Intense Heat Stress Induce Human Umbilical Vein Endothelial Cells Apoptosis Through Mitochondrial Apoptosis Pathway

BACKGROUND AND AIMSExposure to high ambient temperature, the can appear 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. The heat wave that affected Europe in the summer of2003led to an unprecedented45000excess deaths, of which one-third was attributed to heatstroke. It is predicted that global warming will cause an increase in the frequency and severity of heat waves with an associated rise in mortality. The pathogenesis of tissue injury and cell death in heatstroke is not well understood, which may explain the high mortality and disability rate as no specific mechanisms can be targeted for treatment. Heat is the most fundamental factor in the process of heat stroke. Studies in cell lines and animal models suggest that heat can directly induce tissue damage and cell death, and the apoptosis signal or necrosis occurred as the degree of heat stress. Extreme temperatures (49to50℃) cause damage to most cellular structures and their function, resulting in cell death by necrosis in less than5minutes, while the moderate heat stress (less then45℃) mainly activated apoptotic signals. These data imply that apoptosis may play an important role in the physiology/pathophysiology of heat stroke. In recent years, Studies in molecular biology found that heat play an important role in cell transduction process: Mediated apoptosis process through regulating the activation of apoptosis related proteins such as caspase; Reactive oxygen metabolites, protease and cytokines attributed to intense heat stress, which promote cells survive/death during stressful situations by activate/inhibit signal transduction pathway, such as reactive oxygen species of tissue damage attributed to intense heat stress has aroused a vast concern; It also cause inhibition of cell proliferation and apoptosis through induces DNA damage, p53activation. However, on the one hand It is still fewer in studies on heat stress induces apoptosis mechanism, on the other hand most of studies focus on tumor cell lines, studies in normal cell lines is still less. With increase in the frequency and severity of heat waves with an associated rise in mortality, the normal tissue damage caused by intense heat stress urgently need to arouse people’s attention.Endothelial-cell play an important role in tissue damage during acute-phase response to heat stress. Studies in cell lines and animal models have confirmed that intense heat stress can induce endothelial cell apoptosis widely. Currently, little is known about the exact biological effects of endothelial-cell apoptosis during pathology of heatstroke, and the mechanism of endothelial cell apoptosis induces by heat stress is not well understood, mainly because of no related research. Our preliminary clinical trials found that patients with Severe heatstroke appear serious vascular endothelial cell injury. Studies in cell lines found that cytotoxic effect directly cause by heat stress on endothelial-cell can lead to Cell proliferation inhibition. However, the injury mechanism of HUVEC cells induces by heat stress need to further research.The purpose of this study is to investigate the mechanism of apoptosis induced by intense heat stress, including signaling pathway, upstream signal molecule and interaction between the signal molecule. These studies promote the understanding of induced endothelial cell apoptosis mechanism at molecular level. As to provide theoretical basis for clinical heatstroke prevention and treatment.METHODS AND RESULTS1. cytotoxic effect directly cause by heat stress induces early apoptosis of cellsHUVEC cells were treated with43℃and37℃(control) for2h, and the cells were further incubated(0,1,3,6,9h) for different time as indicated, the cytotoxic effect of heat stress were tested using both the LDH and wst-1. Apoptosis was analyzed by flow cytometry using Annexin V-FITC/PI staining. The cell damage appeared at early after heat stress:cells damage in a time-dependent manner after heat stress (F:109.459). The number of early apoptotic cells was increased markedly to the treatment of HUVEC cells in2h at43℃and induced apoptosis to approximately30%by6h (F:959.117, P:0.001)2. Intense heat stress induces early wave of apoptosis by triggering the mitochondrial pathway in HUVEC cellsProteins were extracted from cells treated with heat stress(0,1,3,6,9h) or without heat stress, respectively. Western blot analysis of time-dependent effects of heat stress on the activation of UPR:p-PERK,P-eIF2a,ATF4,XBP1-s, GRP78and GADD153. RT-PCR analysis of the transcription of the XBP-1s target gene EDEM. The results showed as follows:phosphorylation PERK were tested at Oh after heat stress, Then phosphorylation were inhibited gradually, phosphorylation eIF2a were tested at1h after heat stress, which were inhibited in time-dependent manner after heat stress.ATF4were tested at3h after heat stress, peak at6h after heat stress, while were inhibited obviously. ATF6activation peak at Oh after heat stress, then decreased gradually, and following gradually decreased to the basic level. The expression of XBP-1s occur at1h and peak at3h after heat stress, then decreased gradually, EDEM mRNA level change trend consistent with expression of XBP-1s. GRP78activation at oh after heat stress and decreased gradually at3h after heat stress. There is no obvious increase for expression of GADD153in cells treated with heat stress, these results suggest that intense heat stress triggers the UPR to protect cells against ER stress, which active three UPR transducer pathways:ERK-eIF2a-ATF4,IRE1-XBP-1S and ATF6. Their activation receded at later stage, but heat stress not induces ER apoptosis pathway.Enzymatic activity of caspase-9,-3,-8and-4was measured in cell lysates using fluorogenic substrate Ac-LEHD-AFC, Ac-DEVD-AMC, Ac-ATAD-AFC and Ac-IETD-pNA, respectively. The loss of⊿Ψm was measured by JC-1and flow cytometry. Extraction of mitochondria and cytoplasm protein,respectively. The expression levels of cytochrome C were determined by Western Blots, caspase-9and-3activation were detected at3h after heat stress treatment. The levels of caspase-9and-3activities were increased by more than2fold and3fold, respectively (caspase-9F:47.903, P:0.001; caspase-3F-.65.602, P:0.001). Caspase-8and-4was not activated (caspase-4F:2.146, P:0.149; caspase-8F:0.604, P:0.669). The release of cytochrome c from mitochondria occur at3,6and9h. These results indicate that intense heat stress might induce the mitochondrial pathway leading to caspase-9/caspase-3-mediated early apoptosis in HUVEC cells.3. Mitochondrial localization of p53promotes mitochondrial apoptosis pathwaysHUVEC cells were transfected with scrambled siRNA (Scr) or P53siRNA (P53). The P53siRNA transfected cells expressed reduced level of p53protein, and were less susceptible to intense heat stress-induced early apoptosis compared with transfected with scrambled siRNA cells. When compared with transfected with scrambled siRNA cells, P53siRNA transfected HUVEC cells showed a drastic reduction in caspase-9activity and the release of cytochrome c. The loss of⊿Ψm was alleviated in P53siRNA transfected HUVEC cells compared with transfected with scrambled siRNA cells. Highly enriched mitochondria fractions were purified from untreated or heat-treated HUVEC cell were prepared by sucrose gradient centrifugation and analyzed by Western blot Exposure of HUVEC cells to heat stress for2h, and further incubating for different time exhibited a significant amount of p53accumulation at the mitochondria in a time-dependent manner, which occurs at Oh after cells were treated with43℃and peaked at6h. P53mitochondrial translocation was further detected by immunofuorescence staining and laser scanning confocal microscope. These findings thus suggest that intense heat stress induce rapid mitochondrial translocation of p53, which may activate the apoptotic program.4.The imbalance of calcium homeostasis activite mitochondrial permeability transition pore (MPTP)opening promotes mitochondrial apoptosis pathwaysFree cytosolic calcium were mark using the cell-permeable calcium-sensitive fluorescent dye Fluo-3/AM. Free cytosolic calcium levels was analyzed by flow cytometry. Ca2+distribution in cell was analyzed by laser scanning confocal microscope, stained with Ca2+(green), MitoTracker (red). After heat stress,[Ca2+]c increased at Oh and peak at1h after heat stress, then decreased gradually. Ca2+distribution in cell was analyzed by laser scanning confocal microscope for control,1h and6h after heat stress, stained with Ca2+(green) and MitoTracker (red) overlap at6h after heat stress, image show fluorescent Orange. The activity of MPTP was evaluated by the uptake of amitochondrial membrane permeability fluorescence dye calcein. Calpain activity was measured using Ac-LLY-AFC as the substrate provided in the calpain activity assay kit. MPTP was activated at1h after heat stress, mitochondria fluorescence intensity decreased gradually a time-dependent manner (F:40.293, P:0.001). while calpain activity not show. Futher study found that BAPTA-AM completely inhibited MPTP activation at1h after heat stress.HUVEC cells were pretreated with CsA followed by treated with intense heat stress (43℃) for2h.we found that CsA significantly inhibited cytochrome c release from mitochondria, loss of⊿Ψm and caspase-9activity (caspase-9F:4.958, P:0.008;⊿Ψm F:3.668, P:0.021)5.Bax Conformational Change and Mitochondrial Translocation mediated mitochondrial apoptotic pathwayMitochondria and cytoplasm proteins were extracted from cells treated with heat stress(0,1,3,6,9h) or control, respectively. Western blot measure Bax protein distribution in cell. We transfected pEGFP-C3-Bax into HUVEC cells, and the GFP-Bax distribution in cell after heat stress were measured by laser scanning confocal microscope. Bax protein in cytoplasm transfer to mitochondria at3,6and9h after heat stress, stained with GFP-Bax (green) and MitoTracker (red) overlap at6h after heat stress, image show fluorescent Orange, conformation-specific Bax was immunoprecipitated from whole-cell lysates using the6A7antibody and subjected to Western blot analysis using a polyclonal anti-Bax antibody, we found that intense heat stress resulted in a marked increase in Bax that had undergone a conformational change at3,6,9h after heat stress. HUVEC cells were transfected with Bax-siRNA to lower Bax protein expression. The Bax-siRNA significantly inhibited cytochrome c release from mitochondria, loss of⊿Ψm and caspase-9activity (caspase-9F:11.810, P:0.008;⊿Ψm F:7.878, P:0.021)6.Both p53mitochondrial translocation and MPTP activation mediated Bax Conformational Change and Mitochondrial TranslocationHUVEC cells were pretreated with PFT and CsA, respectiveyy, then treated with intense heat stress (43℃) for2h followed by further incubated for6h. Mitochondria and cytoplasm proteins were extracted from cells treated with heat stress or control, respectively. Western blot measure Bax protein distribution in cell. Immunoprecipitated from whole-cell lysates using the6A7antibody were measured by Western blot. Results suggest that both PFT and CsA significantly inhibited Bax conformational change and mitochondrial translocation.7. ROS generation via heat stress mediated p53mitochondrial translocation and imbalance of calcium homeostasis, as well as activation of mitochondrial apoptotic pathwayROS(O2,H2O2and NO) were measured using fluorescence probe DHE,DHR and DAF-FMDA. Heat stress induce rise of two kinds of free radicals:O2and H2O2(O2-F:225.788, P:0.008; H2O2F:256.770, P:0.021).But02-significantly increase at Oh after heat stress, while H2O2significantly increase at0.5h after heat stress, both free radicals increase in a time-dependent manner.HUVEC cells were pretreated with MnTBAP,PFT, BAPTA-AM, respectively, then treated with intense heat stress (43℃) for2h followed by further incubated for0/6h. We found that MnTBAP Completely suppressed p53mitochondrial translocation and imbalance of calcium homeostasis at Oh after heat stress.In addition, MnTBAP also significantly inhibited cells apotosis induce by heat stress, Bax conformational change and mitochondrial translocation, cytochrome c release, loss of⊿Ψm and caspase-9activity (caspase-9F:6.386, P:0.003;⊿Ψm F:6.827, P:0.002). While both PFT and BAPTA-AM cannot change ROS generation induce by heat stress.CONCLUSION1. Intense heat stress induces mitochondrial apoptotic pathway,and triggers the UPR to protect cells against ER stress, which active three UPR transducer pathways: ERK-eIF2a-ATF4,IRE1-XBP-1S and ATF6. Their activation receded at later stage, but heat stress not induces ER apoptosis pathway. 2. p53play an important role in intense heat stress induced apoptosis in HUVEC cells, which promotes mitochondrial apoptosis pathways through transcription-dependent.3. The imbalance of calcium homeostasis is also play an important role in intense heat stress induced apoptosis in HUVEC cells, which mediated mitochondrial apoptosis pathways through activating mitochondrial permeability transition pore (MPTP)opening.4.Bax is the central link during intense heat stress induced cell apoptosis, both p53mitochondrial translocation and imbalance of calcium homeostasis through activating bax conformational change and mitochondrial translocation to triggers mitochondrial apoptotic pathway.5. ROS generation via intense heat stress play the upstream signal molecule, which triggers both p53mitochondrial translocation and imbalance of calcium homeostasis followed by mediated the mitochondrial apoptotic pathway.