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Objective:To compare the differentiated endothelial cells from the embryonic stem cells in vitro with human umbilical vein endothelial cells (HUVECs).Methods:Induction of the stem cells HUES9 to endothelial cells follows 2 steps.Stem cells were treated with CHIR99021 (10 μmol/L) and bone morphogenetic protein 4 (25 ng/mL) for 3 days to keep mesoderm state,then subsequent exposure them to VEGF165 (200 ng/mL) and Forskolin (2 μmol/L) to differentiate into endothelial cells.The morphology of differentiated endothelial cells were compared with HUVECs.The surface marker CD144 on differentiated cells and HUVECs were detected.The capabilities of two types of endothelial ceils in migration and angiogenesis were examined.Results:The differentiated endothelial cells show the same morphology with HUVECs.After 6 days of differentiation,the efficiency reached 73.4％.The positive percentage of CD144 for the differentiated endothelial cells and HUVECs was 86.6％ and 94.4％,respectively.Both of them show capabilities of migration and angiogenesis,especially when they were treated with SB431542 to inhibit TGF-β signal pathway.Conclusion:The method for induction of stem cells to endothelial cells is productivity and it can be used for further study.
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Objective:To explore bromodomain and extra-terminal (BET) inhibition in the regulation of vascular endothelial cells activation and early atherosclerosis formation and its potential molecular mechanisms.Methods:1.Human umbilical vein endothelial cells (HUVEC) and mouse heart endothelial cells (MHEC) were isolated,and tumor necrosis factor α (TNFα) was used to activate in flammatory genes transcription in the presence or absence of JQ1,a specific BET inhibitor.The groups are as follows:(1)Normal control group;(2) TNFα(25 ng/mL)group;(3) TNFα+JQ 1 group.The gene mRNA and protein expression of inflammatory cytokines were measured by both real-time PCR and flow cytometry (FCM).2.LDL receptor-deficient (LDLR-/-) mice were randomly divided into 2 groups:JQ1 group (n=8,JQlintraperitoneal,50 mg/kg,daily) and control group (n=8,DMSO,daily).After 8 weeks feeding with high cholesterol diet,vascular cell adhesion molecule-1 (VCAM-1) expression in aortic arch was measured by immunohistochemistry.The activity of nuclear factor kappa B (NF-κB) signaling was monitored by 5XκB luciferase reporter assay in HEK293.Results:TNFα dramatically induced the mRNA and protein expression of inflammatory genes and JQ 1 significantly downregulated the induction of them (E-selectin,P-selectin,VCAM-1,IL-8)(P＜0.01).Immunohistochemistry detection indicated that JQ1 significantly downregulated the expression of VCAM-1 in aortic arch induced by 8 weeks high cholesterol diet feeding comparing to control group.In addition,BET bromodomain inhibition downregulated TNFα upregulated NF-κB transcriptional activity (P＜0.01).Conclusions:Our study demonstrated that BET bromodomain was involved in NF-κB mediated inflammatory genes expression;inhibition of BET bromodomain suppressed vascular endothelial activation in vitro,and attenuated early atherogenesis in vivo.
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OBJECTIVE@#To explore the effect of ROCK inhibitor Y-27632 on the matrix metalloproteinase 2 and 9 (MMP2 and MMP9) gene expression and activity in tumor necrosis factor α (TNF-α)-treated human umbilical vein endothelial cell (HUVEC). @*METHODS@#HHUVEC was divided into 3 groups, a control group, a TNF-α group, and a TNF-α plus Y-27632 group. The expressions of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), MMP2 and MMP9 were examined by real-time PCR. The MMP2/9 activity was measured by gelatin zymography. @*RESULTS@#Compared to the control group, the mRNA expressions of ICAM-1, VCAM-1, MMP2 and MMP9 were increased TNF-α-treated cells, which were suppressed by ROCK inhibitor (P<0.01). The MMP2/9 activity was elevated in TNF-α-treated cells, which was reversed by ROCK inhibitor (P<0.05). @*CONCLUSION@#ROCK inhibitor can suppress TNF-α-induced inflammation in endothelial cells through down-regulation of MMP2/9.