Abstract:Aim To investigate the role of miR-221 in homocysteine (Hcy)-induced injury of human coronary artery endothelial cells (HCAEC) mediated by cyclin D1. Methods HCAECs were cultured and divided into four groups. The control group was treated with serum-free medium, Hcy group was treated with medium containing 1 mmol/L Hcy, Hcy＋NC (negative control) group was treated with medium containing 1 mmol/L Hcy after transfection with NC inhibitor, and Hcy＋miR-221 group was treated with medium containing 1 mmol/L Hcy after transfection with miR-221 inhibitor. Fluorescence quantitative PCR was used to detect the expression of miR-221, Western blot was used to detect the expression of cyclin D1, MTS was used to detect the OD490 nm level of cell viability, flow cytometry was used to detect cell cycle, and double luciferase reporter gene experiment was used to verify miR-221 targeting cyclin D1. Results Compared with the control group, the expression level of miR-221 and the proportion of G0/G1 phase of HCAEC were significantly increased, while the level of OD490 nm, the proportion of S phase and G2/M phase and cyclin D1 expression level were significantly decreased in Hcy group. Compared with Hcy group and Hcy＋NC group, the expression level of miR-221 and the proportion of G0/G1 phase of HCAEC were significantly decreased, while the level of OD490 nm, the proportion of S phase and G2/M phase and cyclin D1 expression level were significantly increased in Hcy＋miR-221 group. The 1224-1231 base of mRNA 3′UTR of cyclin D1 gene was the binding site of miR-221, and miR-221 reduced the fluorescence activity of wild-type cyclin D1 double luciferase reporter gene. Conclusions The expression of miR-221 increases during Hcy-induced HCAEC injury, and inhibition of miR-221 expression can reduce Hcy-induced HCAEC injury. Targeting cyclin D1 is a possible molecular mechanism.

Abstract:Aim To investigate the effect of cladribine (2-CdA) on the viability, migration, cell cycle and secretion activity of human umbilical vein endothelial cells, and to evaluate the influence of 2-CdA on cardiovascular system in clinical application. Methods The effect of different concentrations of 2-CdA on the growth of endothelial cells was detected by CCK-8. The cell migration ability was detected by scratch test. The cell cycle was analyzed by flow cytometry. The cell apoptosis was detected by acridine orange and ethidium bromide. The content of nitric oxide (NO) was determined by Gries method. ELISA method was used to detect vascular endothelial growth factor (VEGF) level. Results In the concentration range of 0.4 to 40 mol/L, 2-CdA had an obvious inhibitory effect on endothelial cells, and IC50 was 4.126 mol/L. With the increase of drug concentration and the prolongation of action time, the inhibitory effect of 2-CdA increased. High concentration of 2-CdA for a long time could induce endothelial cell apoptosis. After 5 μmol/L 2-CdA acted on endothelial cells, the migration ability of cells was inhibited, and the cell cycle was significantly blocked in S phase. The staining results of acridine orange and ethidium bromide showed that high concentration of 2-CdA could induce apoptosis of endothelial cells. After the action of 5 μmol/L 2-CdA on endothelial cells, NO and VEGF contents were reduced. Conclusion 2-CdA has the toxic effect of endothelial cells, resulting in cell cycle arrest and cell apoptosis.

Abstract:Aim To explore the effect of dasatinib on proliferation, migration, cell cycle and apoptosis of human umbilical vein endothelial cells cytologically and molecularly to offer help on clinical application of dasatinib. MethodsExperimental grouping:dasatinib group (dasatinib concentration was 50 nmol/L) and LY294002 group (PI3K inhibitor,concentration was 20 μmol/L), combined treatment group (dasatinib treatment concentration was 50 nmol/L, the concentration of LY294002 for 20 μmol/L) and mock group (DMSO was 0.1%). Cell viability was measured by CCK8. The migration ability of human umbilical vein endothelial cells was measured by scratch assay. Apoptosis and cell cycle were analyzed by flow cytometry. The expression and phosphorylation of Akt protein were determined by Western blot. Results The cell viability of human umbilical vein endothelial cells decreased gradually following the increasing concentration (1~400 nmol/L) and prolonged exposure to dasatinib (50 nmol/L, 24~96 h). Dasatinib and LY294002 (an inhibitor of PI3K), both inhibited cell viability of human umbilical vein endothelial cells. Dasatinib (50~100 nmol/L) weakened the migration capability of human umbilical vein endothelial cells. In addition, compared with control, dasatinib (50 nmol/L) induced apoptosis and cell cycle arrest (P＜0.05) of human umbilical vein endothelial cells. The phosphorylation of Akt was inhibited by dasatinib and LY294002, and LY294002 had more powerful inhibition of p-Akt than dasatinib. Conclusion Dasatinib could facilitate the injures of human umbilical vein endothelial cells via PI3K/Akt pathway, mainly to suppress the proliferation and migration of human umbilical vein endothelial cells, alter the cell morphology, blocked the G1-S transition and induced apoptosis.

Abstract:Aim To analyze the effect of Toll like receptor 3 (TLR3) agonist PolyI∶C on the proliferation and apoptosis of human umbilical cord blood-derived endothelial progenitor cells (EPC) and its mechanism. Methods Endothelial progenitor cells were treated with different concentrations of PolyI∶C sequentially, and then the phase of cell cycle and cell apoptosis were tested by flow cytometry. CCK-8 assay was used to detect the effect of TNF-α and IL-1β on the apoptosis of endothelial progenitor cells, and the effect of Caspase 8 inhibitior, Caspase 9 inhibitior and IL-1 receptor 1 neutralizing antibody (anti-IL-1R1) on PolyI∶C-induced apoptosis. Results Compared with the control group, PolyI∶C at high concentrations of 1 and 10 g/L significantly decreased the proportion of cells in S phase and G2/M phase.Moreover, PolyI∶C down-regulated the gene expression of cyclin A, B1, D1 and E, inducing cell cycle arrest in G0/G1 phase. Additionally, PolyI∶C induced the apoptosis of endothelial progenitor cells in a dose-dependent manner. Caspase 8 and Caspase 9 inhibitors did not reduce PolyI∶C-induced apoptosis of endothelial progenitor cells. TNF-α had no effect on apoptosis of endothelial progenitor cells. IL-1β induced cell apoptosis in a dose-dependent manner. Moreover, when endothelial progenitor cells pre-treated with anti-IL-1R1, then re-stimulated with PolyI∶C, the cell apoptosis induced by PolyI∶C was decreased. Conclusions PolyI∶C at high concentrations inhibited endothelial progenitor cells proliferation by inducing cell cycle arrest in G0/G1 phase and inducing cell apoptosis of endothelial progenitor cells. PolyI∶C induced the apoptosis of endothelial progenitor cells through up-regulating the expression of IL-1β via activating TLR3. Endogenous and exogenous apoptosis pathway and TNF-α did not contribute to PolyI∶C-induced cell apoptosis.

Abstract:Aim To investigate the effect and mechanism of aspirin on human umbilical vein endothelial cells （HUVEC） senescence exposed to high glucose condition. Methods The cultured HUVEC were treated with concentrations of glucose at 5.5, 33 mmol/L and glucose at 33 mmol/L with aspirin at 0.01, 0.1, 1 and 3 mmol/L for 48 h. SA-β-gal staining was used to evaluate senescence. Telomerase activity was detected by PCR-ELISA. Cell cycle and the level of reactive oxygen species （ROS） were detected by flow cytometry. Results Endothelial cells exhibited the characteristic of senescence, increased the number of β-gal positive cells, decreased telomerase activity significantly, enhanced the proportion of HUVEC in the G0/G1 phase and reduced that in the S phase and increased the level of ROS after exposure to high glucose （33 mmol/L, P<0.05）. All these phenomena were reversed by 0.01, 0.1 and 1 mmol/L aspirin remarkably （P<0.05）. However, there was no significant difference between 3 mmol/L aspirin group and high glucose group. Conclusions 0.01, 0.1 and 1 mmol/L aspirin delay endothelial cell senescence exposed to high glucose. This positive effect may be associated with decreasing oxidative stress.

Abstract:Aim To investigate the protective effect of 5,7-Dihydroxyl-8-nitrio chrysin （8-NOChR） on the growth of vascular endothelial cell damaged by lysophosphatidyl choline （LPC） and its possible molecular mechanism. Methods Human umbilical vein endothelial cells （HUVEC） were incubated in vitro, the damaged effect of LPC on HUVEC cells and the rivalry effect of various concentration of 8-NOChR on HUVEC cells induced by LPC were evaluated by MTT assay. The colony formations were detected by plate colony formation assay. Distribution of cell cycle was analyzed by flow cytometry. Western blot assay was used to measure proteins related to cell cycle. Results MTT showed the viability of HUVEC treated with LPC decreased in a dose and time dependent manner compared with the cells treated with NS group and 0.1%DMSO group （P<0.05）, while 8-NOChR could increase the viability and the colony formations of cells treated with LPC in a dose and time-dependent manner compared with the LPC group （P<0.05）. FCM indicated the G2/M accumulation of HUVEC treated with LPC combining 8-NOChR decreased in dose-dependent, and the expression of cyclin A and cyclin B protein were activated （P<0.05）, and protein level of P53 and P21 were down-regulated （P<0.05）, while the expression of CDK1 protein showed no change with the same treatment（P>0.05）. Conclusion 8-NOChR could significantly prevent the damaged HUVEC induced by LPC, which correlated with activation of cyclin A and cyclin B protein and down-regulation of P53 and P21 protein.

Abstract:AimTo investigate the role of Valsartan on Angiotensin Ⅱ(AngⅡ)-induced senescence of human umbilical endothelial cell senescence and gene expression of p16INK4a.MethodsHUVEC were cultured in vitro and intervened by AngⅡ(10-6mol/L) and Valsartan(AngⅡ type 1 receptor blocker).HUVEC were divided into 3 groups, the control group, AngⅡ group, Valsartan group.β-gal staining was used to identify cell aging status.Flow cytometry was used for analyzing the cell cycle changes; The positive cell rate of p16INK4a was detected by immunocytochemical staining, and the expressions of p16INK4a protein were determined by Western blot.ResultsCompared with the control cells, the positive cell number of β-gal staining was significantly higher in AngⅡ-induced cells 81.24%±6.46%; the cell cycle was at G0-G1 88.36%±6.45%. In Valsartan group, p16INK4a protein expression decreased evidently (p<0.05) compared to that in the AngⅡ group, which suggests that p16INK4a activity plays an important role in regulating vascular endothelial cell senescence lifespan in vitro.ConclusionCell Endothelial cell senescence is induced by AngⅡ.One of its molecular mechanisms might be associated with increasing the expression level of p16INK4a in aging cell, and then up-regulating the amount of cells blocking in G1 phase of cell cycle.Valsartan could antagonize the process effectively and delay endothelial cell aging significantly.

Abstract:AimTo investigate the effect of extractive of pericarpium trichosanthis (EPT) on cell cycle of rat vascular smooth muscle (VSMC) proliferation induced by platelet-derived growth factor-BB (PDGF-BB) and to probe especially into its mechanism.MethodsVSMC from the thoracic aorta of SD rats were cultured by tissue explant method.Effect of EPT (10, 20 and 30 mg/L) on PDGF-BB-induced VSMC proliferation was assessed by 3H-TdR method, the cell cycle was analyzed by flow cytometry, expression of c-fos and c-myc mRNA in VSMC were detected by real-time quantitative reverse transcription-polymerase chain reaction (real-time RT-PCR).ResultsPDGF-BB could significantly increase the rate of 3H-TdR incorporation (p<0.01) and the percentage of S phase cells, and degrade the G0/G1 phase cell percentage in the cell cycle (p<0.01).At the same time, PDGF-BB could up-regulate c-fos and c-myc mRNA expression (p<0.01).Addition of EPT (10, 20 and 30 mg/L) markedly inhibited the PDGF-BB-induced proliferation of the VSMC (p<0.01), decreased the S phase cell percentage and upgraded the G0/G1 phase cell percentage in the cell cycle, EPT could also depress the elevated expression of c-fos and c-myc mRNA indcued by PDGF-BB.ConclusionsEPT could inhibit the VSMC proliferation induced by PDGF-BB through preventing the transformation of the G0/G1 phase cell to S phase cell in the cell cycle.The mechanism may be related to its down-regulatory effect on c-fos and c-myc mRNA expressions.

Abstract:Aim To investigate the effect of lycopene in endothelial progenitor cells of human peripheral blood and its underlying molecular mechanism. Motheds The promotion survival effect of lycopene in different concentrations on endothelial progenitor cells was measured by thiazolyl blue(MTT) assay.Cell cycle was analyzed by flow cytometry.The expression levels of cyclin-dependent kinase 4(CDK4) and cyclin D1 were detected by reverse transcription polymerase chain reaction(RT-PCR) and real-time polymerase chain reaction(real-time PCR) and Western blot. Results The MTT assay showed that lycopen promoted survival effect on endothelial progenitor cells in a time and dose-dependent manner.Flow cytometry analysis revealed that G1 phase of endothelial progenitor cells was decreased after exposed to different concentrations of lycopene.The expression levels of CDK4 and cyclin D1 were increased significantly after treated with lycopene in a dose-dependent manner using RT-PCR,real-time PCR and Western blot. ConclusionThe promotion survival effect of lycopene in endothelial progenitor cells is related to G1 phrase reducing through increased expression levels of CDK4 and cyclin D1.

Abstract:AimTo explore the possibility of homocysteine-induced vascular smooth muscle cell (VSMC) proliferation via N-methy-D-aspartate (NMDA) receptor and its possible terminal target molecules.MethodsVSMC proliferation was induced by homocysteine, and the VSMC was treated with NMDA receptor antagonist MK801.The rate of proliferation of VSMC were detected by the way of MTT.Cell cycle distribution were determined by flow cytometer.The cyclin D1 mRNA expression in cultured VSMC were measured by RT-PCR.ResultsHomocysteine significantly stimulated VSMC proliferation, promoted VSMC convert from the G0/G1 to S phase, and increased cyclin D1 mRNA expression. MK801, however, inhibited the proliferation of VSMC by homocysteine, the conversion from G0/G1 to S phase and the expression of cyclin D1 mRNA were synchronously inhibited.All these effects showed significant dose-dependent manner.ConclusionThe proliferation-promoting effect of homocysteine on VSMC might be partly mediated by NMDA receptor and ultimately achieved through the cyclin D pathway.