Effect of homocysteine on the apoptosis of cultured human umbilical vein endothelial cells

AIM: To investigate the effect of homocysteine(Hcy) on the apoptosis of endothelial cells (EC). METHODS: First-passaged human umbilical vein endothelial cells (hUVEC) were cultured with M199 containing 3 mmol/L Hcy. hUVEC apoptosis was detected as follow: demonstration of nuclear changes by Hoechst 33258 staining, agarose gel electrophoresis of DNA fragments, detection of apoptotic cells by flow cytometry following Annexin V-PI doubled stain, Western blot for P53 and Bax protein detection and colorimetry detecting caspase-3 activity. RESULTS: Compared with control, homocysteine induced characteristic apoptotic changes in hUVEC. The chromosomal DNA of hUVEC appeared "DNA ladder" by agarose gel electrophoresis. Apoptotic cells were increased significantly (P<0.01, n=3). Hcy promoted the expression of protein Bax, P53 (P<0.01, n=3) and enhanced the activity of caspase 3 (P<0.05, n=3). CONCLUSION: Homocysteine induces apoptosis in cultured hUVEC.     

Effects of folic acid and vitamin B12 on human umbilical vein endothelial cells against homocysteine-induced apoptosis

AIM: To investigate the effect of folic acid and vitamin B₁₂ on homocysteine (Hcy)-induced apoptosis of human umbilical vein endothelial cells (HUVECs) through mammalian sterile 20-like kinase 1 (MST1). ME-THODS: HUVECs were cultured in the absence (control group), or presence of 100 μmol/L Hcy alone (Hcy group) or 100 μmol/L Hcy plus 30 μmol/L folic acid and vitamin B₁₂ (intervention group) for 72 h. The effect of Hcy on the apoptosis of HUVECs was analyzed by flow cytometry. The transfection efficiency of DNA methyltransferase 1 (DNMT1)-overexpressing adenovirus was observed under fluorescence inverted microscope. The mRNA and the protein levels of DNMT1 and MST1 were determined by RT-qPCR and Western blot. The DNA methylation level of MST1 promoter was detected by methylation-specific PCR. RESULTS: Compared with control group, the apoptotic rate (P<0.01) and the expression of MST1 at mRNA (P<0.01) and protein (P<0.05) levels in the HUVECs were significantly increased, while the mRNA levels of DNMT1 was decreased in Hcy group (P<0.01). In addition, folic acid and vitamin B₁₂ treatment significantly inhibited Hcy-mediated apoptosis of HUVECs (P<0.01), increase in MST1 mRNA level (P<0.01) and decrease in DNMT1 mRNA level (P<0.01). Meantime, the mRNA level of MST1 was positively correlated with the apoptotic rate of the HUVECs (r=0.943 9, P<0.001). The expression of DNMT1 at mRNA and protein levels was significantly increased after the transfection of DNMT1-overexpressing adenovirus into HUVECs (P<0.01), and a large amount of green fluorescent protein expression was observed. Meanwhile, the DNA methylation level of MST1 promoter was increased (P<0.01), while the protein level of MST1 was decreased (P<0.01).CONCLUSION: Up-regulation of MST1 promotes Hcy-induced apoptosis of HUVECs, while folic acid and vitamin B₁₂ exert an anti-apoptosis effect, which might be regulated by hypermethylation of MST1 promoter region.     

Effects of carvedilol on metabolic mechanism of endogenous NOS inhibitor in human umbilical vein endothelial cells treated with oxidized free radical

AIM: To observe the effects of oxidized free radical (OFR) on dimethylarginine dimethylaminohydrolase (DDAH) activity and concentration in human umbilical vein endothelial cells (HUVECs), and to investigate the metabolic mechanism of endogenous NOS inhibitor and the role of carvedilol. METHODS: HUVECs of 3-6th passage, cultured with modified Jaffes method, were divided into three groups: (1) cells cultured with equivalent DMEM medium as control; (2) OFR intervention groups, 0.01 mmol/L, or 0.1 mmol/L OFR was added respectively; (3) drug intervention groups: 0.1 mmol/L OFR plus 10 μmol/L carvedilol. ADMA, nitric oxide (NO), endothim (ET), L-citrulline concentrations and the activity of NOS in conditioned medium were measured after 24 h exposure. ADMA concentration in the conditioned medium was determined by high-performance liquid chromatography. Western blotting was performed to evaluate DDAH expression. RESULTS: Compared with control group, ADMA and ET concentrations were increased, while the level of NO and the activity of NOS decreased and relevant to the concentrations of OFR. We assayed DDAH activity by determining L-citrulline formation from ADMA. The concentration of L-citrulline was decreased, while the DDAH expression had no obvious change. With the role of carvedilol, ADMA, ET concentrations were decreased, while the level of NO, L-citrulline and the activity of NOS increased. CONCLUSION: Endothelial dysfunction induced by OFR is associated with the increase in ADMA concentration and reduction of DDAH activity, but not DDAH expression. Carvedilol promotes the degradation of ADMA through increasing activity of DDAH and improving endothelium function.     

Effect of propolis water extract on apoptosis of human umbilical vein endothelial cells in vitro

AIM: To explore the effect of water extract propolis (WEP) from Taishan on apoptosis of human umbilical vascular endothelial cells (HUVECs) induced in vitro by tumor necrosis factor-α (TNF-α). METHODS: HUVECs were collect by digestion-perfusion and cultivated. TNF-α at concentration of 50 μg/L was administrated to induce the apoptosis of HUVECs. After injury, HUVECs were treated with WEP at concentrations of 50, 100, and 200 mg/L, respectively, for 24 h. Apoptosis was evaluated by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) and flow cytometry (FCM). RESULTS: Apoptosis index in injured group was significantly higher than that in control group, and decreased significantly after treating with WEP at concentrations of 50, 100, and 200 mg/L, respectively (P<0.01). CONCLUSION: WEP may be useful for protection of vascular endothelial cells by inhibiting apoptosis.     

Cisplatin induces apoptosis of human renal tubular epithelial cells through p66Shc-mitochondrial signal pathway

AIM:To evaluate the roles of p66Shc-mitochondrial signal pathway in apoptosis of human renal tubular epithelial cells induced by cisplatin. METHODS:The human renal tubular epithelial cells were cultured in vitro. The levels of p66Shc and phospho-p66Shc(Ser36) protein were detected by Western blotting. The cells were divided into control group, cisplatin group and cisplatin+p66ShcS36A(p66Shc with Ser mutating into Ala at position 36) group. The effects of p66Shc on cisplatin-induced cellular reactive oxygen species (ROS), mitochondrial ROS and apoptosis were measured by confocal microscopy. The expression of the proteins related to apoptosis mitochondrial signal transduction pathway was analyzed by Western blotting. RESULTS:Cisplatin induced p66Shc phosphorylation, but did not affect the expression of p66Shc. Cisplatin enhanced apoptosis and production of both cellular and mitochondrial ROS, release of cytochrome C and expression of caspase-9, which were inhibited by the transfection of p66ShcS36A. CONCLUSION: Cisplatin induces apoptosis of human renaltubular epithelial cells through p66Shc-mitochondrial signal pathway.     

Effect of activated protein C on apoptosis in human umbilical vein endothelial cells induced by lipopolysaccharide

AIM:To study the effect of activated protein C (APC) at different concentrations on apoptosis of human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS).METHODS:The HUVECs were induced by LPS (1.0 mg/L) as apoptotic model that was administered by different concentration of APC (10 μg/L or 50 μg/L). Meanwhile, the control group and induced apoptosis group induced by LPS (1.0 mg/L) stimulation were also set up. The changes of cellular ultrastructures were observed under electron microscope. The DNA ladder and TUNEL fluorescent staining were measured in cells. Annexin-Ⅴ/PI double staining was used to measure the cell apoptosis rate by flow cytometry. Cell survival rate was measured by MTT assay. The proliferating cell nuclear antigen (PCNA) expression levels in cells were also measured by Western blotting to reflect the proliferation of the cells.RESULTS:There were significant apoptotic changes in the cells induced by LPS, but the apoptotic changes were reduced and apoptosis rates were decreased in the cells treated with APC. Meanwhile, cell survival rate and the protein levels of PCNA were increased after APC treatment, particularly at the concentration of 50 μg/L, which showed difference when compared with those induced apoptosis group by LPS (P<0.05).CONCLUSION:APC can inhibit HUVECs apoptosis induced by LPS and promote cell proliferation, thus protect the cells from injury.     

Proliferation and apoptosis of human umbilical vein endothelial cells induced by oxidized low density lipoprotein

AIM:To investigate proliferation and apoptosis of cultured endothelial cell (ECV-304 cell line) induced by varied concentrations of oxidized low density lipoprotein (ox-LDL). METHODS:Cell morphology, Typan blue test, MTT test, LDH release test, flow cytometry and micro-molecular weight DNA fragment gel electrophoresis of apoptosis were used for the detection of the cytotoxic effects of ox-LDL on ECV-304 cell line.RESULTS:0.1, 1, 10 mg/L ox-LDL could promote proliferation of ECV-304 cells. When the concentration of ox-LDL reached up to 100 mg/L and above, the distinct cytotoxic effect appeared. Further study showed that the apoptosis rate of endothelial cells, induced by ox-LDL of 150 mg/L and 200 mg/L for 12 hours, are 15.86% and 21.89%, respectively. 18 h and above hours after incubation, the apoptosis rate began to decrease and rate of necrosis increased. CONCLUSION:ox-LDL has strong cytotoxic effects on endothelial cells and could give rise to different pathologic process, such as proliferation, apoptosis prophase, apoptosis and necrosis.     

Tanshinone IIA prevents high glucose-induced human umbilical vein endothelial cell apoptosis

AIM: To investigate the effect of tanshinone IIA on the apoptosis of human umbilical vein endothelial cells(HUVECs) after high glucose treatment.METHODS: The cell viability was determined by MTT assay. The cell apoptotic rate was examined by flow cytometry with Annexin V-FITC/PI double staining. The expression of Bcl-2 and Bax, and the release of mitochondrial cytochrome C(Cyt C) were analyzed by Western blotting.RESULTS: Tanshinone IIA significantly inhibited high glucose-induced decrease in cell viability and increased the cell apoptosis. Additionally, after tanshinone IIA treatment, Bax expression and the release of mitochondrial Cyt C were significantly inhibited, while Bcl-2 expression was increased.CONCLUSION: Tanshinone IIA prevents high glucose-induced endothelial cell apoptosis via mitochondria-dependent pathway.     

Effect of lipopolysaccharide on apoptosis of human umbilical vein endothelial cells

AIM: To explore the mechanism of lipopolysaccharide (LPS) on vascular endothelial cell(VEC) damage. METHODS: By using cytometry techniques, we studied the effects of LPS on apoptosis of human umbilical vein endothelial cells (HUVECs) in vitro. RESULTS: LPS was able to induce apoptosis of HUVECs in a time-dose-dependent fashion.CONCLUSION:Apoptosis might play a role in LPS-induced damage of vascular endothelial cells.     

Mechanism underlying the antagonistic effect of folic acid on homocysteine-induced apoptosis in endothelial cell

AIM: To investigate the effects of homocysteine (Hcy) on apoptosis in SV40-transformed aortic rat endothelial cell line and the anti-apoptosis effects of folic acid. METHODS: Cells were treated with different concentrations of Hcy and folic acid, apoptosis was detected by TUNEL and annexin-V/ PI staining methods. Immunohistochemical assay was used to examine the expression of Bax and Bcl-2 in all groups. RESULTS: Both annexin-V/PI staining and TUNEL method showed that Hcy increased endothelial apoptosis in a dose-dependent manner, while folic acid reduced cell apoptosis. Hcy increased expression of Bax and Bcl-2 in endothelial cells, and folic acid decreased it. Bax/Bcl-2 ratio in 0.5 mmol/L Hcy and 5.0 mmol/L Hcy group were upregulated compared with control group (P<0.05 and P<0.01, respectively). Addition of 0.1 mmol/L folic acid decreased Bax/Bcl-2 ratio compared with the corresponding group without folic acid (P<0.05). Correlation analysis showed a strong relation between Bax/Bcl-2 ratio and apoptotic rate (P<0.01). CONCLUSION: Folic acid attenuates the apoptosis induced by Hcy in endothelial cells. Hcy may promote endothelial cell apoptosis via upregulation of Bax /Bcl-2 ratio, which can be partially antagonized by folic acid.     

Artesunate induces apoptosis of human hepatocellular carcinoma HepG2 cells by increasing generation of reactive oxygen species

AIM: To investigate the effect of reactive oxygen species (ROS) on the apoptosis of HepG2 cells induced by artesunate. METHODS: The effect of artesunate on the viability of HepG2 cells was measured by MTT assay. The morphological changes of the apoptotic cells were observed by the method of Hoechst 33258 fluorescence staining.The apoptosis of HepG2 cells was analyzed by flow cytometry. DCFH-DA was used to detect the changes of ROS generation during the process of apoptosis. The protein levels of Bax, Bcl-2, cleaved caspase-3 and cytochrome C (Cyt C) were determined by Western blot. HepG2 cells were pretreated with apocynin and then Western blot was used to detect the expression of p47^phox and p22^phox, and ROS changes were analyzed by flow cytometry. RESULTS: Compare with control group, the cell viability was obviously inhibited after treatment with artesunate for 24 h (P<0.05). The nuclei were densely stained, and the proportion of apoptotic cells was increased (P<0.05). ROS was increased significantly (P<0.05). The results of Western blot demonstrated that the expression level of Bax was increased, Bcl-2 was decreased, the ratio of Bax/Bcl-2 was increased, and the protein levels of cleaved caspase-3 and Cyt C were increased. Pretreatment with apocynin reduced the expression of p47^phox and p22^phox and the generation of ROS in the artesunate treatment group. CONCLUSION: Artesunate induces the apoptosis of HepG2 cells. The possible mechanism may be related to the increase in the generation of ROS.     

Effect of 27nt-miRNA on apoptosis of human umbilical vein endothelial cells induced by oxidized low-density lipoprotein

AIM To investigate the effect of 27nt-miRNA （27nt-miR） on apoptosis of human umbilical vein endothelial cells （HUVECs） induced by oxidized low-density lipoprotein （Ox-LDL） and its underlying mechanism. METHODS HUVECs were cultured in vitro and grouped as below： normal control group, Ox-LDL group, 27nt-miR+Ox-LDL group, anti-27nt-miR+Ox-LDL group and negative control+Ox-LDL group. The cells in Ox-LDL group were treated with Ox-LDL at 40 mg/L for 48 h, while those in normal control group were untreated but cultured normally. The cells in 27nt-miR+Ox-LDL group, anti-27nt-miR+Ox-LDL group and negative control+Ox-LDL group were transfected with their corresponding lentiviral vectors under the same procedure, followed by treatment with Ox-LDL at 40 mg/L for 48 h to induce apoptosis. The cell viability was measured by CCK-8 assay. The migration capacity was detected by scratch assay. The caspase-3 activity was measured by caspase-3 activity assay kit. The apoptotic rate was analyzed by Hoechst 33258 and flow cytometry. The mRNA and protein expression levels of Bcl-2, Bax and caspase-3 were determined by RT-qPCR and Western blot. RESUITS： Compared with negative control+Ox-LDL group, the cell viability and migration ability were significantly decreased by over-expression of 27nt-miR in the HUVECs （P<0.05）, while the activity of caspase-3 and apoptosis induced by Ox-LDL were significantly increased （P<0.05）. Furthermore, the mRNA and protein expression levels of Bax and caspase-3 were significantly up-regulated （P<0.05）, and the mRNA and protein expression level of Bcl-2 was down-regulated in 27nt-miR+Ox-LDL group （P<0.05）. Meanwhile, all the above indexes showed an opposite tendency in anti-27nt-miR+Ox-LDL group. CONCLUSION 27nt-miR promotes Ox-LDL-induced apoptosis and inhibits the viability and migration of HUVECs in vitro, possibly through regulating the expression of apoptotic/anti-apoptotic proteins such as Bax,caspase-3 and Bcl-2.     

Inhibition of HMGB1 expression promotes apoptosis of hemangioma endothelial cells

AIM: To investigate the effect of inhibiting high-mobility group box protein 1 (HMGB1) expression on the viability and apoptosis of hemangioma endothelial cells (HemECs). METHODS: Human HemECs were isolated and cultured, and HMGB1 small interfering RNA (HMGB1-siRNA) was transfected into the cells. The cell viability was detected by CCK-8 assay. The apoptosis and reactive oxygen species (ROS) content were analyzed by flow cytometry. The protein levels of HMGB1, NF-κB p65, p-IκBα, cyclin D1 and survivin were determined by Western blot. RESULTS: The protein expression of HMGB1 in the HemECs transfected with HMGB1-siRNA was significantly lower than that in blank control group (P<0.05). Compared with NC group, the cell viability was decreased significantly in the HemECs transfected with HMGB1-siRNA, the apoptotic rate was significantly increased, the content of ROS increased significantly, and the protein levels of NF-κB p65, p-IκBα, cyclin D1 and survivin were significantly decreased (P<0.05). After exposure to NF-κB signaling pathway inhibitor PDTC, the cell viability was inhibited, the apoptosis was increased, ROS content, and the protein levels of NF-κB p65, p-IκBα, cyclin D1 and survivin were down-regulated significantly, as compared with si-HMGB1 group (P<0.05). CONCLUSION: Inhibition of HMGB1 reduces the viability of HemECs and induces apoptosis by increasing the content of ROS and down-regulating the activity of NF-κB signaling pathway.     

Effects of Chinese yellow wine on homocysteine-induced dysfunction in rat endothelial progenitor cells

AIM: To investigate whether Chinese yellow wine has influences on homocysteine (Hcy)-induced dysfunction in rat endothelial progenitor cells (EPCs). METHODS: Rat bone marrow was extracted to harvest mononuclear cells (MNCs) by density gradient centrifugation. The MNCs were plated on fibronectin-coated culture dishes, and were induced into EPCs by EGM-2 complete medium supplemented with cell growth factor. The adherent cells were collected 7 d later for all studies. EPCs were characterized as adherent cells double positive for DiI-ac-LDL uptaking and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. The viability, migration, apoptosis and in vitro vasculogenic activity of the EPCs were determined by MTT assay, Transwell chamber assay, apoptosis kit and in vitro vasculogenesis kit, respectively. RESULTS: Compared with control group, the viability, migration and in vitro vasculogenic capacity of the EPCs in Hcy group were significantly decreased (P<0.01). Compared with Hcy group, yellow wine group and red wine group both significantly improved the viability, migration and in vitro vasculogenic capacity of Hcy-induced EPCs (P<0.01). Compared with control group, yellow wine group and red wine group both significantly improved the above-mentioned functions of EPCs (P<0.05). However, no significant difference of apoptosis in all groups was observed. CONCLUSION: Hcy may result in dysfuction of EPCs. Treatment with yellow wine improves Hcy-induced EPC functions.     

Necroptosis mediates high glucose-induced injury in human umbilical vein endothelial cells

AIM: To explore whether necroptosis contributes to the high glucose (HG)-induced damage in human umbilical vein endothelial cells (HUVECs). METHODS: The protein levels of receptor-interacting protein 3 (RIP3) and cleaved caspase-3 were detected by Western blot. The intracellular levels of reactive oxygen species (ROS) were determined by DCFH-DA staining followed by photofluorography. Mitochondrial membrane potential (MMP) was measured by rhodamine 123 staining followed by photofluorography. RESULTS: Treatment of HUVECs with HG at different concentrations (10, 20 and 40 mmol/L glucose) for 24 h gradually enhanced the expression levels of RIP3. Treatment of HUVECs with HG (40 mmol/L glucose) for different time (3 h, 6 h, 9 h, 12 h and 24 h) also up-regulated the expression levels of RIP3, peaking at 9 h. Pretreatment of HUVECs with 20 μmol/L Z-VAD-FMK (an inhibitor of caspase) for 30 min before exposure to HG enhanced the expression level of RIP3. Pretreatment of HUVECs with 100 μmol/L necrostatin-1 (an inhi-bitor of necroptosis) for 1 h before exposure to HG alleviated the HG-induced injuries, such as a decrease in cell viability, an increase in ROS generation and dissipation of MMP, but up-regulated the protein level of cleaved caspase-3. CONCLUSION: Necroptosis mediates HG-induced injury in HUVECs. There is a negative interacting between necroptosis and apoptosis.     

PPARδ agonist GW501516 inhibits ox-LDL-or high glucose-induced apoptosis in human umbilical vein endothelial cells

AIM: To investigate the inhibitory effect of peroxisome proliferator-activated receptor δ (PPARδ) agonist GW501516 on the apoptosis induced by oxidized low-density lipoprotein(ox-LDL) or high concentration of glucose in human umbilical vein endothelial cells (HUVECs). METHODS: Cell apoptosis was induced by ox-LDL or high concentration of glucose in HUVECs and was examined by flow cytometry.The HUVECs were treated with GW501516 at different concentrations. The viability of HUVECs was analyzed by MTT assay. RESULTS: The apoptosis rate of HUVECs treated with ox-LDL was 21.3%, while those of HUVECs treated with ox-LDL combined with low, medium and high concentrations of GW501516 were 17.47%, 9.72% and 3.94%, respectively. The apoptosis rate of HUVECs treated with glucose was 22.60%, while those of HUVECs treated with glucose combined with different concentrations of GW501516 were 20.23%, 17.01% and 9.38%, respectively.The results indicated that ox-LDL or glucose induced apoptosis of HUVECs and GW501516 decreased the apoptotic rates induced by ox-LDL or glucose in a dose-dependent manner. The results of MTT assay showed that glucose or ox-LDL decreased the viability of HUVECs and GW501516 attenuated the effect of glucose or ox-LDL on HUVECs. CONCLUSION: GW501516 inhibits the apoptotic effects of ox-LDL and glucose on HUVECs and increases the viability of the cells.     

CREG prevents human umbilical vein endothelial cells from apoptosis by inhibiting p38 MAPK activation

AIM: To study the effect of cellular repressor of E1A-stimulated genes(CREG) and its mechanism on apoptosis of human umbilical vein endothelial cells (HUVECs) induced by etoposide (VP-16).METHODS: Primary HUVECs were cultured. RetroviraI eukaryotic expression vectors pLNCX-CREG and pLXSN-shRNA-CREG were transfected into HUVECs. The stable cell clone was selected and obtained by screening with G418 (800 mg/L) and the puromycin (2.5 mg/L), respectively. CREG expression was detected by Western blotting. The cells with overexpression of CREG (H-C) and those with CREG down-regulation (H-S) were pretreated with apoptotic inducer VP-16 at 100 μmol/L for 6 h. The apoptotic rates of the 3 kinds of cells were analyzed by TUNEL and flow cytometry with annexin V/PI dualstaining. Furthermore, the protein levels of phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) in the 3 kinds of cells were analyzed by Western blotting. The p38-specific inhibitor SB203580(20 μmol/L)was used to investigate the effects of p-p38 expression on apoptosis. RESULTS: Western blotting showed that CREG expression was obviously increased up to 160% in H-C compared to HUVECs. However, CREG expression in H-S cells was identified to be down-regulated to 70% compared with HUVECs. TUNEL assay and annexin V/PI dual-color FACS showed that the apoptotic rate was dramatically increased in H-S cells,but decreased in H-C cells. Subsequently, Western blotting exhibited that p-p38 expression was increased in H-S cells compared to HUVECs and H-C cells. When the H-S was pretreated with SB203580, the apoptotic rate was decreased. CONCLUSION: CREG overexpression might prevent HUVECs from apoptosis by inhibiting p38 MAPK activition.     

Role of juglone in regulating oxidative stress and apoptosis in ovarian cancer SKOV3 cells

AIM: To study the cytotoxicity of juglone on ovarian cancer SKOV3 cells. METHODS: The activity of SKOV3 cells was detected by MTT assay. The cells apoptosis was examined by flow cytometry. The level of reactive oxygen species (ROS) was measured by DCF-DA staining. The protein levels of cytochrome C (Cyt C) and activated caspase-3 were evaluated by Western blot. RESULTS: MTT assay showed that juglone significantly inhibited the growth of SKOV3 cells in dose- and time-dependent manners (P<0.05). The early apoptotic rate and late apoptotic rate of SKOV3 cells in 50 μmol/L juglone group at 24 and 48 h were higher than those in control group (P<0.01).Moreover, juglone induced ROS accumulation, and increased the protein levels of Cyt C and activated caspase-3.CONCLUSION: Juglone inhibits the cell growth and induces the apoptosis of SKOV3 cells by ROS accumulation.