Animals ; Endothelium, Vascular ; drug effects ; Humans ; Pyrazines ; pharmacology

OBJECTIVESTo investigate the effects of glucose and free fatty acids (FFAs) on the proliferation and cell cycle of human vascular endothelial cells in vitro, and to examine whether the combined presence of elevated FFAs and glucose may cross-amplify their individual injurious effects.

METHODSCultured human vascular endothelial cells (ECV304) were incubated with various concentrations of glucose and/or FFAs (palmitate and/or oleate) for 24 - 96 h. Morphologic alterations were observed using a phase contrast microscope and an electron microscope. Inhibition of proliferation was measured by a colorimetric 3-[4, 5-dimethyl thiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Cell viability was determined using trypan blue exclusion. Distribution of cells along phases of the cell cycle was analyzed by flow cytometry.

RESULTSGlucose 15 or 30 mmol/L, palmitate (PA) 0.25 or 0.5 mmol/L, and oleate (OA) 0.5 mmol/L inhibited proliferation and accelerated death of endothelial cells in a dose-and-time-dependent manner. After treatment with elevated glucose and/or FFAs, the G(0)/G(1) phase cells increased, whereas S phase cells decreased, suggesting that high glucose and/or FFAs mainly arrested endothelial cells at G(0)/G(1) phase. The inhibitive rates of proliferation and population of dead cells in endothelial cells incubated with glucose plus FFAs (glucose 30 mmol/L + PA 0.25 mmol/L, glucose 30 mmol/L + OA 0.5 mmol/L, glucose 30 mmol/L + PA 0.25 mmol/L + OA 0.5 mmol/L) increased more markedly than those treated with high glucose or FFAs (PA and/or OA) alone.

CONCLUSIONBoth high ambient glucose and FFAs can inhibit proliferation and accelerate death of endothelial cells in vitro. These changes were cross-amplified in the combined presence of high levels of glucose and FFAs.

Cell Division ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Endothelium, Vascular ; cytology ; Fatty Acids, Nonesterified ; pharmacology ; Glucose ; pharmacology ; Humans

AIMTo compare the differences of pharmacological characteristics of the endothelial target for acetylcholine (ETA) between rat aorta and tail artery.

METHODSDifferences in the endothelium-dependent relaxation induced by acetylcholine (ACh: 10(-8) - 10(-4) mol/L) were studied using isolated rat tail artery helical strips and aortic rings, so that the pharmacological characteristics of ETA in small artery can be observed.

RESULTSACh-induced endothelium-dependent relaxation was observed both in rat tail artery strips and in aortic rings precontracted with potassium chloride (60 mmol/L) in a concentration-dependent manner. In tail artery this effect was partially blocked by L-N(omega)-Nitro-arginine methyl ester (L-NAME: 10(-4) mol/L) or methylene blue (MB: 10(-5) mol/L), together with indomethacin (Indo: 10(-4) mol/L), but in aorta it was completely blocked by L-NAME or MB.

CONCLUSIONIt is different of the pharmacological characteristics of ETA between big artery and small artery. A non-NO and non-PGI2 relaxing factor, together with nitric oxide (NO) and prostacyclin (PGI2), mediates endothelium-dependent vasorelaxation induced by ACh in small artery, but NO may be the principal endothelial vasodilator substance in big artery.

Acetylcholine ; pharmacology ; Animals ; Aorta ; drug effects ; Arteries ; drug effects ; Endothelium, Vascular ; drug effects ; physiology ; In Vitro Techniques ; Male ; Rats ; Rats, Wistar ; Vasodilation ; drug effects

OBJECTIVETo investigate the vasorelaxant effect of taurine (Tau) in rat aortic rings and the mechanism.

METHODThe isolated thoracic aortic rings of male Wistar rats were mounted on the organ bath. The effect of Tau 10, 20, 40, 80 mmol x L(-1) on the rings with endothelium intact or endothelium denuded precontracted by the phenylephrine (1 micromol x L(-1)) or KCl (60 mmol x L(-1)), and the effect of Tau on the vessel reaction induced by various drugs were recorded with biological signal analytical system.

RESULTTaurine completely relaxed the contractions induced by KCl and phenylephrine in a concentration-dependent manner in endothelium-intact and endothelium-denuded rat aorta. Taurine attenuated the contraction to PE both in the absence and presence of calcium, but had no significant effect on the contraction induced by caffeine. The relaxant effect of taurine was significantly inhibited by pretreatment of endothelium-denuded aorta with potassium channel antagonists glibenclamide and tetraethylamine but not by BaCl2 or 4-aminopyridine.

CONCLUSIONTaurine induces an endothelium-independent relaxation in rat aortic rings. The mechanisms may involve the reduction in Ca2+-influx and Ca2+-release and the participation of the potassium channels (KATP and KCa, but not Kir or KV).

Animals ; Aorta ; drug effects ; physiology ; Endothelium, Vascular ; drug effects ; physiology ; Male ; Models, Animal ; Muscle Relaxation ; drug effects ; Rats ; Rats, Wistar ; Taurine ; pharmacology ; Vasodilation ; drug effects ; Vasodilator Agents ; pharmacology

AIMTo examine the effect of HO-1 inducer hemin on hydrogen peroxide (H2O2) caused decrease in contraction of isolated rat aortic rings, and to elucidate the underlying mechanism.

METHODSThe thoracic aortic rings with endothelium of male Sprague-Dawley rats were mounted on a bath system. Isometric contractions of aortic rings were measured.

RESULTS(1) After intraperitoneal injection of HO-1 inducer hemin, HO-1 activity of thoracic aorta and COHb concentration in rat blood enhanced. And it also prevented the decrease in contraction responses to PE which pretreatment of arteries with 300 micromol/L H2O2. (2) Pretreatment of ATP-sensitive potassium channel inhibitor glibenclamide, but not GC inhibitor methylene blue, could partly abolish the protection of hemin in arteries with H2O2 exposure. (3) Hemin could not influence the shift of concentration-response curve to [Ca2+]o in arteries with H2O2 exposure. (4) In Ca(2+) -free K-H solution, exposure of H2O2 reduced caffeine and PE-induced constriction in the rat aortic rings. After pretreatment of hemin, could prevent the decrease in contraction responses to caffeine and PE.

CONCLUSIONIncrease in HO-1 activity could prevent the H2O2 induced decrease in contraction responses to PE in intact aortic rings. The mechanism might be involved in activation of ATP-sensitive potassium channel and mobilization of intracellular calcium stores, but had no relationship with the GC pathway.

Animals ; Aorta, Thoracic ; drug effects ; physiology ; Endothelium, Vascular ; Heme Oxygenase-1 ; pharmacology ; Hydrogen Peroxide ; adverse effects ; KATP Channels ; drug effects ; Male ; Rats ; Rats, Sprague-Dawley ; Vasoconstriction ; drug effects

Pingyangmycin (bleomycin A5 hydrochloride, PYM) is one of the anti-neoplastic agents which have been commonly used to treat venous malformations. However, the underlying mechanism by which PYM treats venous malformations remains poorly understood. It was reported that venous endothelial cells could recruit neutrophils via adhesion molecules (E-selectin, ICAM-1, ICAM-3, VCAM-1) during the acute/chronic inflammation and subsequent histological fibrosis after sclerotherapy with PYM. This study explored if the expression of E-selectin, ICAM-1, ICAM-3 and VCAM-1 in human venous malformation endothelial cells could be affected by PYM. HVMECs were cultured from human venous malformation tissue. Expressions of E-selectin, ICAM-1, ICAM-3 and VCAM-1 on HVMECs in response to PYM were analyzed by cell ELISA. The relative levels of mRNA expression in the cells were semi-quantified. The results showed that PYM up-regulated the expressions of E-selectin, ICAM-3, VCAM-1 and ICAM-1 in both time- and concentration-dependent manner. Our findings suggested that PYM could induce the expression of adhesion molecules in HVMECs, which might be a possible mechanism by which sclerotherapy by intralesional injection of PYM treats venous malformations.
Bleomycin ; analogs & derivatives ; pharmacology ; Cell Adhesion Molecules ; genetics ; metabolism ; Cells, Cultured ; Endothelial Cells ; drug effects ; metabolism ; Endothelium, Vascular ; drug effects ; metabolism ; Gene Expression ; drug effects ; genetics ; Humans

OBJECTIVETo establish an in vitro pig iliac artery endothelial cells (PIECs) senescence model using carbamide peroxide (CP).

METHODSMTT assay and DAPI staining were used to define the optimal concentration of CP for inducing to the PIECs senescence model. Cellular morphology, MTT assay, EdU labeling, SA-β-gal staining and cell scratch test were performed to analyze the cell growth kinetic, proliferative activity, aging ratio and migratory activity difference post CP induction. PI signal staining flow cytometry was used to analyze the cell cycle distribution difference of cells before and after CP induction.

RESULTSThe optimal CP concentration was 40 µmol/L to induce PIECs senescence. After 1 h treatment with 40 µmol/L CP, the PIECs presented typical aging form with lager and more rounded shapes. Compared with control group, the proliferative activity and the migratory distance of CP group were significantly decreased; the SA-β-gal staining positive ratio was significantly increased; the data of mitotic cycle distribution with flow cytometry analysis showed that most cells were arrested at G(1)/G(0) phase.

CONCLUSIONCP could efficiently induce pig iliac artery endothelial cell senescence in vitro.

Animals ; Cells, Cultured ; Cellular Senescence ; drug effects ; Endothelial Cells ; cytology ; drug effects ; Endothelium, Vascular ; cytology ; drug effects ; Models, Cardiovascular ; Peroxides ; pharmacology ; Swine ; Urea ; analogs & derivatives ; pharmacology

Oxidized low density lipoprotein (oxLDL) can trigger intracellular production of reactive oxygen species and lipid peroxidation (LPO), and is thought to contribute to initiation and progression of atherosclerosis. In order to understand the correlation between oxLDL and macromolecular damage, we measured levels of LPO-derived miscoding etheno-DNA adducts and LPO-modified proteins in cultured human vascular endothelial and smooth muscle cells after incubation with oxLDL for up to 48 h. A semi-quantative analysis method for 1, N6-ethenodeoxyadenosine (ɛdA) by immunohistochemistry was applied. After oxLDL stimulation, ɛdA-stained nuclei were significantly increased in both endothelial and smooth muscle cells. Similarly, 4-hydroxy-2-nonenal (4-HNE)-modified proteins, as analyzed by immunohistochemistry and Western blotting, were also 3-5 fold increased. It was concluded LPO-derived etheno-DNA adducts and LPO-modified proteins are strongly induced by oxLDL in human vascular endothelial and smooth muscle cells. This macromolecular damage may contribute to the dysfunction of arterial endothelium and the onset of atherosclerosis.
DNA ; metabolism ; Endothelium, Vascular ; cytology ; drug effects ; metabolism ; Humans ; Lipid Peroxidation ; drug effects ; Lipoproteins, LDL ; pharmacology ; Muscle, Smooth ; cytology ; drug effects ; metabolism ; Proteins ; metabolism

OBJECTIVETo examine the effect of cinobufacini on rat thoracic aorta and its mechanism.

METHODSIsolated rat thoracic aorta was perfused and isometric tension was recorded by organ bath technique before and after cinobufacini treatment.

RESULTCinobufacini induced contraction of isolated thoracic aorta with or without endothelium in a concentration-dependent manner (at concentration of 2.5,5.0,7.5,10.0 g/L). The vasoconstriction effect of cinobufacini was more potent in endothelium-denuded aorta ring [(16.3+/-3.39)%, (52.5+/-7.70)%, (60.9+/-8.84)%, (69.2+/-11.34)%] than in endothelium-intact aorta ring [(6.2+/-2.07)%, (14.7+/-4.91), (17.6+/-5.86)%, (20.3+/-6.78)% (P<0.01)]. Its contractile effect was attenuated in Ca(2+)-free solution (about 1/10 of that in buffer with 1.25 mmol/L CaCl(2)) or by the treatment with verapamil (10(-7)mol/L), an L-type calcium channel antagonist. Cinobufacini induced contraction on the endothelium-intact rat aorta was augmented by pretreatment with L-NAME (10(-4)mol/L), a nitric oxide synthase inhibitor.

CONCLUSIONCinobufacini contracts rat thoracic aorta by opening the voltage-dependent Ca(2+) channel and increasing Ca(2+) influx into vascular smooth muscle. Cinobufacini can also stimulate the release of vascular relaxant factor, nitric oxide, from the endothelium and thus antagonize cinobufacini-induced contraction.

Animals ; Aorta, Thoracic ; drug effects ; Bufanolides ; pharmacology ; Endothelium, Vascular ; drug effects ; metabolism ; In Vitro Techniques ; Male ; Nitric Oxide ; biosynthesis ; Rats ; Rats, Sprague-Dawley ; Vasoconstriction ; drug effects ; Vasoconstrictor Agents ; pharmacology

AIMTo establish the quality control methods for recombinant human endostatin.

METHODSBiological activity was determined by endothelial cell migration assays. Peptide mapping was tested by trypsin digestion and RP-HPLC. Purity was determined by non-reduced SDS-PAGE and RP-HPLC. Other tests including molecular weight, isoelectrical point, etc. were done according to the National Requirements for Biological Products (2000).

RESULTSThe method of bioassay was established and used for determining activity of endostatin. Specific activity of the three batchs of drug substance was 1.45 x 10(6), 1.57 x 10(6) and 2.73 x 10(6) u.mg-1 proteins. Peptide mappings of the three batches of drug substance were completely identified. Both purity results of the products tested by SDS-PAGE and RP-HPLC were more than 99%.

CONCLUSIONThe established methods can effectively control the quality of recombinant human endostatin.

Cell Movement ; drug effects ; Cells, Cultured ; Endostatins ; pharmacology ; Endothelium, Vascular ; cytology ; Humans ; Quality Control ; Recombinant Proteins ; pharmacology ; Technology, Pharmaceutical ; methods