OBJECTIVETo observe the effects of Danhong Injection (丹红注射液) and its main components, including daiclzein and hydroxysafflor yellow A (HSYA), on the anticoagulation, fibrinolysis, anti-apoptosis in hypoxia model of vein endothelial cells (VECs).

METHODSVECs were prepared and were put in a hypoxia environment, which consisted of mixed gas of 95% N and 5% CO mixed gas, when reached confluent culture. Five groups used different treatments, including normal control group, hypoxia group, daiclzein group, HSYA group and Danhong Injection group. The VECs were identified by fluorescence double labeling methods. The morphology was observed by a phase contrast microscopy. The effects of Danhong Injection, daiclzein and HSYA on 6 keto prostaglandin F1α (6-keto-PGF1α) level was measured by the method of radioimmunoassay (RIA). Superoxide dismutase (SOD) activity was tested by water soluble tetrazolium salt. The content of malondialdehyde (MDA) was measured by thiobarbituric acid. The activities of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) were measured by the method of chromogenic substrate. The contents of endothelin (ET) and nitric oxide (NO) were detected by non-equilibrium RIA and enzymelinked immunosorbent assay. Cells apoptosis rate was determined by flow cytometry.

RESULTSCompared with the normal control group, the floating cells number, PAI activity, ET and MDA contents, and cells apoptosis rate in the culture solution of hypoxia group were all significantly increased, whereas the 6-keto-PGF1α and NO contents, and t-PA and SOD activities were decreased significantly (P<0.01). Compared with the hypoxia group, Danhong Injection markedly increased the 6-keto-PGF1α content and SOD activity, regulated PAI and t-PA activities, ET and NO contents, and decreased MDA content and cells apoptosis rate (P<0.05 or P<0.01).

CONCLUSIONSDanhong Injection and its main components played an important role in protecting primary VECs from hypoxic damage by regulating the secretion and vasomotor function of VECs. The function of Danhong Injection was most remarkable.

6-Ketoprostaglandin F1 alpha ; metabolism ; Animals ; Apoptosis ; drug effects ; Blood Coagulation ; drug effects ; Cell Count ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; drug effects ; metabolism ; Endothelins ; metabolism ; Factor VIII ; metabolism ; Fibrinolysis ; drug effects ; Fluorescent Antibody Technique ; Humans ; Infant, Newborn ; Injections ; Malondialdehyde ; metabolism ; Nitric Oxide ; metabolism ; Plasminogen Inactivators ; metabolism ; Rabbits ; Superoxide Dismutase ; metabolism ; Tissue Plasminogen Activator ; metabolism ; Umbilical Veins ; cytology

BACKGROUND/AIMS: Chronic liver disease leads to liver fibrosis, and although the liver does have a certain regenerative capacity, this disease is associated with dysfunction of the liver vessels. C-reactive protein (CRP) is produced in the liver and circulated from there for metabolism. CRP was recently shown to inhibit angiogenesis by inducing endothelial cell dysfunction. The objective of this study was to determine the effect of CRP levels on angiogenesis in a rat model of liver dysfunction induced by bile duct ligation (BDL). METHODS: The diameter of the hepatic vein was analyzed in rat liver tissues using hematoxylin and eosin (H&E) staining. The expression levels of angiogenic factors, albumin, and CRP were analyzed by real-time PCR and Western blotting. A tube formation assay was performed to confirm the effect of CRP on angiogenesis in human umbilical vein endothelial cells (HUVECs) treated with lithocholic acid (LCA) and siRNA-CRP. RESULTS: The diameter of the hepatic portal vein increased significantly with the progression of cirrhosis. The expression levels of angiogenic factors were increased in the cirrhotic liver. In contrast, the expression levels of albumin and CRP were significantly lower in the liver tissue obtained from the BDL rat model than in the normal liver. The CRP level was correlated with the expression of albumin in hepatocytes treated with LCA and siRNA-CRP. Tube formation was significantly decreased in HUVECs when they were treated with LCA or a combination of LCA and siRNA-CRP. CONCLUSION: CRP seems to be involved in the abnormal formation of vessels in hepatic disease, and so it could be a useful diagnostic marker for hepatic disease.
Angiogenic Proteins/genetics/metabolism ; Animals ; Bile Ducts/surgery ; C-Reactive Protein/*analysis/genetics/metabolism ; Cells, Cultured ; Disease Models, Animal ; Hepatic Veins/abnormalities ; Hepatocytes/cytology/metabolism ; Human Umbilical Vein Endothelial Cells ; Humans ; Lithocholic Acid/pharmacology ; Liver/metabolism/pathology ; Liver Cirrhosis/etiology ; Liver Diseases/metabolism/*pathology ; Male ; Microscopy, Fluorescence ; Mitochondria/drug effects/metabolism ; RNA Interference ; RNA, Small Interfering/metabolism ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Serum Albumin/genetics/metabolism

Connexin43 has been shown to play a pivotal role in wound healing process. Wound repair is enhanced by acute downregulation of connexin43, by increasing proliferation and migration of keratinocyte and fibroblast. Angiogenesis is also a central feature of wound repair, but little is known about the effects of connexin43 modulation on functions of endothelial cells. We used connexin43 specific small interference RNA (siRNA) to reduce the expression of connexin43 in human umbilical vein endothelial cell (HUVEC), and investigated the effects of connexin43 downregulation on intercellular communication, viability, proliferation, migration and angiogenic activity of HUVEC. Treatment of siRNA markedly reduced the expression of connexin43 by -80% in HUVEC (P < 0.05), and decreased the intercellular communication by -65% (P < 0.05). The viability, proliferation, migration and angiogenic activity of HUVEC decreased significantly (P < 0.05), compared with that of the normal cells. The results suggest that temporally downregulation of connexin43 expression at early stage of wound to inhibit the abnormal angiogenesis characterized with leaky and inflamed blood vessels, maybe a prerequisite for coordinated normal healing process.
Cell Movement ; Cell Proliferation ; Cell Survival ; Connexin 43 ; metabolism ; Down-Regulation ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Neovascularization, Physiologic ; Umbilical Veins ; cytology ; Wound Healing

OBJECTIVETo explore the new mechanism of propranolol for treatment of hemangioma and the effects of propranolol on proliferation of hemangioma-derived mesenchymal stem cells ( Hem- MSCs).

METHODSWe isolated Hem-MSCs from hemangioma in the proliferating phase by their selective adhesion to plastic culture dishes. Immunofluorescence staining was used to examine the expression of marker antigens in Hem-MSCs. Human umbilical vein endothelial cells(HUVECs) were used as control. Indiuction of multi-lineage differentiation including osteogenesis and adipogeneis was performed with appropriate medium to identify the multi-lineage differentiation potential. MTT cell counting was used to observe the effects of different concentrations of propranolol on proliferation of Hem-MSCs.

RESULTSHem- MSCs were fibroblast-like morphology. All of them expressed vimentin, most expressed α-SMA,CD133, some expressed Glutl, and none of them expressed VEGF. Osteogenic, adipogenic differentiations of Hem- MSCs were induced successfully. Effects of low concentration of propranolol on proliferation of Hem-MSCs were not obvious, while high concentration of propranolol can inhibit the proliferation of Hem-MSCs.

CONCLUSIONSThe cells we isolated from hemangioma are Hem-MSCs. High concentration of propranolol can inhibit the proliferation of Hem-MSCs.

Adipogenesis ; Antigens ; metabolism ; Cell Differentiation ; Cell Proliferation ; drug effects ; Cells, Cultured ; Endothelium, Vascular ; cytology ; Fibroblasts ; cytology ; Hemangioma ; pathology ; Humans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Osteogenesis ; Propranolol ; pharmacology ; Umbilical Veins ; Vimentin ; metabolism

OBJECTIVETo explore the protective effects of danshen (Salvia Miltiorrhiza) on vascular endothelial cells in hypertension patients in the gestation period.

METHODSThe umbilical vein endothelial cells pre-incubated with Danshen solution at different concentrations (0, 100, 200, and 300 mg/L) were randomly divided into 3 groups, i.e., the blank control group (8 cases), the normal control group (14 cases, cultured in the serum from 14 normal pregnant women), and the observation group (14 cases, cultured in the serum from 14 pregnant women with severe pre-eclampsia). The levels of nitric oxide (NO) and endothelin-1 (ET-1) in each culture supernatant were detected respectively.

RESULTSThe ET-1 level was higher in 300 mg/L Danshen solution group than in 0 mg/L and 100 mg/L Danshen solution groups (P <0.05). The NO level was lower in the observation group than in the blank control group and the normal control group (P <0. 05). The NO level was higher in 200 mg/L Danshen solution group than in 0 mg/L Danshen solution group (P <0.05). The NO level was higher in 300 mg/L Danshen solution group than in 0 mg/L, 100 mg/L, and 200 mg/L Danshen solution groups (P <0.05).

CONCLUSIONDanshen could increase the secretion of NO from in vitro umbilical vein endothelial cells cultured in the serum from patients with pre-eclampsia, and reduce the secretion of ET-1.

Cells, Cultured ; Culture Media ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; cytology ; drug effects ; secretion ; Endothelin-1 ; metabolism ; Female ; Humans ; Nitric Oxide ; metabolism ; Phenanthrolines ; pharmacology ; Pre-Eclampsia ; blood ; metabolism ; Pregnancy ; Salvia miltiorrhiza ; chemistry ; Serum ; chemistry ; Umbilical Veins ; cytology

OBJECTIVETo study the protective effect of different solvent extracts from Platycladi Cacumen Carbonisatum (PCC) on LPS-induced human umbilical vein endothelial cell damage, and discuss the effective extracts from PCC for protecting vascular endothelial cells and their possible active substances.

METHODHUVECs were cultured in vitro; And LPS was adopted to establish the human umbilical vein endothelial cell damage model. MTT colorimetric method was used to determine cell activity; Xanthine oxidase method was adopted to detect the activity of superoxide dismutases (SOD) in the cell culture fluid; The TBA method was adopted to determine the content of malondialdehyde (MDA); The nitrate reductase method was used to detect the content of nitric oxide (NO); And UPLC/Q-TOF-MS was used to analyze the difference in flavonoids components among different solvent extracts from PCC.

RESULTCompared with the model group, N-butanol extract (100 mg x L(-1)) and ethylacetate extract (100, 50 mg x L(-1)) could significantly enhance the cell activity (P < 0.05), significantly reduce MDA and NO content, and increase SOD activity (P < 0.05). Among the four solvent extracts, the content of total flavonids were the highest in ethyl acetate extract, the lowest in water extract and equivalent in N-butanol and petroleum benzene extract. In terms of the contents of quercitrin and myricitrin, N-butanol extract were second only to ethyl acetate extract.

CONCLUSIONEthylacetate extract from PCC has a notable antagonistic effect in the damage induced by LPS to HUVECs, and thus is the most effective extract from PCC in protecting vascular endothelial cells. Quercitrin, myricitrin or multiple flavonoids that it contains may be their active substances for protecting vascular endothelial cells. Its mechanism may be related to the decrease in the production of NO and the inhibition of lipid peroxidation in cells.

Cupressus ; chemistry ; Endothelial Cells ; drug effects ; metabolism ; Humans ; Lipopolysaccharides ; adverse effects ; Malondialdehyde ; metabolism ; Nitric Oxide ; metabolism ; Plant Extracts ; isolation & purification ; pharmacology ; Protective Agents ; isolation & purification ; pharmacology ; Superoxide Dismutase ; metabolism ; Umbilical Veins ; cytology

OBJECTIVETo evaluate the effect of irbesartan on the proliferation, apoptosis, and VEGF mRNA expression of human umbilical vein cell line EA.hy926 in vitro.

METHODSThe human umbilical vein cell line EA.hyY926 were treated with various concentrations of irbesartan for 24 h. The cell proliferation after the treatment was detected by CCK8 assay, flow cytometry and FITC Annexin V/PI kit were used to detect changes in the cell apoptosis. RT-PCR was used to evaluate the expression of VEGF mRNA.

RESULTSThere were no changes in cell shape with various concentration of irbesartan. CCK-8 assay showed a greater rate of the cell proliferation in irbesartan group than that in control group with a dose-independent manner after 24 h treatment. After incubation with irbesartan, cell proliferation rate was significant (P < 0.05). FCM analysis showed no significantly changes in the cell apoptosis. Irbesartan increased the proliferation of EA.hy926 cells. At concentration of 1 x 10(-4), 1 x 10(-5), 1 x 10(-6) mol/L, VEGF mRNA expression enhanced either (P < 0.05).

CONCLUSIONIrbesartan could promote the proliferation and up-regulated VEGFmRNA expression in EA.hy926 cell line. This result suggested that in addition to antihypertensive effect, angiotensin receptor antagonist might be a novel therapeutic approach to chronic ischemic heart disease as heart failure.

Apoptosis ; drug effects ; Biphenyl Compounds ; pharmacology ; Cell Line ; Cell Proliferation ; drug effects ; Humans ; RNA, Messenger ; genetics ; Tetrazoles ; pharmacology ; Umbilical Veins ; cytology ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

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

Our previous report has demonstrated that 5-formylhonokiol (FH), a derivative of honokiol (HK), exerts more potent anti-proliferative activities than honokiol in several tumor cell lines. In present study, we first explored the antiangiogenic activities of 5-formylhonokiol on proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) for the first time in vitro. Then we investigated the in vivo antiangiogenic effect of 5-formylhonokiol on zebrafish angiogenesis model. In order to clarify the underlying molecular mechanism of 5-formylhonokiol, we investigated the signaling pathway involved in controlling the angiogenesis process by western blotting assay. Wound-healing results showed that 5-formylhonokiol significantly and dose-dependently inhibited migration of cultured human umbilical vein enthothelial cells. The invasiveness of HUVEC cells was also effectively suppressed at a low concentration of 5-formylhonokiol in the transwell assay. Further F-actin imaging revealed that inhibitory effect of 5-formylhonokiol on invasion may partly contribute to the disruption of assembling stress fiber. Tube formation assay, which is associated with endothelial cells migration, further confirmed the anti-angiogenesis effect of 5-formylhonokiol. In in vivo zebrafish angiogenesis model, we found that 5-formylhonokiol dose-dependently inhibited angiogenesis. Furthermore, western blotting showed that 5-formylhonokiol significantly down-regulated extracellular signal-regulated kinase (ERK) expression and inhibited the phosphorylation of ERK but not affecting the total protein kinase B (Akt) expression and related phosphorylation, suggesting that 5-formylhonokiol might exert anti-angiogenesis capacity via down-regulation of the ERK signal pathway. Taken together, these data suggested that 5-formylhonokiol might be a viable drug candidate in antiangiogenesis and anticancer therapies.
Actins/metabolism ; Angiogenesis Inhibitors/*pharmacology ; Animals ; Antineoplastic Agents, Phytogenic/pharmacology ; Biphenyl Compounds/*pharmacology ; Blotting, Western ; Cell Line, Tumor ; Cell Movement/drug effects ; Cell Proliferation/drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; Embryo, Nonmammalian/drug effects/metabolism ; Endothelium, Vascular/*drug effects/metabolism ; Extracellular Signal-Regulated MAP Kinases/*antagonists & inhibitors/metabolism ; Humans ; Lignans/*pharmacology ; Neovascularization, Physiologic/*drug effects ; Signal Transduction/*drug effects ; Umbilical Veins/cytology ; Wound Healing ; Zebrafish/embryology/metabolism