OBJECTIVE: To study hesperetin-induced vasorelaxation after depolarizing contraction in human umbilical veins (HUVs) to elucidate the role of L-type Ca²⁺ channel (LTCC) and related signaling pathway. METHODS: Isometric tension recording was performed in HUV rings pre-contracted with K⁺. Hesperetin relaxing mechanism was investigated using a LTCC opener (BayK8644) and blockers of cyclic nucleotides and phosphodiesterases (PDEs). Whole-cell patch-clamping in A7r5 cells, a rat vascular smooth muscle cell line, was performed to study the effect of hesperetin on LTCC current. RESULTS: After depolarizing precontraction, hesperetin induced HUV relaxation concentration-dependently and endothelium-independently; 1 mmol/L hesperetin reduced denuded HUV ring tension by 68.7% ± 4.3% compared to matching vehicle, osmolality, and time controls (P<0.0001). Importantly, hesperetin competitively inhibited BayK8644-induced contraction, shifting the half maximal effective concentration of BayK8644 response from 1.08 nmol/L [95% confidence interval (CI) 0.49-2.40] in vehicle control to 11.30 nmol/L (95% CI 5.45-23.41) in hesperetin (P=0.0001). Moreover, hesperetin elicited further vasorelaxation in denuded HUV rings pretreated with inhibitors of soluble guanylyl cyclase, adenylyl cyclase, PDE3, PDE4, and PDE5 (P<0.01), while rings pretreated with PDE1 inhibitors could not be relaxed by hesperetin (P>0.05). However, simultaneously applying inhibitors of soluble guanylyl cyclase and adenylyl cyclase could not inhibit hesperetin's effect (P>0.05). In whole-cell patch-clamping, hesperetin rapidly decreased LTCC current in A7r5 cells to 66.7% ± 5.8% (P=0.0104). CONCLUSIONS Hesperetin diminishes depolarizing contraction of human vascular smooth muscle through inhibition of LTCC, and not cyclic nucleotides nor PDEs. Our evidence supports direct LTCC interaction and provides additional basis for the use of hesperetin and its precursor hesperidin as vasodilators and may lead to future vasodilator drug development as a treatment alternative for cardiovascular diseases.
Hesperidin/pharmacology* ; Humans ; Calcium Channels, L-Type/metabolism* ; Umbilical Veins/physiology* ; Muscle Contraction/drug effects* ; Animals ; Rats ; Calcium Channel Blockers/pharmacology* ; Vasodilation/drug effects* ; Muscle Relaxation/drug effects*

This study investigated the changes in human umbilical vein endothelial cells (HUVECs) induced by overexpression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) and its role in cellular injury. Recombinant NOSTRIN-expressing and empty vectors were transfected into cultured HUVECs, and factor VIII-related antigen was examined by using immunohistochemical analysis. Growth curves were generated for both transfected and untransfected cells and these indicated that the proliferative ability of cells overexpressing NOSTRIN was significantly decreased. The expression of NOSTRIN and eNOS proteins was detected by using Western blot analysis, endothelial NOS (eNOS) activity was assayed by using spectrophotometry, and NO2 (-)/NO3 (-) levels were measured using nitrate reductase. Immunohistochemical analysis demonstrated that all groups expressed NOSTRIN in the plasma membrane and cytoplasm, and Western blot analysis confirmed that NOSTRIN levels were significantly higher in cells transfected with the NOSTRIN plasmid (P<0.01). The activity of eNOS and the levels of NO2 (-)/NO3 (-) were significantly decreased in NOSTRIN overexpressing cells as compared with empty vector and untransfected cells (P<0.01 and P<0.01, respectively). Morphological and ultrastructural changes were observed under light and electron microscopy, and it was found that NOSTRIN-overexpressing cells were elongated with deformities of the karyotheca, injury to the plasma membrane, increased lipids in the cytoplasm, and shortened microvilli. This study showed that overexpression of NOSTRIN had a significant effect on eNOS activity in HUVECs and resulted in significant cellular damage.
Apoptosis ; physiology ; Cell Line ; Cell Proliferation ; Cell Survival ; physiology ; Endothelial Cells ; pathology ; physiology ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Umbilical Veins ; metabolism ; pathology ; Up-Regulation

OBJECTIVETo study the effect of anticolchicine cytotoxicity of Dan Gua-Fang, a Chinesea Chinese), a Chinese herbal compound prescription on endothelial cells of vein (ECV304) cultivated in mediums of different glucose concentrations as well as the proliferation of those cells in the same conditions, in order to reveal the value of Dan Gua-Fang in preventing and treating endothelial damage caused by hyperglycemia in diabetes mellitus.

METHODSThe research was designed as three stages. The growing state and morphological changes were observed when ECV304 were cultivated in the culture mediums, which have different glucose concentrations with or without Dan Gua-Fang and at the same time with or without colchicine.

RESULTS(1) Dan Gua-Fang at all concentrations reduced the floating cell population of ECV304 cultivated in hyperglycemia mediums. (2) Dan Gua-Fang at all concentrations and hyperglycemia both had a function of promoting "pseudopod-like" structure formation in cultivated ECV304, but the function was not superimposed in mediums containing both hyperglycemia and Dan Gua-Fang. (3) Colchicine reduced and even vanished the "pseudopod-like" structure of the endotheliocyte apparently cultivated in mediums of hyperglycemia or with Dan Gua-Fang. The "pseudopod-like" structure of the endotheliocyte emerged quickly in Dan Gua-Fang groups after colchicine was removed, but it was not the case in hyperglycemia only without Dan Gua-Fang groups. (4) Dan Gua-Fang reduced the mortality of cells cultivated in mediums containing colchicine. The cell revived to its normal state fast after colchicine was removed.

CONCLUSIONDan Gua-Fang has the functions of promoting the formation of cytoskeleton and fighting against colchicine cytotoxicity.

Cell Culture Techniques ; Cell Line ; Cell Shape ; drug effects ; Colchicine ; adverse effects ; antagonists & inhibitors ; Culture Media ; adverse effects ; pharmacology ; Cytoprotection ; drug effects ; Cytotoxins ; adverse effects ; antagonists & inhibitors ; Drug Antagonism ; Drug Combinations ; Drug Evaluation, Preclinical ; Drug Synergism ; Drugs, Chinese Herbal ; adverse effects ; pharmacology ; Endothelial Cells ; drug effects ; physiology ; Glucose ; pharmacology ; Humans ; Umbilical Veins ; cytology ; drug effects ; Up-Regulation

The purpose of this study is to determine 1) whether morphine postconditiong (MPostC) can attenuate the intercellular adhesion molecules-1 (ICAM-1) expression after reoxygenation injury and 2) the subtype(s) of the opioid receptors (ORs) that are involved with MPostC. Human umbilical vein endothelial cells (HUVECs) were subjected to 6 hr anoxia followed by 12 hr reoxygenation. Three morphine concentrations (0.3, 3, 30 microM) were used to evaluate the protective effect of MPostC. We also investigated blockading the OR subtypes' effects on MPostC by using three antagonists (a micro-OR antagonist naloxone, a kappa-OR antagonist nor-binaltorphimine, and a delta-OR antagonist naltrindole) and the inhibitor of protein kinase C (PKC) chelerythrine. As results, the ICAM-1 expression was significantly reduced in the MPostC (3, 30 microM) groups compared to the control group at 1, 6, 9, and 12 hours reoxygenation time. As a consequence, neutrophil adhesion was also decreased after MPostC. These effects were abolished by coadministering chelerythrine, nor-binaltorphimine or naltrindole, but not with naloxone. In conclusion, it is assumed that MPostC could attenuate the expression of ICAM-1 on endothelial cells during reoxygenation via the kappa and delta-OR (opioid receptor)-specific pathway, and this also involves a PKC-dependent pathway.
Animals ; Benzophenanthridines/pharmacology ; Endothelial Cells/cytology/*drug effects/*metabolism ; Endothelium, Vascular/cytology ; Humans ; Intercellular Adhesion Molecule-1/genetics/*metabolism ; Morphine/*pharmacology ; Naloxone/pharmacology ; Naltrexone/analogs & derivatives/pharmacology ; Narcotic Antagonists/pharmacology ; Narcotics/*pharmacology ; Protein Isoforms/metabolism ; Protein Kinase C/antagonists & inhibitors/metabolism ; Receptors, Opioid/metabolism ; Reperfusion Injury/*metabolism ; Signal Transduction/physiology ; Umbilical Veins/cytology

OBJECTIVETo explore the correlation between circulating endothelial microparticles (EMPs) and flow-mediated dialation (FMD) in patients with coronary artery disease (CAD), and investigate the role of NADPH oxidase in endothelial cell dysfunction caused by EMPs.

METHODSFifteen patients with CAD and 15 at high risks of CAD were tested for the level of EMPs and FMD and other biochemical indices, and the correlation between the indices were analyzed. EMPs obtained from cultured human umbilical vein endothelial cells (HUVECs) were phenotyped and used to stimulate the HUVECs, whose ROS and NO production was tested.

RESULTSEndothelial dilation function could be damaged by circulating EMPs in CAD patients. Dysfunction of HUVECs caused by 10(5)/ml EMPs could be reversed by pretreatment with 20 micromol/L apocynin, a NADPH oxidase inhibitor.

CONCLUSIONEndothelial dialation function of the endothelial cells can be damaged by circulating EMPs in patients with CAD in association with NADPH oxidase activation.

Adult ; Aged ; Cell-Derived Microparticles ; drug effects ; metabolism ; Cells, Cultured ; Coronary Disease ; pathology ; Endothelial Cells ; cytology ; drug effects ; physiology ; Female ; Humans ; Male ; Middle Aged ; NADPH Oxidases ; metabolism ; Umbilical Veins ; cytology

OBJECTIVETo investigate the magnetic resonance (MR) signal changes of superparamagnetic iron oxide (SPIO) and its biological effects on endothelial cells.

METHODSThe citric-acid coated SPIO was synthesized by co-precipitation method. The human umbilical vein endothelial cells (HUVECs) were incubated with SPIO for 24 h in culture medium at iron concentration of 0.01, 0.05, 0.10, 0.15 mg/ml (experimental groups), and the cells incubated without SPIO served as control groups. The uptake efficiency of intracellular iron was measured by Prussian blue staining, and the cell viability was monitored by Calcein-AM method. The cell cytoskeleton (F-actin and tubulin), adherence and migration capacity were measured by immunofluorescence staining. The iron oxide nanoparticles distribution and the cellular organelle change were monitored by transmission electron microscopy (TEM). Quantification of particle uptake was measured by atomic absorption spectrometry. The MR signal of endothelial cells after labeling was monitored by Philips 3.0 T MR scanner.

RESULTSSPIO was uptaken by HUVECs in a concentration-dependence manner. Compared with the control group, cell viability was decreased along with the increase of iron concentration. Compared with the control group, the cell cytoskeleton was markedly disorganized and the FAK spot was bigger and sparser.The nanoparticles were mainly existed in lysosomes, and the higher concentration of SPIO, the more lysosomes and vacuoles presented in the cells. The iron content per cell was (55.86 +/-9.935) pg when the SPIO concentration was 0.15 mg/ml. The MR image showed that the cells labeled with SPIO resulted in the decrease of MR signal.

CONCLUSIONThe cells labeled with SPIO can be detected by MR. The cell viability, cytoskeleton, adherence and migration capacity of HUVECs are affected by citric-acid coated SPIO in a concentration-dependent manner.

Cells, Cultured ; Contrast Media ; pharmacology ; Endothelial Cells ; cytology ; physiology ; Ferrosoferric Oxide ; chemistry ; pharmacology ; Humans ; Image Enhancement ; methods ; Magnetic Resonance Imaging ; methods ; Magnetite Nanoparticles ; chemistry ; Spectrophotometry, Atomic ; Umbilical Veins ; cytology

OBJECTIVE: To determine the effect of pregnancy-associated plasma protein A (PAPP-A) on the function of vascular endothelial cells (VEC). METHODS: Human umbilical vein endothelial cell (HUVEC) line, derived from human umbilical vein, was cultured in vitro with PAPP-A at 0, 50, 100, and 200 ng/mL for 0, 12, 24, and 48 hours, respectively. Nitric oxide (NO) levels and endothlin-1 (ET-1) levels were determined by spectrophotometer and immunehistory. RESULTS: The NO levels of HUVECs in the PAPP-A groups were significantly lower than those in the control group (P<0.05). The ET-1 levels of HUVECs in the PAPP-A groups were significantly higher than those in the control group (P<0.05). The changes were all dose-dependent. CONCLUSION PAPP-A may affect the function of vascular endothelial cells by reducing the secretion of NO and increasing the level of ET-1.
Cell Line ; Endothelial Cells ; cytology ; metabolism ; physiology ; Endothelin-1 ; biosynthesis ; Female ; Humans ; Nitric Oxide ; biosynthesis ; Pregnancy-Associated Plasma Protein-A ; pharmacology ; Umbilical Veins ; cytology ; metabolism

OBJECTIVE: To evaluate the effect of intermittent high glucose on the apoptosis of human umbilical vein endothelial cells (HUVECs) and its mechanism. METHODS: Intermittent high glucose and constant high glucose were applied to HUVEC-12 for 7 days. Flow cytometer and fluorescent staining with Hoechst 33258 were used to detect apoptosis of HUVEC-12. The superoxide dismutase (SOD) activity and the content of malonaldehyde (MDA) in culture solution were detected with colorimetry, and the changes of p-JNK level were examined by Western blot. RESULTS: The apoptosis rate was obviously higher in the intermittent high glucose group than that in the constant high glucose group (P < 0.05). The SOD activity was significantly lower in the intermittent high glucose group (P < 0.05), but MDA level was higher than those of constant high glucose(P < 0.05). SP600125, the inhibitor of JNK, decreased the apoptosis rate induced by intermittent high glucose (P < 0.05). Antioxidant (Vitamin C) inhibited the p-JNK, decreased the apoptosis rate (P < 0.05). CONCLUSION Intermittent high glucose is easier to worsen the proapoptotic effects on HUVECs than that of constant high glucose, which may account for the increased oxidative stress, and then activates JNK signal transduction pathway.
Apoptosis ; drug effects ; Cells, Cultured ; Endothelial Cells ; pathology ; Glucose ; pharmacology ; Humans ; JNK Mitogen-Activated Protein Kinases ; genetics ; physiology ; Signal Transduction ; Umbilical Veins ; cytology ; pathology

OBJECTIVETo evaluate the functional regulation of endothelial Myosin light chain kinase (MLCK) in extravascular migration of fibrosarcoma HT1080 cells.

METHODSAn in vitro model of fibrosarcoma cell transmigration across a monolayer of HUVEC cultured on collagen gel was applied to observe extravascular migration of HT1080 cells,and were the electrical resistance of HUVEC monolayer and endothelial MLC phosphorylation in extravascular migration of HT1080 cells.

RESULTHT1080 cells migrated through endothelial cells into collagen gel, the electrical resistance of a HUVEC monolayer was reduced and endothelial MLC phosphorylation was enhanced in extravascular migration of fibrosarcoma cells. Endothelial MLCK inhibitor (ML-7) blocked extravascular migration of HT1080 cells and inhibited reduction of electrical resistance of a HUVEC monolayer and enhancement of endothelial MLC phosphorylation in extravascular migration of HT1080 cells in a dose-dependent manner.

CONCLUSIONEndothelial MLCK regulates fibrosarcoma cell transendothelial migration through MLC phosphorylation, leading to cytoskeletal reorganization and endothelial cell constriction, then fibrosarcoma cells migrate into extravascular tissue through the gaps between endothelial cells.

Cell Line, Tumor ; Cell Movement ; drug effects ; Endothelial Cells ; cytology ; enzymology ; Fibrosarcoma ; pathology ; physiopathology ; Humans ; Myosin-Light-Chain Kinase ; metabolism ; physiology ; Neoplasm Invasiveness ; Umbilical Veins ; cytology

The changes of pressure in local circulation flow field and the alterations of biorheological characteristics in Endothelial cells (ECs) would follow the geometric changes of cardiovascular wall structures and would further result in distinct pathophysiological changes of endothelial cellular proliferation and vitality. This experiment is designed to observe the effects of pressure shift on ECs proliferation, apoptosis, and expression of apoptosis-associated protein, to elucidate the influences of pressure shift on the vitality of ECs, and to shed light on the dose-effect relationship concerned. By adopting flow cytometery, transmission electron microscopy, real-time RT-PCR and Western blotting, we set the levels of pressure loading ECs groups and set down the non-activated cultured ECs,single shear stress loading ECs as the control group for studies on the ultra-structure alterations, on the distribution of cell cycle and the changes of proliferation and apoptosis in ECs. We also investigated the changes of the expression of Caspase-3 gene and the changing regularity of P53, Bcl-2 and Fas protein at the translation level. When ECs being exposed to decreased pressure shift (-40 cmH2O), distinct apoptosis in ECs could be observed and a pattern of duration-dependence was seen. The expressions of P53, Bcl-2 and Fas proteins are essential for regulating the genesis and process of ECs apoptosis induced by -40 cmH2O pressure.
Apoptosis ; physiology ; Apoptosis Inducing Factor ; metabolism ; Caspase 3 ; metabolism ; Cell Proliferation ; Cells, Cultured ; Endothelial Cells ; cytology ; Fas Ligand Protein ; metabolism ; Humans ; Pressure ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rheology ; Stress, Mechanical ; Tumor Suppressor Protein p53 ; metabolism ; Umbilical Veins ; cytology