| ObjectiveDiabetes-related cardiovascular complications have been identified as the major direct cause of death in patients with diabetes mellitus, including both macrovascular and microvascular diseases. In these cardiovascular complications, endothelial cells （ECs）of blood vessels are characterized as the first targets. ECs dysfunction is mainly caused by cell injury and apoptosis,which initially impairs endothelial integrity and blood vessel function. Thus, it is extremely important to protect against ECs injury and apoptosis. Up until now, despite incontrovertibility of evidence implicating hyperglycemia in ECs damage, the mechanisms involved remain ambiguous.Peroxisome proliferator-activated receptor （PPAR）-y coactivator-1α （PGC-1α） is a key regulator in energy metabolism and mitochondrial function. Increasing evidences suggest that PGC-la expression is highly associated with glucose metabolism.Obvious decreases in PGC-la expression were observed in adipose tissue from insulin-resistant subjects and in skeletal muscle from type 2 diabetics. PGC-la is a key regulator in hepatic gluconeogenesis of rodents in diabetic model. PGC-la is an essential regulator for mitochondrial function and the metabolism of reactive oxygen species （ROS）, while changes in the structure and function of mitochondrial are pivots in cell apoptosis; excessive ROS will cause endogence cell apoptosis.Up-regulated PGC-1α can increase cell apoptosis by ROS purified enzyme pathway in nerve cells. Recent studies show that the decreased PGC-la expression was causally related to hyperglycemia-induced vascular smooth muscle cells （VSMCs）proliferation and migration. So we hypothesize that PGC-la might be the important pathological physiological factors in endothelial cell injury and apoptosis in the diabetic vascular complications. The functional role of PGC-la in endothelium and whether it participates in high glucose-induced cell apoptosis and possible mechanism of action are not extensively studied. Therefore, in this study, we silenced PGC-la expression by using a recombinant adenovirus expressing PGC-la small hairpin RNA（Ad-shPGC-la） to determine whether PGC-la plays a functional role in high glucose induced apoptosis in human umbilical vein endothelial cells （HUVECs）, and to further explore the precise mechanisms, so as to get a better understanding of the pathogenesis of diabetes vascular complication and provide theoretical basis for early prevention.Methods1.To culture and treat cells: In sterile environment, the umbilical cords of full term infants of healthy puerperants, from caesarean delivery, were collected. Then the human umbilical vein endothelial cells （HUVECs） were collected and cultivatedunder proper conditions, and those at passages 3-8 were used in proceeding experiments. In order to create appropriate conditions for in vitro experiments of diabetes, we first divided the cultured umbilical vein endothelial cells into five groups—a group under fundamental conditions （control）（M199 medium containing 5.5mmol/L glucose, 20% FBS, 2% L-glutamine,100U/mL penicillin and streptomycin antibiotics）, a mannitol group （mannitol 25 mmol/1） , and three groups under the treatments of 11 mmol/L glucose, 15mmol/L glucose, and 25mmol/L glucose respectively, and they were all incubated for 48 hours. Then we incubated cells under the optimal glucose concentration for 12h, 24h, 48h, 72h, 96h respectively. Cell counting kit-8 （CCK-8） was used to examine the cell viability, and Western Blot was used to examine PGC-1α protein expression.2.To explore the effect of down-regulation of PGC-la on high glucose-induced cell injury and apoptosis in HUVECs: （1） The umbilical vein endothelial cells were infected by Ad-shPGC-la and control shRNA （Ad-Scr）, under the multiplicity of infection （MOI） for 10, 20 and 40 for 24h. After the infection, the Western Blot and Quantitative Real-time PCR （qPCR） were used to examine the PGC-1α protein expression and the mRNA level. （2） Ad-shPGC-1α and Ad-Scr infected the group of cells under fundamental conditions and high-glucose induced cells, respectively.CCK-8 was used to examine the cell viability in each group （Groups in experiment:the control group , the high-glucose induced group, the Ad-sh PGC-1α infected the group of cells under fundamental conditions and high-glucose induced group, the Ad-Scr infected the group of cells under fundamental conditions and high-glucose induced group）. Flow cytometry, accompanied by FITC-Annexin V/PI double staining, were used to examine the cell aptotsis in each group.3.To investigate the influence of down-regulation of PGC-la on high glucose-induced alterations in inner mitochondrial membrane permeability and mitochondrial membrane potential. （1） Calcein-AM staining was used to examine the permeability of the inner mitochondrial membrane: The umbilical vein endothelial cells, with Calcein-AM and CoC12, were placed in 37℃ cell incubator for 10 min. Then we observed the cells with laser scan confocal microscope and analyzed fluorescence intensity with microplate reader. （2） The JC-1 staining was used to measure the potential of mitochondrial membranes: The umbilical vein endothelial cells, with JC-1 staining, was incubated in 37℃ for 15 min. Laser scan confocal microscope was used for observation, and ImageJ for analysis of fluorescence intensity. The depolarization of mitochondrial membranes was measured by the ratio of the red and green lights. （Groups in experiment: the control group, the high-glucose induced group, the Ad-shPGC-1α infected high-glucose induced group, the Ad-Scr infected high-glucose induced group）4.To further explore the influence of down-regulation of PGC-1α on high glucose-induced alterations in mitochondrial membrane permeability: We examined the release of high-glucose induced cytochrome c （cyt c） on endothelial cells and the change in the activity of caspase. Western blot was used to examine the Cyto-c protein expression in the mitochondrial and cytosol and protein expression of caspase-3 and caspase-9. （the experimental groups are the same as those in 3）.5. For the close relationship among Bcl-2 /Bax、ROS、ERS and cell apoptosis,to investigate whether they are also related to the PGC-la in high-glucose induced endothelial cell apoptosis: （1） Bcl-2 and Bax protein levels were examined by Western Blot. （2） H2DCF-DA was used to examin ROS of the cells, Fluorescence intensity was analyzed with microplate reader. The ROS of cells was measured by fluorescence intensity. （3） GRP78, a maker protein of endoplasmic reticulum stress（ERS）, was examined by Western Blot （the experimental groups are the same as those in 3）.6. For the key role of the Voltage-Dependent Anion Channel （VDAC） in the cell apoptosis, to learn whether PGC-1α is relevant to VDAC in the process of high-glucose induced endothelial cell apoptosis: We used Western Blot to examine the protein expression of VDAC 1-3 in each group of cells; VDAC inhibitor was chosen to suppress the activity of VDAC in the experimental cells, and the flow cytomety was used to examine the cell apoptosis in each group. （Groups in experiment: the control group, the group under fundamental conditions with inhibitor,Ad-shPGC-1α infected high-glucose induced group, Ad-shPGC-la infectedhigh-glucose induced group with inhibitor group）.Results1. Under different glucose concentrations for 48 hours, the cell viability in mannitol group and the control group are roughly the same, without significant difference（P>0.05）. The viability of cells treated in different concentrations glucose all decreases, showing significant difference from that in the control group （P<0.01）.This viability inhibitory effect was the most obvious at 15 mmol/L. Moreover, high glucose （15 mmol/L） inhibited cell viability in a time-dependent manner. Compared with control group, the cell proliferations at12, 24, 48, 72 and 96 h have decreased significantly （P<0.01）. The cell viability in the group incubated for 48 hours has the most obvious decrease. At the same time, the PGC-la protein expression examined by Western Blot showed that PGC-1α protein expression is related to the decrease in the cell proliferation of high-glucose induced umbilical vein endothelial cells. The PGC-la protein expression decreased in groups of different glucose concentrations and different times, showing significant difference from that in the control and to the greatest extent in the group of 15mmol and 48h. Therefore, in the following experiments, we incubated glucose induced cell group at 15 mmol/L for 48 h.2. The effect of down-regulation of PGC-la on high glucose-induced cell injury and apoptosis in HUVECs.（1） The HUVECs infected by adenovirus encoding a small hairpin RNA of PGC-la（Ad-shPGC-1α） suggests that compared with the control group, the PGC-la protein expression and mRNA level all decreases under different multiplicity of infection（MOI）, with statistical significance（P<0.01）. The group at MOI of 20 shows the lowest PGC-la protein expression and mRNA level. Meanwhile, in the Ad-Scr infected HUVECs, no change was found in PGC-la protein expression and mRNA level, without significant difference from the control group （P>0.05）.（2） The viability of HUVECs examined by CCK-8 indicates that no cell viability decrease was detected in the non-glucose-induced umbilical vein endothelial cells,either Ad-sh PGC-1α or Ad-Scr infected, without significant difference from the control group （P>0.05）, while in the glucose-induced endothelial cells, cell viability significantly decreased compared to the control group （P<0.01）. In the Ad-sh PGC-la infected high-glucose induced cells, the cell viability further decreased, with significant difference compared to the high-glucose induced group （P<0.01）. If the high-glucose induced endothelial cells were infected by Ad-Scr, compared to the control group, the cell viability significantly decreased （P<0.01）, but showed no difference from the high-glucose induced group （P>0.05）.（3） Cell apoptosis examined by flow cytometry: in non-high-glucose induced umbilical vein endothelial cells, Ad-sh PGC-la or Ad-Scr infected groups, compared with the control group, did not show any significant changes in the rate of cell apoptosis （P>0.05）. In the high-glucose induced umbilical vein endothelial cells, the rate of cell apoptosis increased significantly compared with the control group（P<0.01）. In Ad-sh PGC-1α infected high-glucose induced cells, the rate of cell apoptosis further increased, with significant difference compared to high-glucose induced group （p<0.01）. In Ad-Scr infected high-glucose induced group, the rate of cell apoptosis increased significantly, compared to the control group （p<0.01）, and did not change compared to high-glucose induced group （p>0.05）.3. The influence of down-regulation of PGC-1α on high glucose-induced alterations in inner mitochondrial membrane （IMM） permeability and mitochondrial membrane potential （MMP）:（1） We firstly measured the inner mitochondrial membrane permeabilization using the Calcein-AM staining. The results show that in the single high-glucose induced group of the umbilical vein endothelial cells, after being incubated with Calcein-AM pins and CoCl2, the cell injury of IMM permeability increase, the green fluorescence in the membrance was destroyed by the CoC12 outside the membrane. Compared to the control group, the green fluorescence decreased, showing statistical significance（P<0.01）. The injury of the Ad-shPGC-1α infected high-glucose induced mitochondrial membrane was exacerbated, and the green fluorescence in the membrane further decreased, showing significant difference from the high-glucose induced group （P<0.01）. In the Ad-Scr infected high-glucose induced group, the green fluorescence in the membrane decreased, with huge difference from the control group（P<0.01）, but no significant difference from the pure high-glucose induced group（P>0.05）.（2） We detected the MMP by JC-1 staining. After high glucose treatment, the red fluorescence of JC-1 dramatically decreased, while the green fluorescence increased,resulting in an attenuated ratio of red/green intensity, with significant difference fromthe control group （P<0.01）. In the Ad-shPGC-la infected high-glucose induced group,the red fluorescence further decreased, while the green fluorescence increased, and their ratio further decreased, showing significant difference from the high-glucose induced group （P<0.01）. In the Ad-Scr infected high-glucose induced group, the ratio between red and green fluorescence decreased,with significant difference from the control group （P<0.01） and no difference from the pure high-glucose induced group（P>0.05）.4. To further explore the influence of down-regulation of PGC-la on high glucose-induced alterations in inner mitochondrial membrane permeability, we examined the release of the cytochrome c in the high-glucose induced umbilical vein endothelial cells and the change in the activity of caspase. The results from Western Blot indicate that, compared to the control group, the high-glucose induced group showed increase in the cytoplasm cyt c protein expression; meanwhile, the cell cyt c in mitochondria significantly decreased, the ratio of cyt c in cytoplasm and mitochondria increased, showing significant difference from the control group（P<0.01）. In the Ad-shPGC-la infected high-glucose induced group, the cell cyt c protein expression in cytoplasm further increased, while that in mitochondrials further decreased, and the their ratio further increased, showing significant difference from those in the high-glucose induced group （P<0.01）. In the high-glucose induced group,cells caspase-3 and caspase-9 protein expression significantly increased, with huge difference from those in the control group （P<0.01）. In the Ad-shPGC-la infected high glucose induced group, the caspase-3 and caspase-9 protein expression significantly increased, showing notable difference from the high-glucose induced group （P<0.01）. In the Ad-Scr infected high-glucose induced group, the cell cyt c and caspase protein expression showed significant difference from those in the control group （P<0.01）, but no difference from the high-glucose induced group （P>0.05）.5. To investigate whether Bcl-2/Bax、ROS、ERS are also related to the PGC-la in high-glucose induced endothelial cell apoptosis:（1） First we observed the expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax. Although lower Bcl-2 expression and higher Bax expression were observed following high glucose treatment, with significant difference from the control group （P<0.01）, no differences were noted following infection with Ad-shPGC-1α or Ad-Scr compared to high glucose-treated cells （P>0.05）（2） In the experiment observing the creation of ROS, we found that compared to the control group, the high-glucose induced group showed significant increase in the ROS in the umbilical vein endothelial cells, and significant difference in the fluorescence intensity from the control group （P<0.01）. In the Ad-shPGC-1α infected high-glucose induced group and the Ad-Scr infected high-glucose induced group, the ROS generation significantly increased, and the fluorescence intensity also shows significant difference from that in the control group （P<0.01）, but no significant difference from that in the high-glucose induced group （P>0.05）.（3） In the experiment for the GRP78 expression, the Western Blot results indicates that compared to the control group, the high-glucose induced group shows an increase in the GRP78 protein expression, with significant difference （P<0.01）. In the Ad-Scr infected high-glucose induced group, the GRP78 expression increased, showing significant difference from that in the control group （P<0.01）, but no difference from that in the high-glucose induced group （P>0.05）. However, in the Ad-sh PGC-la infected high-glucose induced group, GPR78 expression significantly decreased,showing notable difference from that in the high-glucose induced group （P<0.01）.6. To investigate the relationship between the apoptosis of PGC-la in high-glucose induced umbilical vein endothelial cell and VDAC, we first examined the protein expression of VDAC in cells in each experiment group. Compared with the control group, the high-glucose induced group showed increase in the expression of VDAC13,but in the Ad-shPGC-la infected high-glucose induced group, only VDAC1 expression increased, VDAC2-3 showed no significant changes. This result suggeststhat VDAC1 may be a key element for down-regulation of PGC-1α in accelerating apoptosis induced by high glucose. To further verify this hypothesis, we submitted the Ad-shPGC-la infected umbilical vein endothelial cells to high-glucose medium in the presence of 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid（DIDS）, an inhibitor of VDAC1. After treatment with DIDS, in the foundmental condition cells, no changewas found in the cell apoptosis, with no difference from the control group（P>0.05） ,while in the Ad-shPGC-la infected high-glucose induced cells, the increased apoptosis was markedly inhibited, showing significant difference from that in the Ad-shPGC-la infected high-glucose induced group （P<0.01）.Conclusion1.PGC-la expression in HUVECs parallels with high glucose-decreased cell viability.The proliferation rate of high glucose-induced HUVECs decreases, and meanwhile PGC-la expression decreases.2.The down-regulation of PGC-la further decreases the proliferation of high glucose-induced HUVECs,accelerating their apoptosis,but has no effect on the proliferation and apoptosis of HUVECs under normal conditions.3. The down-regulation of PGC-la augments the injury of the mitochondrial membrane of high glucose-induced HUVECs. The membrane’s permeability increases, and the depolarization of the membrane potentials decreases.4. The down-regulation of PGC-1α increases the release of cytochrome c in high glucose-induced HUVECs and the activity of caspases, further indicating that the down-regulation of PGC-1α exacerbates the changes of the mitochondrial membrane’s permeability.5. The increase of apoptosis of the HUVECs caused by the down-regulation of PGC-1α is independent of Bcl-2 and Bax proteins,and independent of ROS and ERS as well.6. The down-regulation of PGC-1α to exacerbate the apoptosis of high glucose-induced HUVECs is achieved by increasing the activity of voltage dependent anion channel VDAC1.In summary, under the high glucose condition, PGC-1α deficiency activates the mitochondria-dependent pathway, increases the activity of mitochondrial membrane voltage dependent anion channel VDAC1, and exacerbates the injury and apoptosis of high glucose-induced HUVECs. |
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