Blood compatible heteratom-doped carbon dots for bio-imaging of human umbilical vein endothelial cells

Carbon dots have unique advantages in biological applications owing to their excellent optical prope rties.However,the biosafety evaluation of carbon dots has limitations owing to cytotoxicity in vitro,and the re is little pre-safety evaluation before in vivo and clinical applications.Whether the carbon dots are or not suitable for applications in vivo,evaluation analysis can be made based on hemolysis and changes in erythrocyte morphology.In this work,a green fluorescent N,S-doped carbon dots(N,S-CDs)were obtained by hydrothermal method,tobias acid,and m-phenylenediamine as precursors.N,S-CDs not only possessed excellent dispersibility,uniform particle size,high quantum yield(37.2%)and stable photoluminescence property but also retain their photostability and stro ng fluorescence intensity in the acid/alkaline solutions,different ionic strengths(NaCl)and under 365 nm UV illumination.Moreove r,the N,S-CDs displayed low cytotoxicity and high cellular uptake efficiency in human umbilical vein endothelial cells(HUVEC)and excellent blood compatibility to the erythrocyte.It is foreseeable that N,S-CDs could be further studied as a promising biological imaging agent in vivo.     

A tag-less method for direct isolation of human umbilical vein endothelial cells by gravitational field-flow fractionation

The analysis of cellular and molecular profiles represents a powerful tool in many biomedical applications to identify the mechanisms underlying the pathological changes. The improvement of cellular starting material and the maintenance of the physiological status in the sample preparation are very useful. Human umbilical vein endothelial cells (HUVEC) are a model for prediction of endothelial dysfunction. HUVEC are enzymatically removed from the umbilical vein by collagenase. This method provides obtaining a good sample yield. However, the obtained cells are often contaminated with blood cells and fibroblasts. Methods based on negative selection by in vitro passages or on the use of defined marker are currently employed to isolate target cells. However, these approaches cannot reproduce physiological status and they require expensive instrumentation. Here we proposed a new method for an easy, tag-less and direct isolation of HUVEC from raw umbilical cord sample based on the gravitational field-flow fractionation (GrFFF). This is a low-cost, fully biocompatible method with low instrumental and training investments for flow-assisted cell fractionation. The method allows obtaining pure cells without cell culture procedures as starting material for further analysis; for example, a proper amount of RNA can be extracted. The approach can be easily integrated into clinical and biomedical procedures.     

An on-line method for determining ATPase bioactivity and its application to human umbilical vein endothelial cell membrane

Based on the determination of inorganic phosphate (the product of ATP dephosphorylation by ATPase), a method for on-line determination of bioactivity of ATPase on cell membrane is presented, in which on-line sampling of cell membrane and separation of inorganic phosphate is employed in a flow injection system. The method has been successfully applied to the investigation of variation of ATPase from human umbilical vein endothelial cell membrane; the results show that ATPase activity decreases with elapsed time studied, which may supply important information for the pertinent studies on cell membrane enzymes bioactivity.     

Prostaglandin E2 stimulates angiogenesis by activating the nitric oxide/cGMP pathway in human umbilical vein endothelial cells

Prostaglandin E2 (PGE2), a major product of cyclooxygenase, has been implicated in modulating angiogenesis, vascular function, and inflammatory processes, but the underlying mechanism is not clearly elucidated. We here investigated the molecular mechanism by which PGE2 regulates angiogenesis. Treatment of human umbilical vein endothelial cells (HUVEC) with PGE2 increased angiogenesis. PGE2 increased phosphorylation of Akt and endothelial nitric oxide synthase (eNOS), eNOS activity, and nitric oxide (NO) production by the activation of cAMP-dependent protein kinase (PKA) and phosphatidylinositol 3-kinase (PI3K). Dibutyryl cAMP (DB-cAMP) mimicked the role of PGE2 in angiogenesis and the signaling pathway, suggesting that cAMP is a down-stream mediator of PGE2. Furthermore, PGE2 increased endothelial cell sprouting from normal murine aortic segments, but not from eNOS-deficient ones, on Matrigel. The angiogenic effects of PGE2 were inhibited by the inhibitors of PKA, PI3K, eNOS, and soluble guanylate cyclase, but not by phospholipase C inhibitor. These results clearly show that PGE2 increased angiogenesis by activating the NO/cGMP signaling pathway through PKA/PI3K/Akt-dependent increase in eNOS activity.     

HPLC-UV measurements of metabolites in the supernatant of endothelial cells exposed to oxidative stress

2,8-Dihydroxyadenine (2,8-DHA) was identified by high-performance liquid chromatography with ultraviolet detection as a major metabolite in the supernatant of endothelial cells of the pulmonary artery (PAECs) and aorta (AECs), in addition to hypoxanthine, xanthine, uric acid, and uracil. Under normoxic, hypoxic, and hyperoxic conditions, the concentrations of all the identified metabolites change with time, marking the response of endothelial cells to stress, as a result of changes in cellular metabolism. Thus, the metabolites can serve as stress markers, and their concentrations can indicate the type and the level of cell stress. The results verify that PAECs adapt to survive oxidative stress of hyperoxia. However, AECs can adapt to hypoxia only for a short time and do not survive prolonged hypoxia. The role of the polyamine synthesis pathway in the formation of the unsalvaged adenine, as a possible source of 2,8-DHA, is discussed.     

A rapid method for detection of cell adhesion molecules (CAMs) on human umbilical vein endothelial cells (HUVECs)

Detection of cell surface proteins is widely used as molecular markers for initiation, progression and severity of many diseases. In particular, detection of cell adhesion molecules (CAMs) on endothelial cells is important as it indicates the extent of inflammation associated with several diseases including arthritis, asthma, tumor metastasis, etc. Here, we report, a rapid method for detection of CAMs on endothelial cells by covalently immobilizing TNF-α induced cells on a photoactivated polystyrene microtiter plate at 50 °C in 45 min followed by performing enzyme-linked immunosorbent assay (ELISA) technique at elevated temperature. Our method reduced the time of cell-ELISA to 3 h with results akin to conventional cell-ELISA carried out in 38 h. The method thus described herein could be potentially useful in clinical and research laboratories for rapid detection of cell surface proteins including CAMs on intact cell samples.     

Expression of integrin beta1 and its roles on adhesion between different cell cycle hepatocellular carcinoma cells (SMMC-7721) and human umbilical vein endothelial cells

To investigate expression of integrin β₁ and its roles on adhesion between different cell cycle hepatocellular carcinoma cell (HCC) and human umbilical vein endothelial cells (HUVEC), the synchronous G₁ and S phase HCC were achieved through thymine-2-deoxyriboside and colchicines sequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Expression of integrin β₁ on hepatocellular carcinoma cells was detected with flow cytometer. Further, the adhesive force of HCC to HUVEC and the role of integrin β₁ in this adhesive course were studied by micropipette aspiration technique. The results showed that percentage of each cyclic phases of the controlled HCC (non-synchronous) are: G₂+M phase, 11%; G₁ phase, 54%; S phase, 36%; the synchronous rates of G₁ and S phase HCC amount to 74 and 98%, respectively. The expressive fluorescent intensity of integrin β₁ in G₁ phase HCC is depressed significantly than the values of S phase and controlled HCC. Accordingly, the adhesive forces of G₁ phase HCC to HUVEC was significantly lower than the value of S phase cells (P<0.01), but it has no remarkable difference when compared the adhesive force values of S phase HCC with control; the contribution of integrin β₁ was about 50% in the adhesion of HCC to HUVEC. It suggested that HCC would be synchronized preferably in G₁ and S phase with thymine-2-deoxyriboside and colchicines, the adhesive molecule integrin β₁ expressed in a high lever in HCC and presented differences in vary cell cycle, and integrin β₁ played an important roles in adhesion of HCC to HUVEC. Possibly, S phase HCC take a great action in this adhesive course.     

New constituents from Crinum latifolium with inhibitory effects against tube-like formation of human umbilical venous endothelial cells

Six compounds (1-6) were isolated from the methanol extract of Crinum latifolium by bioassay-guided separation. Among the six isolates, compounds 2 and 6 were new metabolites. Their structures were established as 4-senecioyloxymethyl-3,4-dimethoxycoumarin (2) and 5,6,3'-trihydroxy-7,8,4'-trimethoxyflavone (6) based on spectroscopic analyses. Compound 2 was found to be strongly inhibitory against the in vitro tube-like formation of human umbilical venous endothelial cells (HUVECs) while manifesting no cytotoxicity in tumor cell lines (B16F10, HCT116). Significant inhibitory activity (inhibition percentage, 53.5%) was still observed at concentrations as low as 1 microg/mL. Compound 6 showed a modest inhibitory effect on the tube-like formation of HUVECs. Other compounds, including cycloartenol (1), 4',7-dihydroxy-3'-methoxyflavan (3), 4',7-dihydroxyflavan (4), and 2',4',7-trihydroxydihydrochalcone (5) were found to be nearly inactive.     

Effect of surface charge of magnetite nanoparticles on their internalization into breast cancer and umbilical vein endothelial cells

Internalization of magnetite nanoparticles with diameter of approximately 40 nm into normal and cancer cells was examined by microscopic observation and flow cytometry. Magnetite nanoparticles were synthesized by hydrolysis in an aqueous solution containing ferrous chloride with organic amines as a base. It was demonstrated that the difference in surface charge of magnetite nanoparticles brought about the difference in uptake efficiency. The nanoparticles with positive charge showed higher internalization into human breast cancer cells than the nanoparticles with negative charge, while the degree of internalization of the positively- and negatively-charged nanoparticles into human umbilical vein endothelial cells (HUVEC) was almost the same.     

In situ formation and scanning electrochemical microscopy assisted positioning of NO-sensors above human umbilical vein endothelial cells for the detection of nitric oxide release

Pt microelectrodes (50 μm diameter) were positioned by means of scanning electrochemical microscopy assisted z-approach curves and in situ modified with nickel tetrasulfonated phthalocyanine tetrasodium salt as electrocatalytic layer for the specific oxidative detection of nitric oxide. The thus modified electrodes were then moved over a layer of adherently growing human umbilical vein endothelial cells (HUVEC) in order to amperometrically detect nitric oxide (NO) released from the cells upon stimulation with bradykinin. This approach actually takes advantage of the use of SECM to define a sequential procedure that enables the in situ functionalisation of the SECM tip thus allowing to accurately control the separation between the functionalised SECM tip and the cell population.     

System to screen and purify active ingredients from herbal medicines using hydrogel-modified human umbilical vein endothelial cell membrane chromatography coupled with semi-preparative high-performance liquid chromatography-offline-high-performance liquid chromatography-mass spectrometry

Analytical screening and validation systems based on a combination of cell membrane chromatography and two-dimensional chromatography-tandem mass spectrometry are incapable of providing prepared samples containing the active ingredients found in traditional Chinese medicine; therefore, these samples cannot be directly used in subsequent studies. In this study, a semi-preparative cell membrane chromatography column was developed using a hydrogel-modified carrier and human umbilical vein endothelial cells to optimize prepared conditions, such as hydrogel polymerization, cell fragmentation, and cell membrane volume. This increased the binding ratio of membrane protein and carrier to 15.79 mg/g. The column was systematically evaluated using multitarget tyrosine kinase inhibitors that displayed good specificity and reproducibility. Subsequently, using the column coupled with a semi-preparative high-performance liquid chromatography-offline-high-performance liquid chromatography-mass spectrometry system, 15 active ingredients were screened and purified from Indigo naturalis, and five main components were identified: l -lysine, oxyresveratrol, tryptanthrin, isorhamnetin, and indirubin. Furthermore, the pharmacological effects of the ingredients were confirmed using cell proliferation and apoptosis assays. Results revealed potent proliferation-inhibiting and apoptosis-promoting abilities on human chronic myelogenous leukemic cells and human promyelocytic leukemic cells (p < 0.001). Overall, the system presented screening and purification functions that could be used to prepare I. naturalis samples acting on the epidermal growth factor receptor and vascular endothelial cell growth factor.     

Protective effect of total flavonoids from Spirodela polyrrhiza (L.) Schleid on human umbilical vein endothelial cell damage induced by hydrogen peroxide

In this study, we determined the protective effect of total flavonoids from Spirodela polyrrhiza (L.) Schleid (STF), which is a kind of traditional Chinese medicine, on human umbilical vein endothelial cells (ECV-304) damage induced by hydrogen peroxide (H(2)O(2)). Treated with 1mmol/L H(2)O(2) for 1h, the viability of ECV-304 cells markedly decreased. However, pretreatment with 10-50mug/mL STF resulted in a significant recovery. The survival rate of ECV-304 increased from 21.98% (only treated with 1mmol/L H(2)O(2)) to 64.74% (pretreated with 50microg/mL STF), which accompanied with the amounts of malondialdenhyde (MDA) decreasing from 1.6883nmol/L to 0.9628nmol/L. Furthermore, compared with control group, the 50mumol/L STF pretreatment enhanced the total antioxidant capacity (T-AOC) by 4.49 times, increased the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) by 85.12%, 158.94% and 94.5%,respectively, and increased the content of nitric oxide (NO) by 116.55%.Taken together, STF protect ECV-304 cells against H(2)O(2) damage by enhancing the antioxidant ability and increasing NO production.     

Screening and analyzing the potential bioactive components from Shaofu Zhuyu decoction, using human umbilical vein endothelial cell extraction and high-performance liquid chromatography coupled with mass spectrometry

In this paper, a useful method for screening and analyzing the potential bioactive components in bioassay-guided fraction (SF-11) from Shaofu Zhuyu decoction was developed using human umbilical vein endothelial cell (HUVEC) extraction and high-performance liquid chromatography coupled with Q-TOF/MS spectrometry. In addition, the protective effects on HUVEC damage induced by adrenaline in vitro were also investigated. The results showed that SF-11 significantly inhibited the endothelin (ET) release and reversed the NO secretion of HUVEC (p < 0.05), and promoted the PGI(2) release of HUVEC (p < 0.05). Two effective components, paeoniflorin and typhaneoside, from SF-11 were screened and identified using live cell extract and HPLC coupled with Q-TOF/MS spectrometry. The compounds, paeoniflorin and typhaneoside, showed significantly inhibiting effects on the ET release and reversing of NO secretion of HUVEC (p < 0.05), with similar effects to SF-11, and promoting the PGI(2) release of HUVEC at the concentration of 0.208 and 0.013 micromol/mL, respectively (p < 0.05). These data indicated that the method of live cell extraction coupled with HPLC-MS technology is feasible, rapid and useful for screening and analyzing potential bioactive components from TCMs.     

UV-A induced oxidative stress is more prominent in naturally pigmented aged human RPE cells compared to non-pigmented human RPE cells independent of zinc treatment

To investigate the effects of zinc supplementation on human amelanotic (ARPE-19) and native pigmented retinal pigment epithelial cells (hRPE) under normal light conditions and after ultraviolet A light exposure. hRPE cells, containing both melanin and lipofuscin granules, were prepared from human donor eyes of 60-70 year old patients. Cells of the amelanotic ARPE-19 cell line and pigmented hRPE cells were treated with zinc chloride and subjected to oxidative stress by UV-A irradiation. Intracellular H(2)O(2) formation was measured using a fluorescence oxidation assay. Additionally, apoptosis and viability assays were performed. Control cells were treated identically except for irradiation and zinc supplementation. Under normal light conditions, zinc treated hRPE cells produced less H(2)O(2) than unsupplemented hRPE cells. Viability and apoptosis events did not change. After UV-A irradiation, ARPE and hRPE cells were greatly impaired in all tests performed compared to the non-irradiated controls. No differences were found after zinc supplementation. hRPE cells showed a higher apoptosis and mortality rate than non-pigmented cells when stressed by UV-A light. ARPE cells never showed any zinc related effects. In contrast, without irradiation, zinc supplementation reduced H(2)O(2) production in pigmented hRPE cells slightly. We did not find any zinc effect in irradiated hRPE cells. After UV light exposure, pigmented cells showed a higher apoptosis and mortality than cells lacking any pigmentation. We conclude that cells with pigmentation consisting of melanin and lipofuscin granules have more prooxidative than antioxidative capacity when stressed by UV light exposure compared to cells lacking any pigmentation.     

Expression of integrin β1 and its roles on adhesion between different cell cycle hepatocellular carcinoma cells (SMMC-7721) and human umbilical vein endothelial cells

To investigate expression of integrin β₁ and its roles on adhesion between different cell cycle hepatocellular carcinoma cell (HCC) and human umbilical vein endothelial cells (HUVEC), the synchronous G₁ and S phase HCC were achieved through thymine-2-deoxyriboside and colchicines sequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Expression of integrin β₁ on hepatocellular carcinoma cells was detected with flow cytometer. Further, the adhesive force of HCC to HUVEC and the role of integrin β₁ in this adhesive course were studied by micropipette aspiration technique. The results showed that percentage of each cyclic phases of the controlled HCC (non-synchronous) are: G₂+M phase, 11%; G₁ phase, 54%; S phase, 36%; the synchronous rates of G₁ and S phase HCC amount to 74 and 98%, respectively. The expressive fluorescent intensity of integrin β₁ in G₁ phase HCC is depressed significantly than the values of S phase and controlled HCC. Accordingly, the adhesive forces of G₁ phase HCC to HUVEC was significantly lower than the value of S phase cells (P<0.01), but it has no remarkable difference when compared the adhesive force values of S phase HCC with control; the contribution of integrin β₁ was about 50% in the adhesion of HCC to HUVEC. It suggested that HCC would be synchronized preferably in G₁ and S phase with thymine-2-deoxyriboside and colchicines, the adhesive molecule integrin β₁ expressed in a high lever in HCC and presented differences in vary cell cycle, and integrin β₁ played an important roles in adhesion of HCC to HUVEC. Possibly, S phase HCC take a great action in this adhesive course.     

The toxicity of hydroxylated and carboxylated multi-walled carbon nanotubes to human endothelial cells was not exacerbated by ER stress inducer

The toxicity of hydroxylated or carboxylated MWCNTs to human endothelial cells was modest, and the toxicity was not exacerbated by ER stress inducer.     

Silibinin is a potent sensitizer of UVA radiation-induced oxidative stress and apoptosis in human keratinocyte HaCaT cells

UVA radiation (315-400 nm), which constitutes ca 95% of the UV irradiation in natural sunlight reaching earth surface, is a major environmental risk factor associated with human skin cancer pathogenesis. UVA is an oxidizing agent that causes significant damage to cellular components through the release of reactive oxygen species (ROS) and leads to photoaging and photocarcinogenesis. Here we investigate the effect of silibinin, the flavonolignan from Silybum marianum, on UVA-induced ROS and cell death in human keratinocyte cell line HaCaT. In addition, the effect of silibinin on UVA-induced intracellular ROS-mediated endoplasmic reticulum (ER) stress was also analyzed. UVA irradiation resulted in ROS production and apoptosis in HaCaT cells in a dose-dependent manner, and the ROS levels and apoptotic index were found to be elevated significantly when the cells were treated with 75 μmsilibinin for 2 h before UVA exposure. When the cells were pretreated with 10 mmN-acetyl cysteine, the enhancement of UVA-induced apoptosis by silibinin was compromised. Furthermore, we found that silibinin enhances ER stress-mediated apoptosis in HaCaT cells by increasing the expression of CHOP protein. These results suggest that silibinin may be beneficial in the removal of UVA-damaged cells and the prevention of skin cancer.