BACKGROUNDPrevious studies have shown that resveratrol increases endothelial progenitor cell (EPC) numbers and functional activity. Increased EPC numbers and activity are associated with the inhibition of EPC senescence. In this study, we investigated the effect of resveratrol on the senescence of EPCs, leading to potentiation of cellular function.

METHODSEPCs were isolated from human peripheral blood and identified immunocytochemically. EPCs were incubated with resveratrol (1, 10, and 50 µmol/L) or control for specified times. After in vitro cultivation, acidic β-galactosidase staining revealed the extent of senescence in the cells. To gain further insight into the underlying mechanism of the effect of resveratrol, we measured telomerase activity using a polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (ELISA) technique. Furthermore, we measured the expression of human telomerase reverse transcriptase (hTERT) and the phosphorylation of Akt by immunoblotting.

RESULTSResveratrol dose-dependently inhibited the onset of EPC senescence in culture. Resveratrol also significantly increased telomerase activity. Interestingly, quantitative real-time PCR analysis demonstrated that resveratrol dose-dependently increased the expression of the catalytic subunit, hTERT, an effect that was significantly inhibited by pharmacological phosphatidylinositol 3-kinase (PI3-K) blockers (wortmannin). The expression of hTERT is regulated by the PI3-K/Akt pathway; therefore, we examined the effect of resveratrol on Akt activity in EPCs. Immunoblotting analysis revealed that resveratrol led to dose-dependent phosphorylation and activation of Akt in EPCs.

CONCLUSIONResveratrol delayed EPCs senescence in vitro, which may be dependent on telomerase activation.

Cells, Cultured ; Cellular Senescence ; drug effects ; Endothelial Cells ; cytology ; drug effects ; enzymology ; Humans ; Stem Cells ; cytology ; drug effects ; enzymology ; Stilbenes ; toxicity ; Telomerase ; metabolism

To investigate the effect of total panax notoginseng saponins (tPNS) to induce the differentiation of mononuclear cells (MNC) in cord blood into endothelial cells, the DMEM culture media containing tPNS were used to induce the MNC of cord blood. Then, the morphology of the adherent cells was observed by the light microscopy and the fluorescence microscopy, the changes of cell surface markers (UEA-1), function marker (vWF) and CD31 were detected by flow cytometry. The results showed that the number of adherent cells produced by 250 mg/L tPNS and the positive rate of cells expressing CD31 and UEA-1 were higher than those in the groups of other concentrations (P < 0.05). There was no significant difference in the number of adherent cells expressing CD31 and UEA-1 between 50 ng/ml VEGF + 250 mg/L tPNS and 50 ng/ml VEGF. It is concluded that the traditional Chinese drug tPNS can induce partial MNC in the cord blood to differentiate into endothelial cells. No synergistic effect has been found between tPNS and VEGF.
Cell Differentiation ; drug effects ; Cells, Cultured ; Endothelial Cells ; cytology ; enzymology ; Fetal Blood ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Panax notoginseng ; chemistry ; Saponins ; isolation & purification ; pharmacology

To determine the effect of andrographolide (Andro) on angiogenesis of human umbilical vein endothelial cells (HUVECs).
 Methods: HUVECs were treated with different concentrations of Andro and the cell viability was detected with Cell Counting Kit-8 (CCK-8). HUVECs were treated with half lethal dose (IC50) of Andro. Matrigel was used to make capillary formation of HUVECs and the effect of Andro on capillary formation was evaluated by calculating the percentage of capillary formation. Moreover, the effects of Andro and the supernatant from cultured A549 tumor cells on capillary formation were evaluated by calculating the percentage of capillary formation. The effect of Andro on the expression of matrix metalloproteinase-9 (MMP-9) was determined with Western blot.
 Results: The cell viability of HUVECs decreased with the increase of Andro concentrations. IC50 was 20 μmol/L. The capillary formation of HUVECs was inhibited when treated with 20 μmol/L Andro for 24 hours. Moreover, Andro was able to antagonize the promotion of the capillary formation induced by the supernatant from cultured tumor cells. Andro could suppress the expression of MMP-9 and antagonize the capillary formation.
 Conclusion: Andro inhibits the capillary formation of HUVECs and can antagonize the promotion of angiogenesis induced by the supernatant from cultured tumor cells.
Capillaries ; drug effects ; Cell Survival ; Collagen ; Culture Media ; Diterpenes ; pharmacology ; Drug Combinations ; Human Umbilical Vein Endothelial Cells ; drug effects ; Humans ; Laminin ; Matrix Metalloproteinase 9 ; metabolism ; Neovascularization, Pathologic ; enzymology ; etiology ; prevention & control ; Proteoglycans ; Tumor Cells, Cultured

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

OBJECTIVETo study the protective effect of recombinant adenovirus-mediated human cytosolic glutathione peroxidase (hCGPx) gene transfection on vascular endothelial cells ECV304 from oxidative damage.

METHODSpGEM-T Easy Vector containing hCGPx cDNA and recombinant adenovirus shuttle plasmid pACCMV-pLpA were used to construct the shuttle plasmid pACCMV-hCGPx for cotransfection of 293 cells with pJM17, thereby to obtain the recombinant adenovirus AdCMV-hCGPx. Cultured ECV304 cells were transfected with AdCMV-hCGPx for 24, 48 and 72 h, respectively, with the cells transfected with the empty vector serving as control, and hCGPx gene expression was then examined in the transfected cells. The transfected cell viability and apoptotic cell ratio were evaluated after treatment of the cells with H(2)O(2).

RESULTSThe expression ratio of hCGPx gene was significantly higher in the AdCMV-hCGPx-transfected cells than in those with empty vector transfection (P<0.01). The hCGPx gene-transfected cells showed significantly higher viability and significantly lower apoptotic ratio than the control cells following challenge with H(2)O(2)-induced oxidative damage.

CONCLUSIONhCGPx gene transfer mediated by recombinant adenovirus protects the vascular endothelial cells from oxidative damage in vitro, possibly due to the antioxidative and apoptosis-inhibiting effect of hCGPx.

Adenoviridae ; genetics ; Apoptosis ; drug effects ; Cell Line ; Cell Survival ; drug effects ; Cytosol ; enzymology ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Flow Cytometry ; Genetic Vectors ; Glutathione Peroxidase ; biosynthesis ; genetics ; Humans ; Hydrogen Peroxide ; pharmacology ; Oxidative Stress ; Plasmids ; genetics ; Time Factors ; Transfection

OBJECTIVEThis study aimed at investigating whether notoginsenoside R1 (R1), a unique saponin found in Panax notoginseng could promote angiogenic activity on human umbilical vein endothelial cells (HUVECs) and elucidate their potential molecular mechanisms. In addition, vascular restorative activities of R1 was assessed in a chemically-induced blood vessel loss model in zebrafish.

METHODSThe in vitro angiogenic effect of R1 was compared with other previously reported angiogenic saponins Rg1 and Re. The HUVECs proliferation in the presence of R1 was determined by cell proliferation kit II (XTT) assay. R1, Rg1 and Re-induced HUVECs invasion across polycarbonate membrane was stained with Hoechst-33342 and quantified microscopically. Tube formation assay using matrigelcoated wells was performed to evaluate the pro-angiogenic actions of R1. In order to understand the mechanism underlying the pro-angiogenic effect, various pathway inhibitors such as SU5416, wortmannin (wort) or L-Nω-nitro- L-arginine methyl ester hydrochloride (L-NAME), SH-6 were used to probe the possible involvement of signaling pathway in the R1 mediated HUVECs proliferation. In in vivo assays, zebrafish embryos at 21 hpf were pre-treated with vascular endothelial growth factor (VEGF) receptor kinase inhibitor II (VRI) for 3 h only and subsequently post-treated with R1 for 48 h, respectively. The intersegmental vessels (ISVs) in zebrafish were assessed for the restorative effect of R1 on defective blood vessels.

RESULTSR1 could stimulate the proliferation of HUVECs. In the chemoinvasion assay, R1 significantly increased the number of cross-membrane HUVECs. In addition, R1 markedly enhanced the tube formation ability of HUVECs. The proliferative effects of these saponins on HUVECs were effectively blocked by the addition of SU5416 (a VEGF-KDR/Flk-1 inhibitor). Similarly, pre-treatment with wort [a phosphatidylinositol 3-kinase (PI3K)-kinase inhibitor], L-NAME [an endothelial nitric oxide synthase (eNOS) inhibitor] or SH-6 (an Akt pathway inhibitor) significantly abrogated the R1 induced proliferation of HUVECs. In chemicallyinduced blood vessel loss model in zebrafish, R1 significantly rescue the damaged ISVs.

CONCLUSIONR1, similar to Rg1 and Re, had been showed pro-angiogenic action, possibly via the activation of the VEGF-KDR/Flk-1 and PI3K-Akt-eNOS signaling pathways. Our findings also shed light on intriguing pro-angiogenic effect of R1 under deficient angiogenesis condition in a pharmacologic-induced blood vessels loss model in zebrafish. The present study in vivo and in vitro provided scientific evidence to explain the ethnomedical use of Panax notoginseng in the treatment of cardiovascular diseases, traumatic injuries and wound healing.

Animals ; Blood Vessels ; pathology ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Collagen ; pharmacology ; Disease Models, Animal ; Drug Combinations ; Ginsenosides ; chemistry ; pharmacology ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; enzymology ; physiology ; Humans ; Laminin ; pharmacology ; Neovascularization, Physiologic ; drug effects ; Phosphatidylinositol 3-Kinases ; metabolism ; Protein Kinase Inhibitors ; pharmacology ; Proteoglycans ; pharmacology ; Proto-Oncogene Proteins c-akt ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism ; Zebrafish

BACKGROUNDSalvianolic acid B (Sal B) is a bioactive water-soluble compound of Salviae miltiorrhizae, a traditional herbal medicine that has been used clinically for the treatment of cardiovascular diseases. This study sought to evaluate the effect of Sal B on matrix metalloproteinase-9 (MMP-9) and on the underlying mechanisms in tumor necrosis factor-α± (TNF-α±)-activated human coronary artery endothelial cells (HCAECs), a cell model of Kawasaki disease.

METHODSHCAECs were pretreated with 1-10 αμmol/L of Sal B, and then stimulated by TNF-α± at different time points. The protein expression and activity of MMP-9 were determined by Western blot assay and gelatin zymogram assay, respectively. Nuclear factor-κB (NF-κB) activation was detected with immunofluorescence, electrophoretic mobility shift assay, and Western blot assay. Protein expression levels of mitogen-activated protein kinase (c-Jun N-terminal kinase [JNK], extra-cellular signal-regulated kinase [ERK], and p38) were determined by Western blot assay.

RESULTSAfter HCAECs were exposed to TNF-α±, 1-10 αμmol/L Sal B significantly inhibited TNF-α±-induced MMP-9 expression and activity. Furthermore, Sal B significantly decreased IκBα± phosphorylation and p65 nuclear translocation in HCAECs stimulated with TNF-α± for 30 min. In addition, Sal B decreased the phosphorylation of JNK and ERK1/2 proteins in cells treated with TNF-α± for 10 min.

CONCLUSIONSThe data suggested that Sal B suppressed TNF-α±-induced MMP-9 expression and activity by blocking the activation of NF-κB, JNK, and ERK1/2 signaling pathways.

Benzofurans ; pharmacology ; Blotting, Western ; Cell Line ; Cell Survival ; drug effects ; Coronary Vessels ; cytology ; Endothelial Cells ; drug effects ; enzymology ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; NF-kappa B ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo observe the time-effect relation of extracts from ginseng, notoginseng and chuanxiong on angiotensin II (Ang II)-induced senescence of vascular endothelial cells and explore the feature of Chinese medicine against vascular diseases.

METHODSHuman umbilical vein endothelial cells (HUVECs) cultured in vitro were stimulated with 10(-6) mol/L AngII to induce cell senescence, which were divided into 4 groups, the blank control group, the Ang II model group, the extracts group and the telmisartan group. The β-gal was used to identify senescence of cells, the cell counting kit-8 method was applied to assess the cell viability, the cell function was examined with the level of endothelial nitric oxide synthase (eNOS) and the flow cytometry was used for analyzing the cell cycle changes.

RESULTSCompared with the control cells, the cells positive for β-gal staining was significantly increased in the Ang II model group, and showed cell cycle arrest at G0/G1 phase with decreased S and G2/M phase cell percentage, eNOS expression and cell viability (P<0.05). The extracts and telmisartan treatment of Ang II-induced cells resulted in decreased β-gal positive cells with a reduction in G0/G1 phase cells and an increasing in S, G2/M phase cells and eNOS expression (P<0.05). At 24 h, the extracts were more effective than telmisartan (P<0.05); while telmisartan was more effective at 48 h (P<0.05).

CONCLUSIONExtracts from ginseng, notoginseng and chuanxiong can delay Ang II-induced aging of HUVECs and may play an important role in early senescence.

Cell Cycle Checkpoints ; drug effects ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cellular Senescence ; drug effects ; Down-Regulation ; drug effects ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; enzymology ; Humans ; Nitric Oxide Synthase Type III ; metabolism ; Panax notoginseng ; chemistry ; Plant Extracts ; pharmacology ; Time Factors ; beta-Galactosidase ; metabolism

Angiogenesis is critical and indispensable for tumor progression. Since VEGF is known to play a central role in angiogenesis, the disruption of VEGF-VEGF receptor system is a promising target for anti-cancer therapy. Previously, we reported that a hexapeptide (RRKRRR, RK6) blocked the growth and metastasis of tumor by inhibiting VEGF binding to its receptors. In addition, dRK6, the D-form derivative of RK6, retained its biological activity with improved serum stability. In the present study, we developed a serum-stable branched dimeric peptide (MAP2-dRK6) with enhanced anti-VEGF and anti-tumor activity. MAP2-dRK6 is more effective than dRK6 in many respects: inhibition of VEGF binding to its receptors, VEGF- and tumor conditioned medium-induced proliferation and ERK signaling of endothelial cells, and VEGF-induced migration and tube formation of endothelial cells. Moreover, MAP2-dRK6 blocks in vivo growth of VEGF-secreting colorectal cancer cells by the suppression of angiogenesis and the subsequent induction of tumor cell apoptosis. Our observations suggest that MAP2-dRK6 can be a prospective therapeutic molecule or lead compound for the development of drugs for various VEGF-related angiogenic diseases.
Amino Acid Sequence ; Angiogenesis Inhibitors/*pharmacology ; Animals ; Cell Movement/drug effects ; Cell Proliferation/drug effects ; Colorectal Neoplasms/*pathology/secretion ; Endothelial Cells/cytology/drug effects/enzymology ; Enzyme Activation/drug effects ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Humans ; Mice ; Mice, Nude ; Molecular Sequence Data ; Neovascularization, Pathologic/pathology/prevention & control ; Neovascularization, Physiologic/drug effects ; Peptides/chemistry/*pharmacology ; Protein Multimerization/*drug effects ; Protein Stability/drug effects ; Rats ; Serum ; Vascular Endothelial Growth Factor A/*antagonists & inhibitors/secretion

OBJECTIVETo investigate the inhibitory effect of a tripeptide, a new arginine analog, on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS).

METHODSMacrophages challenged with lipopolysaccharide were cultured to test the inhibitory effect of the arginine analog of different concentrations on iNOS. Primarily cultured human umbilical vein endothelial cells (HUVECs) treated with insulin were used to test the effect of the analog on endothelial NOS (eNOS).

RESULTSThe new arginine analog significantly inhibited NO production in the macrophages in a dose-dependent manner, but had little effect on insulin-stimulated NO production in HUVECs. The new analog could significantly suppress iNOS activity, but had no significant inhibitory effect on constitutive NOS activity or eNOS activity. Western blot analysis showed that the new analog did not significantly affect the protein expression of iNOS.

CONCLUSIONThe new analog is a NOS inhibitor with high selectivity on iNOS.

Arginine ; analogs & derivatives ; pharmacology ; Blotting, Western ; Cell Line ; Cells, Cultured ; Dose-Response Relationship, Drug ; Endothelial Cells ; cytology ; drug effects ; enzymology ; Humans ; Insulin ; pharmacology ; Lipopolysaccharides ; pharmacology ; Macrophages ; drug effects ; enzymology ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; antagonists & inhibitors ; metabolism ; Oligopeptides ; chemistry ; pharmacology