaction of Extracellular Vesicles with Si Surface Studied by Nanomechanical Microcantilever Sensors

We report on the interaction of small (&lt;150 nm) extracellular vesicles (EVs) with silicon surface. The study is conducted by leveraging Si nanomechanical microcantilever sensors actuated in static and dynamic modes, that allow tracking of EV collective adsorption energy and adsorbed mass. Upon incubation for 30 min at about 10 nM concentration, EVs isolated from human vascular endothelial cell (HVEC) lines form a patchy layer that partially covers the Si total surface. Formation of this layer releases a surface energy equal to (8 ± 1) mJ/m2, typical of weak electrostatic interactions. These findings give a first insight into the EV-Si interface and proof the possibility to realize new hybrid biointerphases that can be exploited as advanced models to investigate properties of biological membranes and/or biosensing platforms that take advantage of biomolecules embedded/supported in membranes.

Chlamydia pneumoniae infection promotes monocyte transendothelial migration by increasing vascular endothelial cell permeability via the tyrosine phosphorylation of VE-cadherin

Migration of monocytes into the subendothelial layer of the intima is one of the critical events in early atherosclerosis. Chlamydia pneumoniae (C. pneumoniae) infection has been shown to promote monocyte transendothelial migration (TEM). However, the exact mechanisms have not yet been fully clarified. In this study, we tested the hypothesis that C. pneumoniae infection increases vascular endothelial cell (VEC) permeability and subsequent monocyte TEM through stimulating the tyrosine phosphorylation of vascular endothelial-cadherin (VE-cadherin). Here, we demonstrated that C. pneumoniae infection promoted monocyte TEM in a TEM assay possibly by increasing the permeability of a VEC line EA.hy926 cell as assessed by measuring the passage of FITC-BSA across a VEC monolayer. Subsequently, Western blot analysis showed that C. pneumoniae infection induced VE-cadherin internalization. Our further data revealed that Src-mediated VE-cadherin phosphorylation at Tyr658 was involved in C. pneumoniae infection-induced internalization of VE-cadherin, VEC hyperpermeability and monocyte TEM. Taken together, our data indicate that C. pneumoniae infection promotes monocyte TEM by increasing VEC permeability via the tyrosine phosphorylation and internalization of VE-cadherin in VECs.