Endothelial Cell Line EAhy926 Research Articles

Preparation of Dexamethasone-Loaded Nanoparticles and Therapeutic Adoption for Acute Lung Injury in Septic Mice Through the TLR9 Pathway

This research aimed to better exert the efficacy of dexamethasone (DEX) and fabricate an intercellular adhesion molecule A (ILDMA) monoclonal antibody (mAb)-modified nanostructured lipid carrier (NSLC). The anionic DEX NSLC was fabricated by the aqueous solvent diffusion methodology using DEX as the model drug and in combination with various types of lipids. Using N,N’-succinimidyl carbonate as the link, anti-ILDMA mAb-modified anion DEX NSLC (ILDM/DEX/NSLC) and anti-IgG mAb-modified cation DEX NSLC (IgG/DEX/NSLC) were prepared. The total lipid content was controlled unchanged, 3% mass ratio of glyceryl monostearate (MS) in the original prescription was replaced with 3% mass ratio of octadecylamine (ODA), and ILDM/DEX/ODA-NSLC and IgG/DEX/ODA-NSLC were fabricated in the same way. The four NSLCs prepared in the experiment were round in shape and uniform in size. The nanoparticles with a size of approximately 230 nm were similar, and the zeta potentials were (−29.8±21.5) mV, (−27.9±1.6) mV, (36.8±0.8) mV, and (33.7±2.9) mV, respectively. In vitro drug release demonstrated a cumulative release rate of more than 55% of DEX NSLC. The inhibitory rate of DEX NSLC on the activated human vascular endothelial cell line (EAhy926 cell) was dose dependent, and ILDM/DEX/NSLC could transport DEX to activated endothelial cells more efficiently, thus enhancing the intervention ability on diseased endothelium. For the establishment of a sepsis-induced acute lung injury (ALI) mouse model, ILDM/DEX/NSLC was highly distributed in the lung of the model, and its infiltration effect on inflammatory cells was superior to that of other drugs (P < 0.05). Meanwhile, ILDM/DEX/NSLC could more markedly repair the pathological features in the mouse model than other drugs did (P < 0.05). The nanodrug inhibited the protein level of TLR9 in mouse lung tissue to the maximum extent (P < 0.05), thereby enhancing the survival rate of the mice.

High-affinity Cu(I) chelator PSP-2 as potential anti-angiogenic agent

Copper is an essential trace metal that has been implicated in angiogenesis, the formation of new blood vessels. As tumor growth relies on establishing a functional capillary network for blood supply, copper chelation therapy may hold promise as an anti-cancer strategy by suppressing angiogenesis. To test the anti-angiogenic effect of PSP-2, a recently developed high affinity Cu(I) chelator with low zeptomolar dissociation constant, we utilized the endothelial cancer cell line EAhy926 and assessed changes in cell migration, proliferation, and tube formation in Matrigel. In addition, sprouting was assessed by the chicken and sheep aortic ring assay, and vascular pattern formation was studied in the chorioallantoic membrane of chicken embryos (CAM assay). While incubation with PSP-2 resulted in selective depletion of cellular copper levels, cell migration was not affected and the proliferating activity was even slightly increased. Moreover, the endothelial tube formation assay revealed significant morphological changes in the presence of PSP-2, with thicker tubular walls and an overall decreased meshes area. Similarly, the aortic ring assay and CAM assay showed that PSP-2 evokes significantly longer sprouts with smaller angles at branching points. Altogether, PSP-2 exhibits significant bioactivity at concentrations as low as 5 μM, rendering it a promising anti-angiogenic agent. As EAhy926 cells exhibit both endothelial and tumorigenic properties, the anti-angiogenic effect of PSP-2 might potentially translate also into anti-cancer activity.

Biological activity of Aesculus hippocastanum flower extracts on vascular endothelial cells cultured in vitro

The aim of this study was to evaluate the utility of a hydroalcoholic extract of Hippocastani flos (HF) in wound healing, especially in regards to cell viability, proliferation, reactive oxygen species (ROS) generation and proinflammatory cytokine production using an endothelial cell line [human umbilical vein endothelial cell (HUVEC) line EAhy926]. Antibacterial and anti-hyaluronidase activity was also evaluated. The phytochemical profile was determined using an the UPLC–MS.The UPLC–MS analysis confirmed the presence of nine flavonoids, mainly kaempferol and quercetin derivatives. Specifically, the HF extract contained a high content of polyphenols (88.84 ± 4.13 mg chlorogenic acid equivalents/g dw), including flavonoids (15.68 ± 0.04 mg/quercetin equivalents/g dw). Antioxidant properties were demonstrated using FRAP assays (IC0.5 = 159.82 μg/mL). The HF extract exhibited a level of anti-hyaluronidase activity (IC50 = 0.86 mg/mL) similar to the positive control kaempferol (IC50 = 0.78 mg/mL). The antimicrobial properties of HF were shown against Staphylococcus aureus (MIC, MBC = 12.5 mg/mL). After exposing the endothelial cells to HF extract in vitro, it did not observed any change in the cell viability, proliferation, wound healing or proinflammatory cytokine production, but there was a dose dependent-reduction in ROS generation. In conclusion, HF extract exhibits anti-hyaluronidase activity, antimicrobial properties, and reduces ROS production. These observations may partly justify the use of HF in supporting wound healing.

Gedunin, A Neem Limonoid in Combination with Epalrestat Inhibits Cancer Hallmarks by Attenuating Aldose Reductase-Driven Oncogenic Signaling in SCC131 Oral Cancer Cells.

Aldose Reductase (AR), a polyol pathway enzyme that mediates diabetic complications is implicated in tumour development and progression. This study was undertaken to determine whether gedunin, a neem limonoid prevents the hallmarks of cancer by inhibiting AR and the associated downstream PI3K/Akt/mTOR/ERK/NF-κB signalling axis in the SCC131 oral cancer cell line. The expression of AR and key molecules involved in cell proliferation, apoptosis, autophagy, invasion and angiogenesis was analysed by qRT-PCR, and immunoblotting. ROS generation and cell cycle were analysed by FACS. Alamar blue assay and scratch assay were used to evaluate cell proliferation and migration in the endothelial cell line Eahy926. Gedunin and the AR inhibitor epalrestat inhibited AR expression and ROS generation. Cell cycle arrest at G1/S was associated with cell death by autophagy with subsequent switch over to apoptosis. Furthermore, hypoxia-induced cell migration was inhibited in Eahy926 cells with downregulation of pro-invasive and proangiogenic proteins in SCC131 as well as Eahy926 cells. Co-inactivation of Akt and ERK was coupled with abrogation of IKK/NF-κB signaling. However, the combination of gedunin and epalrestat was more effective than single agents. Inhibition of AR-mediated ROS signalling may be a key mechanism by which gedunin and epalrestat exert their anticancer effects. Our results provide compelling evidence that the combination of gedunin and epalrestat modulates expression of key oncogenic signalling kinases and transcription factors primarily by influencing phosphorylation and subcellular localisation. AR inhibitors such as gedunin and epalrestat are novel candidate agents for cancer prevention and therapy.

Galectin-8 induces endothelial hyperpermeability through the eNOS pathway involving S-nitrosylation-mediated adherens junction disassembly.

The permeability of endothelial cells is regulated by the stability of the adherens junctions, which is highly sensitive to kinase-mediated phosphorylation and endothelial nitric oxide synthase (eNOS)-mediated S-nitrosylation of its protein components. Solid tumors can produce a variety of factors that stimulate these signaling pathways leading to endothelial cell hyperpermeability. This generates stromal conditions that facilitate tumoral growth and dissemination. Galectin-8 (Gal-8) is overexpressed in several carcinomas and has a variety of cellular effects that can contribute to tumor pathogenicity, including angiogenesis. Here we explored whether Gal-8 has also a role in endothelial permeability. We show that recombinant Gal-8 activates eNOS, induces S-nitrosylation of p120-catenin (p120) and dissociation of adherens junction, leading to hyperpermeability of the human endothelial cell line EAhy926. This pathway involves focal-adhesion kinase (FAK) activation downstream of eNOS as a requirement for eNOS-mediated p120 S-nitrosylation. This suggests a reciprocal, yet little understood, regulation of phosphorylation and S-nitrosylation events acting upon adherens junction permeability. In addition, glutathione S-transferase (GST)-Gal-8 pull-down experiments and function-blocking β1-integrin antibodies point to β1-integrins as cell surface components involved in Gal-8-induced hyperpermeability. Endogenous Gal-8 secreted from the breast cancer cell line MCF-7 has similar hyperpermeability and signaling effects. Furthermore, the mouse cremaster model system showed that Gal-8 also activates eNOS, induces S-nitrosylation of adherens junction components and is an effective hyperpermeability agent in vivo. These results add endothelial permeability regulation by S-nitrosylation as a new function of Gal-8 that can potentially contribute to the pathogenicity of tumors overexpressing this lectin.

Ticagrelor suppresses oxidized low‑density lipoprotein‑induced endothelial cell apoptosis and alleviates atherosclerosis in ApoE‑/‑ mice via downregulation of PCSK9.

Although ticagrelor has been demonstrated to possess an anti-atherosclerosis (AS) effect, its underlying mechanism remains unclear. In the present study, it was investigated whether ticagrelor reduces oxidized low-density lipoprotein (ox-LDL)-induced endothelial cell apoptosis, an initial step for the development of AS, and alleviates AS in apolipoprotein-E-deficient (ApoE−/−) mice by inhibiting the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9). The human endothelial cell line EAhy926 was treated with ox-LDL, ox-LDL + ticagrelor (40 µmol/l) and ox-LDL + ticagrelor (60 µmol/l) for 24 h. Cell apoptosis was detected using Annexin V-fluorescein isothiocyanate/propidium iodide staining. The expression levels of PCSK9, apoptosis-associated proteins and signaling pathways were determined by reverse transcription-quantitative polymerase chain reaction and western blotting. ApoE−/− mice fed a high-fat diet were used to induce an AS model. After 20 weeks, ApoE−/− mice were randomly assigned to receive saline or ticagrelor intragastrically for 10 days. The formation of atherosclerotic plaques was detected by hematoxylin and eosin staining. The expression of PCSK9 in the arterial tissues was measured by immunohistochemistry. The results demonstrated that treatment with ticagrelor was able to decrease ox-LDL-induced apoptosis in a concentration-dependent manner (40 µmol/l vs. ox-LDL, 17.58±2.66 vs. 27.25±5.54%; 60 µmol/l vs. ox-LDL, 12.26±1.54 vs. 27.25±5.54%). The mRNA and protein expression level of PCSK9 significantly decreased following treatment with ticagrelor, accompanied with upregulation of B-cell lymphoma (Bcl) 2 and downregulation of Bcl-2 associated X, apoptosis regulator, caspase-3, p38, phosphorylated-(p) p38, p-c-Jun N-terminal kinases (JNK), p-extracellular signal-regulated kinases and the ratio of p-JNK to JNK. Histological analysis of arterial tissues revealed ticagrelor markedly decreased the atherosclerotic plaque area and inhibited the expression of PCSK9. The present results suggested that ticagrelor may alleviate AS via downregulation of PCSK9-mediated endothelial cell apoptosis, which may be JNK-dependent.

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.

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.

Development and evaluation of injectable nanosized drug delivery systems for apigenin

The purpose of this study was to develop different injectable nanosized drug delivery systems (NDDSs) i.e. liposome, lipid nanocapsule (LNC) and polymeric nanocapsule (PNC) encapsulating apigenin (AG) and compare their characteristics to identify the nanovector(s) that can deliver the largest quantity of AG while being biocompatible. Two liposomes with different surface characteristics (cationic and anionic), a LNC and a PNC were prepared. A novel tocopherol modified poly(ethylene glycol)-b-polyphosphate block-copolymer was used for the first time for the PNC preparation. The NDDSs were compared by their physicochemical characteristics, AG release, storage stability, stability in serum, complement consumption and toxicity against a human macrovascular endothelial cell line (EAhy926). The diameter and surface charge of the NDDSs were comparable with previously reported injectable nanocarriers. The NDDSs showed good encapsulation efficiency and drug loading. Moreover, the NDDSs were stable during storage and in fetal bovine serum for extended periods, showed low complement consumption and were non-toxic to EAhy926 cells up to high concentrations. Therefore, they can be considered as potential injectable nanocarriers of AG. Due to less pronounced burst effect and extended release characteristics, the nanocapsules could be favorable approaches for achieving prolonged pharmacological activity of AG using injectable NDDS.

Cell-based evaluation of changes in coagulation activity induced by antineoplastic drugs for the treatment of acute myeloid leukemia.

Idarubicin (IDR), cytarabine (AraC), and tamibarotene (Am80) are effective for treatment of acute myeloid leukemia (AML). In acute leukemia, the incidence of venous thromboembolism or disseminated intravascular coagulation is associated with induction chemotherapy. Procoagulant effects of IDR, AraC, and Am80 were investigated in a vascular endothelial cell line EAhy926 and AML cell lines HL60 (AML M2), NB4 (AML M3, APL), and U937 (AML M5), focusing on tissue factor (TF), phosphatidylserine (PS), and thrombomodulin (TM). IDR induced procoagulant activity on the surface of vascular endothelial and AML cell lines. Expression of TF antigen, TM antigen, and PS were induced by IDR on the surface of each cell line, whereas expression of TF and TM mRNAs were unchanged. Conversely, Am80 decreased TF exposure and procoagulant activity, and increased TM exposure on NB4 cells. In NB4 cells, we observed downregulation of TF mRNA and upregulation of TM mRNA. These data suggest IDR may induce procoagulant activity in vessels by apoptosis through PS exposure and/or TF expression on vascular endothelial and AML cell lines. Am80 may suppress blood coagulation through downregulation of TF expression and induction of TM expression. Our methods could be useful to investigate changes in procoagulant activity induced by antineoplastic drugs.

Toxic effects of dimethyl sulfoxide on red blood cells, platelets, and vascular endothelial cells in vitro.

Dimethyl sulfoxide (DMSO) is widely used in biological studies as a cryoprotective agent for cells and tissues, and also for cryopreserved platelets (PLTs). However, few data on the toxic effects of DMSO following intravenous infusion of cryopreserved PLTs are available. The aim of this study was to explore dose‐related effects of DMSO on red blood cells (RBCs), PLTs and vascular endothelial cells in vitro. The results showed that DMSO treatments had significant effects on RBCs, affecting osmotic fragility and increasing hemolysis. Free hemoglobin (FHb) level of RBCs was 0.64 ± 0.19 g L−1 after incubation for 6 h with 0.6% DMSO, and these levels were elevated compared with controls (0.09 ± 0.05 g L−1). Aggregation of PLTs induced by adenosine diphosphate, thrombin (THR), and thrombin receptor activator peptide (TRAP) were inhibited by DMSO treatment because the THR generation capacity was reduced. The intensity of the cytosolic esterase‐induced fluorescence response from carboxy dimethyl fluorescein diacetate (CMFDA) in PLTs was decreased about 29% ± 0.04% after treatment with DMSO. DMSO also inhibited the proliferation of the vascular endothelial cell line EAhy926 cells by blocking the G1 phase. Apoptosis of EAhy926 cells with 0.6% DMSO stimulation was increased threefold compared to controls. On the basis of these findings, it was concluded that DMSO was toxic to the hematologic system. This should be taken into account when assessing the infusion effects of cryopreserved PLTs or other blood products requiring DMSO as a vehicle, such as cryopreserved stem cells, in order to avoid adverse therapeutic effects.

Targeting delivery of simvastatin using ICAM-1 antibody-conjugated nanostructured lipid carriers for acute lung injury therapy

Acute lung injury (ALI) is a critical illness without effective therapeutic modalities currently. Recent studies indicated potential efficacy of statins for ALI, while high-dose statins was suggested to be significant for attenuating inflammation in vivo. Therefore, a lung-targeted drug delivery system (DDS) delivering simvastatin (SV) for ALI therapy was developed, attempting to improve the disease with a decreased dose and minimize potential adverse effects. SV-loaded nanostructured lipid carriers (SV/NLCs) with different size were prepared primarily. With particle size increasing from 143.7 nm to 337.8 nm, SV/NLCs showed increasing drug-encapsulated efficiency from 66.70% to 91.04%. Although larger SV/NLCs exhibited slower in vitro cellular uptake by human vascular endothelial cell line EAhy926 at initial stage, while in vivo distribution demonstrated higher pulmonary accumulation of the larger ones. Thus, the largest size SV/NLCs (337.8 nm) were conjugated with intercellular adhesion molecule 1 (ICAM-1) antibody (anti-ICAM/SV/NLCs) for lung-targeted study. The anti-ICAM/SV/NLCs exhibited ideal lung-targeted characteristic in lipopolysaccharide-induced ALI mice. In vivo i.v. administration of anti-ICAM/SV/NLCs attenuated TNF-α, IL-6 and inflammatory cells infiltration more effectively than free SV or non-targeted SV/NLCs after 48-h administration. Significant histological improvements by anti-ICAM/SV/NLCs were further revealed by H&E stain. Therefore, ICAM-1 antibody-conjugated NLCs may represent a potential lung-targeted DDS contributing to ALI therapy by statins.

Abstract 16: BcrKinase is a Novel Akt Kinase That Modulates Scavenger Receptor BI- and PDZK1-dependent Actions of HDL in Endothelium

High density lipoprotein cholesterol (HDL) has direct atheroprotective actions on endothelium. These are mediated by scavenger receptor class B, type I (SR-BI) and its adaptor protein PDZK1, and they entail the activation of Akt kinase, which phosphorylates and thereby stimulates endothelial nitric oxide synthase (eNOS). In the present work we sought to determine how PDZK1 couples HDL/SR-BI to Akt and eNOS to modulate endothelial function. Using tandem affinity purification (TAP) following the infection of the human endothelial cell line EAhy926 with adenovirus expressing TAP-tagged PDZK1, we identified Breakpoint Cluster Region (Bcr) kinase as a PDZK1 interacting protein in endothelium. Whereas Bcr is well-known as a component of the Bcr-Abl fusion protein that results from translocation of the Philadelphia chromosome in chronic myelogenous leukemia, little is known of its function in endothelial cells. Bcr contains several distinctive domains including a C-terminal PDZ binding motif and a serine/threonine protein kinase domain. In primary human aortic endothelial cells (HAEC), we determined that endogenous Bcr interacts with PDZK1 in an HDL-dependent manner, and that Bcr is required for HDL-induced activation of eNOS and HDL stimulation of endothelial cell migration, which underlies the ability of the lipoprotein to promote endothelial monolayer integrity. Studies of mutant forms of Bcr with disruption of PDZK1 binding or kinase activity introduced into endothelial cells further revealed that Bcr-PDZK1 interaction and its kinase function are required for HDL activation of Akt and eNOS. Using a novel kinase assay that we recently developed that employs time-resolved Forster resonance energy transfer, we found that via SR-BI and PDZK1, HDL stimulates Bcr kinase activity in endothelial cells more than 20-fold. In addition, using Akt-based peptides in studies of the two known kinases for Akt, mTOR and PDK1, we determined that HDL activates Bcr kinase to directly phosphorylate Akt-Ser473 in an mTOR independent manner, and that Akt-Thr308 is a direct substrate of PDK1. These collective findings have identified Bcr to be a novel kinase for Akt, and they have revealed that Bcr is critically involved in HDL modulation of endothelial cell phenotype.

Cell-based laboratory evaluation of coagulation activation by antineoplastic drugs for the treatment of lymphoid tumors

Objectives:Combining vorinostat, L-asparaginase, and doxorubicin (Dox) led to improved response rates in the treatment of lymphoid tumors. However, deep-vein thrombosis has been noted as one of the most serious side effects with these drugs, and how these regimens cause deep-vein thrombosis is unclear.Methods:We investigated the procoagulant effects of vorinostat, L-asparaginase, and doxorubicin in lymphoid tumors, focusing on tissue factor, phosphatidylserine, and antithrombin. The human vascular endothelial cell line EAhy926 as well as the lymphoid neoplastic cell lines HUT78 (cutaneous T-cell lymphoma), Molt4 (acute T-lymphoblastic leukemia), and Ramos (Burkitt lymphoma) were employed to investigate these procoagulant effects.Results:Vorinostat, L-asparaginase, and doxorubicin induced exposure of phosphatidylserine and procoagulant activity on the surface of lymphoid tumor cells. Vorinostat and doxorubicin also induced phosphatidylserine exposure and increased procoagulant activity on EAhy926 cells. Expression of tissue factor antigen was induced by doxorubicin on the surface of each type of cells, whereas expression of tissue factor mRNA was unchanged. Secretion of antithrombin from HepG2 cells was reduced only by L-asparaginase.Conclusion:These data suggest that vorinostat and doxorubicin may induce procoagulant activity in vessels through apoptosis of tumor cells and through phosphatidylserine exposure and/or tissue factor expression on vascular endothelial cells. L-asparaginase may induce a thrombophilic state by reducing the secretion of anticoagulant proteins such as antithrombin. The laboratory methods described here could be useful to evaluate the procoagulant effects of antineoplastic drugs.

Nucleoside Transport Inhibition by Dipyridamole Prevents Angiogenesis Impairment by Homocysteine and Adenosine.

Adenosine plays an important role in the pathogenesis of homocysteine-associated vascular complications. This study examined the effects of dipyridamole, an inhibitor for nucleoside transport, on impaired angiogenic processes caused by homocysteine and adenosine in human cardiovascular endothelial cell line (EAhy926). The results showed that dipyridamole restored the extracellular adenosine and intracellular S-adenosylhomocysteine concentrations disrupted by the combination of homocysteine and adenosine. Dipyridamole also ameliorated the impaired proliferation, migration and formation of capillary-like tubes of EAhy926 cells caused by the combination of homocysteine and adenosine. Mechanism analysis revealed that dipyridamole induced the phosphorylation of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinases (ERK) and its effect on cell growth was attenuated by the MEK inhibitor, U0126. Dipyridamole protected against impaired angiogenesis caused by homocysteine and adenosine, at least in part, by activating the MEK/ERK signalling pathway, and this could be associated with its effects in suppressing intracellular S-adenosylhomocysteine accumulation. This is the first paper showing that nucleoside transport inhibition by dipyridamole reduced impaired angiogenic process caused by homocysteine and adenosine.

Bioactivity-integrated UPLC/Q-TOF–MS of Danhong injection to identify NF-κB inhibitors and anti-inflammatory targets based on endothelial cell culture and network pharmacology

Ethnopharmacological relevanceDanhong injection (DHI) is a traditional Chinese medicine injection that has been widely used in therapy for cardiovascular diseases. However, neither its mechanism nor its active constituents are clearly understood. Aim of the studyOur research aimed at identifying the anti-inflammatory ingredients and mechanism of DHI by combining high-throughput screening (HTS) with network pharmacology analysis. Materials and methodsThe human endothelial cell line EAhy926 was cultured in vitro. Methyl thiazolyltetrazolium (MTT) and lactate dehydrogenase (LDH) assays were performed to detect cell viability. The expression of Bcl-2 and Bax, interleukin-6 (IL-6), inhibitor of nuclear factor kappa-B kinase (IKK), phosphorylated IKK, phosphorylated NF-κB and phosphorylated IκB-α from the supernatant were determined. Then, we constructed an assay system combining ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF–MS) with an NF-κB activity luciferase reporter to screen DHI for essential anti-inflammatory components, and the results were verified using network pharmacology. ResultsDHI could significantly suppress inflammatory responses, and the mechanism may be via an NF-κB-dependent pathway. We found nine potential anti-inflammatory ingredients: danshensu, protocatechuic acid, protocatechuic aldehyde, caffeic acid, hydroxysafflor yellow A, safflor yellow A, salvianolic acid A salvianolic acid B and salvianolic acid C. Among these compounds, the NF-κB inhibitory activity of SAC is reported here for the first time. ConclusionsDHI plays an important role in suppressing inflammatory responses through inhibiting the NF-κB signaling pathway. The potential NF-κB inhibitors in DHI contribute to the cross-talk of multiple targets in anti-inflammation.

Mitochondria-targeted antioxidants prevent TNFα-induced endothelial cell damage.

Increased serum level of tumor necrosis factor α (TNFα) causes endothelial dysfunction and leads to serious vascular pathologies. TNFα signaling is known to involve reactive oxygen species (ROS). Using mitochondria-targeted antioxidant SkQR1, we studied the role of mitochondrial ROS in TNFα-induced apoptosis of human endothelial cell line EAhy926. We found that 0.2nM SkQR1 prevents TNFα-induced apoptosis. SkQR1 has no influence on TNFα-dependent proteolytic activation of caspase-8 and Bid, but it inhibits cytochrome c release from mitochondria and cleavage of caspase-3 and its substrate PARP. SkQ analogs lacking the antioxidant moieties do not prevent TNFα-induced apoptosis. The antiapoptotic action of SkQR1 may be related to other observations made in these experiments, namely SkQR1-induced increase in Bcl-2 and corresponding decrease in Bax as well as p53. These results indicate that mitochondrial ROS production is involved in TNFα-initiated endothelial cell death, and they suggest the potential of mitochondria-targeted antioxidants as vasoprotectors.

Development of anionic bubble lipopolyplexes for efficient and safe gene transfection with ultrasound exposure in mice

Anionic bubble lipopolyplexes have been developed as anionic ultrasound (US)-responsive gene delivery carriers with biocompatible compounds for efficient and safe transfection in mice. The particles of the anionic bubble lipopolyplexes were approximately 450–600nm with an anionic surface charge. In the absence of US exposure, the bubble lipopolyplexes showed extremely low gene expression in the human vascular endothelial cell line EAhy926. The anionic bubble lipopolyplexes, however, delivered pDNA into cells without endocytosis and showed markedly high gene expression following US exposure. The anionic bubble lipopolyplexes showed little cytotoxicity in EAhy926 cells and little aggregation with erythrocytes. Following intravenous administration into mice, the anionic bubble lipopolyplexes showed high levels of gene expression in the liver, kidney, and spleen only after US exposure to the abdominal area. The level of gene expression in liver non-parenchymal cells was significantly higher than that in parenchymal cells. In addition, the anionic bubble lipopolyplexes did not show any severe hepatic toxicity and did not enhance the production of proinflammatory cytokines. Overall, we have succeeded in preparing anionic bubble lipopolyplexes for efficient and safe transfection with US exposure in mice.

Activation of Coagulation by Lenalidomide-Based Regimens for the Treatment of Multiple Myeloma

We investigated the procoagulant effects of lenalidomide (Len)-based regimens in vitro focusing on tissue factor (TF) and phosphatidylserine (PS). We examined the effects of a pharmacological concentration of Len with or without the corticosteroid dexamethasone (Dex) and the proteasome inhibitor bortezomib (Bor) using the human vascular endothelial cell line EAhy926 and the monocytic cell lines THP-1 and U937. Cell-surface procoagulant activity (PCA) was induced by Dex-containing regimens in all lines. Expression of TF antigen on the cell surface and of TF mRNA was markedly increased by Dex-containing regimens. PS exposure was increased modestly by a Len-based regimen. PS exposure was increased modestly in EAhy926 cells, and markedly increased in THP-1 and U937 cells by Bor-containing treatment. An anti-TF monoclonal antibody almost completely blocked the induced PCA. When Len is given in combination with Dex, PCA may be induced on endothelial cells and monocytes through TF expression and PS exposure.

Action of polystyrene nanoparticles of different sizes on lysosomal function and integrity

BackgroundData from environmental exposure to nanoparticles (NPs) suggest that chronic exposure may increase the incidence of lung, cardiovascular and neurodegenerative diseases. Impairment of cell function by intracellular accumulation of NPs is also suspected. Many types of NPs have been detected in the endosomal-lysosomal system and, upon repeated exposure, alterations of the endosomal-lysosomal system may occur. To identify such effects we compared the effect of carboxyl polystyrene particles (CPS) of different sizes (20-500 nm) on lysosomes of the endothelial cell line EAhy926 after short (24h) and long (72h-96h) exposure times. Lysosomal localization of CPS, as well as lysosomal pH, lysosomal membrane integrity, morphology of the endosomal-lysosomal system and activities of the lysosomal enzymes,cathepsin B and sulfatases, upon exposure to CPS were recorded.ResultsCPS in sizes ≤100 nm showed high co-localization with lysosomes already after 4h, larger CPS after 24h. None of the particles at non-cytotoxic concentrations caused marked changes in lysosomal pH or destroyed lysosomal membrane integrity. At 24h of exposure, 20 nm CPS induced significant dilatation of the endosomal-lysosomal system and reduced activity of lysosomal sulfatases. After 72h, these alterations were less pronounced.ConclusionsDespite accumulation in lysosomes CPS induced only small changes in lysosomes. Upon longer contact, these changes are even less pronounced. The presented panel of assays may serve to identify effects on lysosomes also for other NPs.