| BACKGROUND AND AIMSEnvironmental heat exposure can result in heat-related illnesses, and in extreme cases, can lead to death. The severity ofheat-related illnesses ranges widely, from mild conditions such as heat exhaustion and heat cramps to the serious, sometimes life-threatening condition of heat stroke. In the summer of 2003,the heat wave affecting Europe resulted in an unprecedented 45,000 excessive deaths, one-third of which were due to heat stroke. Given the increasing intensity and frequency of heat waves as well as increasing evidence of global warming, the morbidity of heatstroke also is also likely to increase.However, the pathogenesis of cell death and tissue injury in heatstroke is not well understood, which may clarify the high disability and mortality rate as no specific mechanisms may be targeted for treatment. Therefore, investigate the pathogenesis of multiple organ dysfunction syndrome of heat stroke will play an important in reducing the high mortality and morbidity rate as well as providing experimental evidence and new ideas for the treatment of severe heat stroke.Heat is the most fundamental factor in the pathogenesis of heat stroke and have a major impact on cells function and structure.At the molecular level, exposure to high ambient temperature can cause protein denaturation; At the cellular level,exposure to high ambient temperature can damage the cell membrane, cytoskeleton and nuclei,and ultimately lead to apoptosis or necrosis. Both in vitro and in vivo studies have demonstrated that heat stress can directly induce cell death and tissue injury. It has been reported that exposure to extreme temperatures (49℃-50℃) compromises cellular structures and function, leading to rapid necrotic cell death in less than 5 minutes. In contrast, cell death in animal models subjected to moderate heat stress proceeds by accelerated apoptosis. Thus, apoptosis represents another potential mechanism of cell death in response to heat stroke. Recent molecular studies indicate a critical role for heat stress in signal transduction pathways involved in cell death; for example, induction of the apoptotic cascade through activity of apoptosis-related proteins, including Caspases; Tissue damage by reactive oxygen species (ROS) as a result of intense heat stress is also of great concern, as ROS inhibit cell proliferation and activate apoptosis through induction of DNA damage. In animal level, these studies mostly limited to the appearance of apoptosis, however, the specific start factors, linkage between the various signaling pathways, influence factors and injury mechanisms still need further study. In addition, in vitro experimental studies mainly focused on the tumor cell lines, but research between normal cells and heat stroke also rarely. With increase in the severity and frequency of heat waves with an associated rise in mortality, therefore, investigate the relationship between tissue damage and heat have profound significance.As an important organ of the human body, vascular endothelial cell (VEC) play a vital role in tissue damage during heat stress, which is not only maintaining the integrity of the vascular permeability barrier but also have major affect on the impaction of cells structure and function during acute-phase response to heat stress. Due to a series of acute and complex pathophysiological and changes in the body’s microcirculation during heat stroke, it will cause the damage of VEC and then induce or aggravate Multiple organ dysfunction syndrome (MODS).Meanwhile, it has shown that VEC is the major reaction cell and one of the most common morphological and functional changes cell during heat stroke. And it is also the earliest occurrence in damage. Both in vitro and in vivo studies have demonstrated that elevated temperatures can result in direct injury to vascular endothelium, indicating that targeted endothelial cell damage may be the underlying cause of prominent heatstroke features. Furthermore, it has been observed that acute heat stress-induced endothelial cell damage results in apoptosis, suggesting that apoptotic death of endothelial cells might be a critical event in the pathogenesis of heat stroke. Our preliminary clinical trials have found that patients with severe heat stroke appear serious VEC injury. Our recent work showed that intense heat stress induce intracellular Ca2+ homeostasis, which mediated rapid mitochondrial translocation of p53 may activate the mitochondrial apoptotic pathway in HUVEC cells. However, the underlying mechanisms of heat stress-induced intracellular Ca2+ homeostasis and endothelial cell apoptosis need further research, which will lay the foundation for revealing pathological processes of VEC injury mediated by heat stroke and provide a theoretical basis for clinical prevention and treatment of heat stroke.METHODS AND RESULTS1. heat stress can be directly toxic on HUVEC cell, induce apoptosis and DNA fragmentationTo investigate changes in viability andcytotoxicity following heat stress in HUVEC cells exposed to elevated temperatures, both the WST-1 and LDH assays were employed. HUVEC cells were maintained in standard culture media for 48h at 37℃ prior to a temperature shift to 39℃,41℃,43℃, or 45℃ for the duration of heat stress treatments. Culture media were replaced with fresh media and cells were further incubated at 37℃ for 6 h. Cell viability declined drastically concomitant with a significant increase in cytotoxicity after cells were cultured at elevated temperatures, as indicated by the temperature-dependent reduction in formazan formation and increase in LDH activity, respectively.HUVEC cells apoptosis was analyzed by Hoechst33258 fluorescent nuclear staining:Compare with the control group, HUVEC cells apoptosis was no significant difference in 39℃.With increasing of heat stress temperature(41℃-43℃),there were more positive nuclei stained. Furthermore, using the inhibitor of Caspase-3, we found that it can inhibit apoptosis induced by heat stress significantly, showing the bright blue fluorescence intensity within apoptotic cells was significantly reduced comparing with before.In order to clarify the impact on apoptosis in HUVEC cells and explore the exist mechanism during heat stress, we chose 43℃as a research temperature(heat stress 2h), observing apoptosis in different time points after rewarming (0h,lh,3h,6h,9h). Using the fluorogenic substrate Ac-DEVD-AMC, we observed Caspase-3 activity at 3h,6h, and 9h after a 2hr period of intense heat stress (43℃), which was consistent with the time period during which PARP cleavage occurred. The classical apoptotic DNA laddering pattern was observed between 3hr and 9h following a 2hr period of intense heat stress (43℃), suggesting that heat stress-induced cytotoxicity occurs through activation of apoptosis.2.Intense heat stress lead to mitochondrial damage, induces apoptosis by triggering the mitochondrial pathway in HUVEC cellsIn order to clarify whether heat stress impact on HUVEC cell mitochondria, we observed cell ultrastructure by transmission electron microscopy, suggesting that mitochondrial structure was normal in the control group (37℃); Whereas, in the gtoup of 37℃ heat stress, mitochondrial swelling,cristae membrane bilayer structure disappeared and mitochondrial matrix homogenized(mitochondria lysates, matrix and chromatin mixed together to form a homogeneous dense clumps of matter), showing that heat stress induced mitochondrial injury.In order to identify the mechanistic pathways involved in heat stress-induced apoptosis, we examined alterations in the activity of initiator Caspases (Caspase-4,-8,-9), the expression of GADD153 and the main effector Caspase (Caspase-3). There was no apparent increase in GADD153 expression, Caspase-4 andCaspase-8 activity in cells undergoing heat stress, whereas activation of Caspase-9 and Caspase-3 was detected 3h after heat stress. Caspase-9 and-3 activity increased by more than 2-fold and 3-fold, respectively. Also, Apaf-1 expression was elevated and PARP cleavage was observed 3h after heat stress, consistent with increased Caspase-9 and -3 activity, suggesting that heat stress might induce the mitochondrial apoptotic pathway. We also investigate that Bcl-xl overexpression significantly decreased heat stress-induced apoptosis, further supporting the hypothesis that heat stress-induced apoptosis of HUVEC cells was mediated by the mitochondrial pathway.3. Calcium elevation induced by intense heat stress activates mitochondrial apoptosis pathways in HUVEC cellsTo investigate whether heat stress-induced apoptosis is associated with increased intracellular calcium, we determined the effect of Ca2+ on heat stress-induced apoptosis using flow cytometric analysis of cells were stained with the fluorescent probe Fluo-3/AM. Heat stress caused an increase in the level of intracellular Ca2+within 2hr, which further increased 2hr after the cessation of heat stress, at which time the intracellular calcium concentration was significantly higher than control (P< 0.05 or<0.01). To determine whether increased intracellular calcium induced by heat stress is involved in regulating the activation of proapoptotic proteins through the intrinsic pathway, we measured enzymatic activity of Caspase-9, Caspase-3 and expression of Apaf-1 and cleaved PARP.As shown,the activities of Caspase-9, Caspase-3 and the expression of Apaf-1 and cleaved PARP were clearly elevated 3h after heat stress and continued to increase up to 9h. Pretreatment with the cell-permeant calcium chelator BAPTA-AM significantly decreased apoptosis 6h after intense heat stress.Taken together, these results indicate that intense heat stress induced a time-dependent increase in intracellular calcium elevation, which regulated activation of proapoptotic mediators through the mitochondrial-mediated apoptosis pathway.4. Intense heat stress induces the endoplasmic reticulum stress and IP3R phosphorylation mediated the imbalance of Ca2+ homeostasis in HUVEC cellsTo explore whether intense heat stress induces ER stress and activates the UPR, we investigated the ER stress-related proteins, including phospho-PERK, phospho-eIF2a, ATF4, and GRP78.As shown, GRP78 was activated at Oh and decreased gradually by 3h after cessation of heat stress; PERK phosphorylation was gradually inhibited immediately following a 2hr heat stress; eIF2a phosphorylation was inhibited in a time-dependent manner beginning 3hr after the cessation of heat stress. ATF4 expression was induced by 6hr after heat stress, and declined thereafter. To investigate which Ca2+ channels were relevant to ER stress induced by high temperatures, we evaluated expression of IP3R, RYR, and SERCA in HUVEC cells. As shown, IP3R phosphorylation was observed immediately following heat stress (0h), and increased significantly up to 9h. Since no obviousincrease in RYR or SERCA expression was observed following heat stress, we concluded that IP3R is primarily responsible for mediating the regulatory effects of heat stress on Ca2+ release. It has been reported that the effects of increased cytoplasmic Ca2+ can be counteracted using the selective IP3R antagonist xestospongin B (XeB). Pretreatment of HUVEC cells with 2.5μM XeB reduced the IP3R phosphorylation within 1hr of cessation of intense heat stress;XeB also reduced the release of Ca2+ by approximately 45%, consistent with IP3R mediating the effects of heat stress on Ca2+ release.Taken together, these results suggest that intense heat stress triggers the UPR to protect cells against ER stress, which activates UPR transducer pathway(GRP78,PERK-eIF2a-ATF4) and IP3R related Ca2+ channels mediated the imbalance of Ca2+ homeostasis in HUVEC cells.5. ROS involved in the elevation of calcium-mediated mitochondrial apoptotic pathway induced by intense heat stress in HUVEC cellsWe hypothesized that heat stress-induced apoptosis potentiates theaccumulation of ROS.To test this hypothesis, we monitored intracellular ROS production using the cell-permeant fluorescent dyes DHE, DHR and DAF-FMDA, whose fluorescence is enhanced under ROS (O2-, H2O2, NO) generating conditions. X/XO, H2O2 and SNP -treated HUVEC cells were used as positive controls. Heat stress induced production of both O2- and H2O2 reactive species; however, O2-noticeably increased immediately after heat stress (Oh), while H2O2 increased significantly 0.5h after heat stress; both free radical speciescontinued to increase with time thereafter. As shown, DHE and DHR exhibited similar increases in fluorescence intensity after heat stress. Thus, heat stress induced time-dependent increases in both O2- and H2O2, with the increase in O2- preceding the increase in H2O2; in contrast, NO generation did not change significantly. To explore the relationship between ROS generation and calcium levels, we pretreated cells with the O2" scavenger MnTBAP and the calcium chelator BAPTA-AM. As shown, increases in both O2- and cytoplasmic Ca2+ were completely inhibited by MnTBAP.MnTBAP pretreatment increased viability and reduced cytotoxicity following heat stress in HUVEC cells. The results confirm that while MnTBA also significantly decreased heat stress-induced apoptosis mediated by the mitochondrial pathway, O2- production was not inhibited by BAPTA-AM.Overall, these results indicate that ROS generated by intense heat stress acts as the upstream stimulus for elevation of calcium levels as well as downstream activation of the mitochondrial apoptotic pathway in HUVEC cells.CONCLUSIONOur findings describe a novel mechanism for heat stress-induced apoptosis in HUVEC cells:heat stress triggers the UPR to protect cells against ER stress, which activates UPR transducer pathway (GRP78,PERK-eIF2a-ATF4) and IP3R related Ca2+ channels mediated imbalance of Ca2+ homeostasis in HUVEC cells. Throughout the process, with explosive increasing of ROS (O2-) as a early event, triggering the imbalance of Ca2+ homeostasis as a intermediate links regulated Mitochondrial damage related apoptosis pathway. |
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