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Heat stress induces apoptosis through transcription-independent p53-mediated mitochondrial pathways in human umbilical vein endothelial cell

Cells apoptosis induced by intense heat stress is the prominent feature of heat-related illness. However, little is known about the biological effects of heat stress on cells apoptosis. Herein, we presented evidence that intense heat stress could induce early apoptosis of HUVEC cells through activating mitochondrial pathway with changes in mitochondrial membrane potential(ΔΨm), release of cytochrome c, and activation of caspase-9 and -3. We further revealed that p53 played a crucial role in heat stress-induced early apoptosis, with p53 protein rapidly translocated into mitochondria. Using pifithrin-α(PFT), a p53's mitochondrial translocation inhibitor, we found that pretreated with PFT, heat stress induced mitochondrial p53 translocation was significantly suppressed, accompanied by a significant alleviation in the loss of ΔΨm, cytochrome c release and caspase-9 activation. Furthermore, we also found that generation of reactive oxygen species (ROS) was a critical mediator in heat stress-induced apoptosis. In addition, the antioxidant MnTMPyP significantly decreased the heat stress-induced p53's mitochondrial translocation, followed by the loss of ΔΨm, cytochrome c release, caspase-9 activation and heat stress-mediated apoptosis. Conclusively, these findings indicate the contribution of the transcription-independent mitochondrial p53 pathway to early apoptosis in HUVEC cells induced by oxidative stress in response to intense heat stress.

H eat is likely to be gained by way of infrared radiation from the sun, or direct contact with hot objects, and absorbed when the ambient temperature rises above body temperature, as well as generated as a byproduct of cellular metabolism and the mechanical work of skeletal muscle 1 . Thus, heat stress is widely considered as a major extracellular stimulus 2 . Moreover, an increase in cellular temperature incurs protein denaturation and interrupts critical cellular processes, thus resulting in apoptosis and cell death 3 . It is well established that intense heat stress wrecks damages to the organism mainly by direct toxicity to cell 4 and radiation 5 .Temperaturesgreater than 41.6uC to 42uC,the critical human body temperature point, can lead to cell death from apoptosis even in a few hours 6 ,whereas extreme high temperatures of 49uC to 50uC can give rise to cellular death from necrosis in less than 5 minutes 7 . Therefore, excessive gain of heat from the elevated ambient temperature has been proposed to be a risk factor for the severely life-threatening disorder known as heat stroke characterized by an increased core body temperature that rapidly rises to above 40uC and engenders central nervous system dysfunction 8 . Besides, the mortality thereof is found very high even after adequate hypothermia or other intensive care therapy 9 . For the heat wave that affected Europe in the summer of 2003, 45 000 deaths have been reported of which one-third was attributed to heatstroke 10,11 . With the prospect of increased global temperatures in near future, the mortality and morbidity may continue to rise 11 . However, the pathogenesis of tissue injury and cell death in heat-stroke attributed to heat stress is not well understood. Studies in cell lines and animal models have suggested that endothelial-cell is an early target of heat stress injury and also is a prominent feature of severe heat-stroke 12,13 . Recent studies suggest that endothelial-cell can induce significant apoptosis during acute-phase response to heat stress 14 .Thus,endothelial-cell apoptosis appears to be a mechanism of heat-stroke,but little is known about the biological effects of heat stress on endothelial-cell apoptosis.
The tumor suppressor gene p53 is a key regulator of apoptosis, which has proapoptotic activity through transcription-dependent or independent pathway 15 . Under stressed conditions, p53 protein works as a transcription factor, activating a series of genes such as Puma, Noxa, Bax, and Bid 16 . On the other hand, cytoplasmic p53 directly activates mitochondrial death pathway through a transcription-independent manner 17 . It recently becomes clear that early in the course of p53-dependent apoptosis, caused by death signal stimuli, a fraction of p53 proteins rapidly undergo translocation to mitochondria(detectable at 30 min-1 h), and trigger an early first wave of marked caspase 3 activation, followed by an early wave of apoptosis 18 . This rapid first wave of apoptosis is transcription-independent, and precedes a second slower wave that is transcription-dependent 18 . Besides, such death signal inducers, which induce mitochondrial p53 accumulation and rapid apoptosis, have been confirmed including exposure to oxidative stress, DNA-damaging drugs, c-irradiation or hypoxia 19 . Oxidative stress have also been indentified in numerous studies correlated with p53-directed cell apoptosis 20,21 . In addition, preliminary data have indicated that ROS plays a signaling role in mitochondrial migration of p53 22 and oxidative stress is thought to play a pivotal role in heat stress induced apoptosis 23 . However, the field about how ROS regulates p53-mediated cell apoptosis and heat stress induced apoptosis remains rarely trodden.
Therefore, in this study, we investigated the molecular mechanism of apoptosis induced by intense heat stress. We reported for the first time that intense heat stress can induce early wave apoptosis in HUVEC cells which occurs through the mechanism that intense heat stress induces p53 translocation to mitochondria, and triggers mitochondrial apoptotic pathways. We also found that ROS produced by heat stress might be involved in transcription-independent p53mediated mitochondrial pathways.

Results
Heat shock inhibits cell proliferation in HUVEC cells. To investigate the effect of heat stress on inhibition of HUVEC cells proliferation, we initially examined the effect of heat stress on the proliferation of HUVEC cells. HUVEC cells were maintained in culture media for 48 h and the culture dishes were sealed with parafilm and immersed for 2 h in a circulating water bath thermoregulated at 37uC, 39uC, 41uC, 43uC, or 45uC for the heat stress treatments 2 . Culture media were replaced with fresh media and the cells were further incubated at 37uC for 6 h. As shown in (Figure 1), exposure of HUVEC cells to heat stress for 2 h and further incubation for 6 h registered an inhibition of the growth of HUVEC cells in a temperature-dependent manner.
Intense heat stress induces early wave of apoptosis by triggering the mitochondrial pathway in HUVEC cells. A quantitative evaluation was made, with flow cytometry adopted to assess the apoptotic cells. When cells were exposed to intense heat stress, the number of early apoptotic cells increased markedly under the treatment of HUVEC cells at 43uC in 2 h and apoptosis induced amounted to 27.7% by 6 h (Figure 2). In order to determine the apoptotic pathways involved in intense heat stress-induced early wave of apoptosis,we examined the changes in the activities of initiator caspase (caspase-8,-9, and-4) and effector caspase (caspase-3). As shown in Figure 3a,d caspase-9 and -3 activation were detected at 3 h after heat stress treatment. The levels of caspase-9 and -3 activities were increased by more than 2 fold and 3 fold, respectively. Caspase-8 and -4 were not activated. We also analyzed the changes in mitochondrial membrane potential(DYm) and release of cytochrome c from mitochondria in heat stress-treated HUVEC cells. As shown in Figure 3e the exposure of HUVEC cells to HUVEC cells viability after heat stress was tested by WST-1. Results are expressed as the percentage of cell proliferation relative to the proliferation of control treatment. The data shown are the mean from three independent experiments. Each value is the mean 6 SD of three determinations. The asterisk indicates a significant difference between control and test groups, *p , 0.05. intense heat stress resulted in the reduction of mitochondrial membrane potential in a time-dependent manner followed by significant increases in the release of cytochrome c from mitochondria( Figure 3f). These results indicated that intense heat stress might induce the mitochondrial pathway leading to caspase-9/ caspase-3-mediated early apoptosis in HUVEC cells. To further explore whether heat stress induces apoptosis through the mitochondrial pathway, we infected HUVEC cells stably over-expressBcl-2 or the vector alone (control cells) with several strains. As shown in Figure 4a,b and c, the overexpressing Bcl-2 of cells significantly decreased the heat stress-induced apoptosis, the loss of DYm and cytochrome c release. These data further supports the hypothesis that the mitochondrial pathway may mediate the intense heat stress-induced early wave of apoptosis of HUVEC ells.
P53 activation of the mitochondrial apoptosis pathway. Previous study has reported heat stress induces apoptotic death in cells through mitochondrial pathway 24 . Our study has also found that intense heat stress results in caspase-9 activation. However, the upstream of mitochondrial apoptotic pathway induced by intense heat stress has not been fully illustrated. Nevertheless, It is well known that p53 protein plays a central role in the regulation of apoptosis. To investigate the involvement of p53 in heat stressinduced apoptosis, the effects of the transfection of the P53 siRNA on the cellular features of apoptosis were studied. As shown in Figure 5a, the transfected cells were assayed for P53 protein expression by western blot assay. Of the several tested siRNA target sequences, one siRNA was effective in decreasing protein levels. P53 mRNA level measured by RT-PCR was significantly decreased in P53 siRNA-transfected cells compared to control cells (scrambled siRNA) ( Figure 5b). The P53 siRNA-transfected cells expressed reduced level of p53 protein, and were less susceptible to intense heat stress-induced early apoptosis compared with control cells (Figure 5c). The caspase-9 activation, release of cytochrome c and mitochondrial membrane potential(DYm) in response to intense heat stress were also studied in P53 siRNA transfected HUVEC cells. When compared with control cells, P53 siRNA transfected HUVEC cells showed a drastic reduction in caspase-9 activity ( Figure 5d) and the release of cytochrome c (Figure 5f). The loss of DYm was alleviated in P53 siRNA transfected HUVEC cells compared with control cells (Figure 5e). These results indicate that signaling of heat stress-induced apoptosis is in part dependent on the expression of p53.
Translocation of p53 to Mitochondria in heatstress-treated HUVEC cells. Based on evidence for the crucial role of transcriptionindependent p53-mediated cells apoptosis in this process of early wave apoptosis 25 , we analyzed further the subcellular distribution of p53 in HUVEC cells during early apoptosis induced by intense heat stress. Highly enriched mitochondria fractions e purified from untreated or heat-treated HUVEC cells were prepared by sucrose gradient centrifugation 26 and analyzed by Western blot. Exposure of HUVEC cells to heat stress for 2 h, and further incubation for different times exhibited a significant amount of p53 accumulation at the mitochondria in a time -dependent manner, which occurs at 0 h after cells were treated with 43uC and peaked at 6 h ( Figure 6a). To directly localize p53 in mitochondria of early apoptosing cells, we performed indirect immunofluorescence. P53 mitochondrial translocation was detected by immunofluorescence staining (Figure 6b). These findings thus suggest that intense heat stress induces rapid mitochondrial translocation of p53, which may activate the apoptotic program.
Mitochondrial localization of p53 promotes mitochondrial apoptosis pathways. To verify further that mitochondrial p53 accumulation is associated with heat stress induced cells apoptotic death, we administered pifithrin-a(PFT), a highly selective and potent synthetic p53 inhibitor to show that PFT can block p53's mitochondrial translocation 27 . HUVEC cells were pretreated with PFT for 1.5 h, then treated with intense heat stress (43uC) for 2 h. As shown in Figure 7a, after pretreatment with PFT the heat stress induced mitochondrial p53 translocation displayed a significant reduction. To investigate the downstream of the mitochondrial p53 pathway,we examined cytochrome c release to the cytosol, caspase-9 activation and mitochondrial membrane potential(DYm)in PFT treated HUVEC cells. A significant decrease in cytosolic cytochrome c (Figure 7b), and caspase-9 activation (Figure 7c) were observed. The loss of DYm was also alleviated in pretreated with PFT HUVEC cells compared with control cells (figure 7d). These results indicate that p53 molecules accumulated in response to heat stress translocate into the mitochondria and activate mitochondrial apoptosis pathway.
Intense heat stress increases the generation of ROS in HUVEC cells. Based on the evidence that ROS generation plays an important role in heat stress 28 , we further validated the possibility that heat stress-induced apoptosis prompts ROS accumulation. We utilized the fluorescent dye DCFH-DA, which produces enhanced The role of ROS on heat stress-induced apoptosis. To investigate whether ROS generation via heat stress also involves p53-mediated mitochondrial pathways, we assessed mitochondrial apoptosis related proteins in HUVEC cells which had been pretreated with antioxidant MnTMPyP (10 umol/l) for 1 h, then treated with intense heat stress (43uC). The depletion of ROS by the cell-   permeable ROS scavenger MnTMPyP significantly decreased the heatstress-mediated translocation of p53 proteins to mitochondria, cytochrome c release from mitochondria, caspase-9 activity, and apoptosis ( Figure 9). These data confirm that heat stress treatment of HUVEC cells activates the mitochondria via release of ROS.

Discussion
Exposure to high ambient temperature and an elevation of body temperature has been proposed to be a risk factor for heat-related illness representing a continuum of disorders from minor syndromes such as heat cramps, heat syncope, and heat exhaustion to the severely life-threatening disorder known as heat stroke 29 . Although lots of researches were performed on this during the past decade, the mechanism of heat-related illnesses is not clear 30 . Studies in animal models found that there was widespread apoptosis in spleen, lymph node, thymus, small bowel, and cells of lymphoid origin in the lamina propria 12 . Using in vitro approach, numerous studies have established that apoptosis can be promoted by application of heat stress [31][32][33] . These data imply that apoptosis may play an important role in the physiology/pathophysiology of heat-related illness attributed to heat stress. In our study, we focused on endothelial cells because (1) endothelial cells are exposed to diverse stressors,which serve not only as a barrier between the bloodstream, its elements and toxins, and tissue parenchyma but also as generalized transducing cells that modulate the parenchymal responses to those stimuli 34 ; (2) Endothelial-cell injury in acute-phase response to heat stress are the prominent features of heat stroke 8 ; and (3) Endothelial-cell apoptosis has been shown to increase the extent of injury in cell lines and animal models attributable to heat stress 12,35 . In this work, we investigated the biological effects of intense heat stress on cells apoptosis, and showed that intense heat stress could induce early apoptosis through the transcription-independent mitochondrial p53 pathway, which might involve reactive oxygen species(ROS) attributed to intense heat stress.
Mitochondria are central integrators and transducers for proapoptotic signals, forming the nexus between the non-specific inducer phase and the final execution phase of apoptosis 36 . Among studies about cell lines, Hsu YL et al found that heat stress triggered the mitochondrial apoptotic pathway resulting in caspase-9 activity 24 . A previous study by Milleron RS et al. also found that under intense heat stress, cells underwent early phase of apoptosis (within 4 h of heat stress),which triggered the mitochondrial apoptotic pathway by activating the apical protease that induced mitochondrial outer membrane permeabilization(MOMP), a loss in DYm and caspase-3 activation 37 . Consistently, our results showed that intense heat stress results in a significant increase of release of cytochrome c from mitochondria and decrease of DYm. In addition, our results also revealed the degree of caspase-9 and caspase-3 activity after HUVEC cells were treated with intense heat stress. Caspase-8 and -4 was not activated in this study. Therefore, our results suggested that intense heat stress might initiate mitochondrial other than death receptors or the endoplasmic reticulum (ER) to promote the early wave of apoptosis of HUVEC cells.
P53 is a transcription factor which modulates expression of numerous target genes that control apoptosis,cell cycle arrest, senescence, DNA repair and genetic stability in response to death signal stressor 38 . In addition to its transcriptional route, P53 also promotes apoptosis through transcription-independent mechanisms, primarily signals through the mitochondrial pathway 39 . Mitochondrial p53 targeting occurs in a wide spectrum of cell types and after a variety of stress signals including oxidative stress, DNA damage, irradiation and hypoxic stress 40 . Previous studies demonstrated that early in the course of p53-dependent apoptosis, caused by such stressor, a fraction of wild-type p53 protein rapidly migrates to the mitochondria 41 . Once at the mitochondrion, p53 leads to mitochondrial dysfunction that can directly trigger the release of pro-apoptotic factors from the mitochondrial intermembrane space 42 . Tian XJ et al. 43 recently demonstrated that at the population level, apoptosis appears in two waves, i.e the fast wave mediated by the mitochondrial p53 pathway and the slow wave by the nuclear p53 pathway. We have shown in this study that mitochondrial p53 translocation started within 1 h and was significant at 3 h after intense heat-treatment, followed by early cells apoptosis (peak at27.7% at 6 h after43uC for 2 h).To identify whether mitochondrial p53 accumulation is associated with heat stress-induced early cells apoptosis, we found that knockdown of p53 by P53 siRNA, reduced DYm loss, release of cytochrome c and caspase-9 and -3 activation, as well as early wave of apoptosis ( Figure 5).This shows that P53 plays an important role in intense heat stress-mediated HUVEC cellular early apoptosis. We also further evaluated the effect of Pifithrin-a(PFT) on apoptosis cells induced by intense heat stress. We found that PFT can significantly block p53's mitochondrial translocation, followed by decreased cytochrome c release (Figure 7), caspase-9 activation, as well as alleviated DYm decrease in heat-treated cells. Therefore, it is plausible that the p53 in heattreated HUVEC cells translocates to mitochondria where it triggers mitochondrial apoptotic pathways. These results are consistent with the notion that mitochondrial p53 can trigger fast apoptosis at higher damage levels 43 . Our data also explains the intriguing observation by Milleron RS et al 37 .who showed that intense heat stressinduced cells an early apoptosis through an unknown upstream of mitochondria.
Oxidative damage to cellular compartments has great implication for animal production as well as human health 44 . Under physiological conditions, the redox status of all aerobic cells is balanced by enzyme and non-enzyme systems. However, under certain stressful conditions, increases in generation of ROS and/or decreases in anti-oxidant capacity enhanced oxidative stress 45 . Pucciariello C et al. 46 discovered that acute heat stress could disturb the balance between the ROS production and the antioxidant systems. Several reports link oxidative stress with heat stress and suggest synergistic augmentation of cell death, and report increased ROS generation in heat exposed cells [47][48][49] . What's more, recent studies have revealed that ROS acts as both an up-stream signal that triggers p53 activation and as a downstream factor that mediates apoptosis 21 . In other previous studies, ROS induced p53 (,2%) moves into mitochondria and initiates apoptosis 50 . Our study has shown that intense heat stressmediated oxidative stress works primarily by increasing the production of ROS. We further observed that heat stress generates ROS, which activates caspase-9 rather than caspase-8 and -4. Furthermore, using MnTMPyP, an agent which is a cell-permeable ROS scavenger 51 , able to block caspase-9 activity, P53 moves to mitochondria and engenders apoptosis. All in all, ROS, as the production of acute heat stress, might act as an upstream signal that triggers transcription-independent p53-mediated mitochondrial pathways to promote the early wave of apoptosis of intense heat stress on HUVEC cells.
In conclusion, our data indicate that HUVEC cells undergo apoptosis soon after intense heat stress. Intense heat stress-induced early wave of apoptosis is associated with the mitochondrial apoptosis pathway involving translocation of p53 to mitochondria, which are in turn mediated by ROS generation. Our study provides for the first time an evidence of ROS involved transcriptional-independent p53mediated mitochondrial pathways in heat stress-mediated apoptosis of HUVEC cells.

Methods
Cell culture, treatments and cell proliferation assay. Human umbilical vein endothelial cells (HUVECs) were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Cells passage 3, were grown in media recommended by the manufacturer. HUVEC cells maintained in culture media Culture dishes were sealed with parafilm and immersed for 2 h in a circulating water bath thermoregulated at 37uC 6 0.5uC for the control treatment or at 39uC,41uC, 43uC,or45uC 6 0.5uC for the heat stress treatments. Culture media were replaced with fresh media and the cells further incubated for the indicated times. Cell proliferation was assessed by Premixed WST-1 Cell Proliferation Reagent (Clontech Laboratories Inc., Mountain View, CA, USA) according to the manufacturer's instructions.
Flow cytometric analysis of cell apoptosis using Annexin V-FITC/PI staining. Cell cycle phase were either kept untreated or exposed to 2 h 43uC before being analyzed by flow cytometry. The detection was performed according to the manual of AnnexinV-FITC apoptosis detection kit (invitrogen). About 1 3 10 6 cells were collected, washed with ice-cold PBS, and resuspended in the binding buffer containing suitable amount of Annexin V-FITC. After 10 min incubation in the dark at room temperature, the buffer was removed by centrifugation. The cells were resuspended in reaction buffer containing propidium iodide (PI). Then flow cytometric analysis was immediately performed to detect apoptosis.
Measurements of ROS. Levels of intracellular ROS were assessed by use of a reactive oxygen species assay kit (Beyotime, Nanjing, China). dichlorofluoresceindiacetate(DCFH-DA, Molecular Probes,Beyotime) was used. This reagent enters the cells and reacts with ROS, producing the fluorophoredichlorofluorescein (DCF). Briefly, cell cycle phase was either kept untreated or exposed to 2 h 43uC, and further incubated for different times, then about 3 3 10 5 were harvested, washed with serum free DMEM culture medium and the cells were stained with10 mM dichlorofluoresceindiacetate (DCFH-DA, Molecular Probes,Beyotime) for 30 min at 37uC in the darkness. Then the cells were harvested, washed and resuspended in serum-free DMEM culture medium for three times. The cells' fluorescence intensity was determined using flow cytometer 52 . 200 mM H 2 O 2 -treated HUVEC cells were as a positive control.
Assay for caspase activity. After treatment with 43uC for 2 h, cells were harvested, and cell lysates were prepared at 280uC for 30 min and then incubated with the following appropriate caspase substrates at 37uC using a Quadruple MonochromatorMicroplate Reader (Infinite M1000, Tecan US, NC, USA). Caspase activities were measured by cleavage of the following fluorogenic peptide substrates 53,54 : Ac-LEHD-AFC for caspase-9, Ac-DEVD-AMC for caspase-3, Ac-ATAD-AFC for caspase-8 and Ac-LEVD-AFC for caspase-4. Activities of caspases are represented as relative cumulative fluorescence of the kinetic reaction and compared to untreated controls.
Immunofluorescence analysis. HUVEC cells were either left without or with heat stress for 2 h and further incubated for 3 h, then removed of the culture medium. All cells were labeled by adding pre-warmed (37uC) staining solution containing 200 nM MitoTrackerH probe and incubated for 20 minutes under 37uC. The cells were washed with PBS twice, subsequently fixed in 3.5% paraformaldehyde, permeabilized in KB solution and 0.2% Triton X-100 at room temperature. After the blocking solution was washed out, cells were incubated with a primary antibody (rabbit anti-human p53, 15500) (diluted in KB) for 30-45 min at 37uC and subsequently f washed with KB twice. After incubated for 30-45 min at 37uC with secondary antibody (diluted in KB), cells were washed with KB again before mounted onto slide with mounting solution containing 0.2 mg/ml DAPI and sealed with nail polisher. The slide were stored at 4uC in a dark box and observed under a fluorescent microscope.
Mitochondrial membrane potential assay. The mitochondrial membrane potential was detected using5,59,6,69-tetrachloro-1, 19,3,39 tetraethylbenzimidazolcarbocyanine iodide (JC-1; Molecular Probes, Eugene, OR, USA). Following treatment with heat stress, cells were rinsed once with complete culture medium and incubated with JC-1 for 30 min at 37uC, centrifuged, washed twice with cold PBS, transferred to a 96-well plate (10 5 cells/well), and assayed in a fluorescence plate reader with the following settings:excitation at 490 nm, emission at 540 nm and 590 nm, Changes in the ratio between the measurement at test wave lengths of 590 nm (red) and 540 nm (green). Fluorescence intensities are indicative of changes in the mitochondria membrane potential 55 .
siRNA transfection. Small-interfering RNA (siRNA) for P53 was designed and synthesized by Guangzhou RiboBio (RiboBioInc, China). The sequence of each gene and their scrambled are shown in follows. For p53,sense and antisense siRNAs are 59-GAAAUUUGCGUGUGGAGUA (dTdT)-39 and 59 -UACUCCACACGCAAAU-UUC(dTdT)-39, respectively. For scrambled control, sense and antisense siRNAs are 59-UGGUUUACAUGUCGACUAA(dTdT)-39 and 59-UUAGUCGACAUGUA-AACCA(dTdT)-39, respectively. Twenty-four hours prior to transfection, HUVEC cells were plated onto a 6-well plate (Nest, Biotech,China) at 30-50% confluence, which were then transfected into cells using TurboFect TM siRNA Transfection Reagent (Fermentas, Vilnius, Lithuania) according to the manufacturer's protocol. Cells were collected after 48-72 h for the further experiments. The mRNA and protein levels of P53 were estimated by RT-PCR and Western blotting. After 2 days of incubation at 37uC in a humidified atmosphere of 5% CO2, cells were exposed to heat stress.
Western blot analysis. HUVEC cells were pretreated with or without 43uC for 2 h, and further incubated for different times as indicated. Western blot was carried out as described 56,57 with monoclonal Anti-p53 antibodies(151000; Cell signaling technology,Danvers, USA) and Polyclonal Anti-Cytochrome C antibodies(151000; Cell signaling technology,Danvers, USA). An HRP-conjugated anti-rabbit IgG antibody was used as the secondary antibody (ZhongshanInc, China). Signals were detected using enhanced chemiluminescence reagents (Pierce, Rockford, IL, USA).
RNA isolation, reverse transcription, and qRT-PCR. Total RNA was extracted from the p53siRNA and ScrsiRNA HUVEC cells using Trizol(Takara, Shiga, Japan). For P53, RNA was transcribed into cDNA and amplified with Specific sense: 5-ACACGCTTCCCTGGATTG-3; antisense primer: 5-CAAGAAGCCCAGAC-GGAAAC-3. Reverse transcription and QPCR were performed in accordance with manufacturer's instructions (Takara, Shiga, Japan). The PCR reaction for each gene was repeated three times. GAPDH was used as inner control for normalizing p53 expression. Differential expression of p53 was calculated using the 2-DDCt method.
Statistical analysis. All data were analyzed for statistical significance using SPSS 13.0 software (SPSS, Chicago, IL, USA). Data were expressed as means 6 SD from at least 3 independent experiments performed in duplicate. Statistical comparisons of the results were made using analysis of One-way ANOVA. p , 0.05 was considered statistically significant.