title: Heat stress induces apoptosis through a Ca²⁺-mediated mitochondrial apoptotic pathway in human umbilical vein endothelial cells.
abstract: BACKGROUND
  Heat stress can be acutely cytotoxic, and heat stress-induced apoptosis is a prominent pathological feature of heat-related illnesses, although the precise mechanisms by which heat stress triggers apoptosis are poorly defined.
  
  
  METHODS
  The percentages of viability and cell death were assessed by WST-1 and LDH release assays. Apoptosis was assayed by DNA fragmentation and caspase activity. Expression of cleaved PARP, Apaf-1, phospho-PERK, Phospho-eIF2a, ATF4, XBP-1s, ATF6, GRP78, phospho-IP3R, RYR and SERCA was estimated by Western blot. The effect of calcium overload was determined using flow cytometric analysis with the fluorescent probe Fluo-3/AM. The generation of ROS (O2-, H2O2, NO) was labeled by confocal laser scanning microscopy images of fluorescently and flow cytometry.
  
  
  RESULTS
  In this study, we found that heat stress in HUVEC cells activated initiators of three major unfolded protein response (UPR) signaling transduction pathways: PERK-eIF2a-ATF4, IRE1-XBP-1S and ATF6 to protect against ER stress, although activation declined over time following cessation of heat stress. Furthermore, we show that intense heat stress may induce apoptosis in HUVEC cells through the calcium-mediated mitochondrial apoptotic pathway, as indicated by elevation of cytoplasmic Ca2+, expression of Apaf-1, activation of caspase-9 and caspase-3, PARP cleavage, and ultimately nucleosomal DNA fragmentation; Reactive oxygen species (ROS) appear to act upstream in this process. In addition, we provide evidence that IP3R upregulation may promote influx of Ca2+ into the cytoplasm after heat stress.
  
  
  CONCLUSIONS
  Our findings describe a novel mechanism for heat stress-induced apoptosis in HUVEC cells: following elevation of cytoplasm Ca2+, activation of the mitochondrial apoptotic pathway via the IP3R upregulation, with ROS acting as an upstream regulator of the process.
authors.affiliation: Department of intensive care unit, Guangzhou General Hospital of Guangzhou Military Command, Key Laboratory of Tropical Zone Trauma Care and Tissue Repair of PLA, Guangzhou, P.R. China, and Southern Medical University, Guangzhou, P.R. China.
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of epilepsy surgery, Guangdong Sanjiu Brain Hospital, Guangzhou, P.R. China.
authors.forename: Hongping
authors.initials: H
authors.lastname: Tan
authors.affiliation: Department of intensive care unit, The Third Affiliated Hospital of Southern Medical University, Guangzhou, P.R. China.
authors.forename: Zhengtao
authors.initials: Z
authors.lastname: Gu
authors.affiliation: Department of intensive care unit, Guangzhou General Hospital of Guangzhou Military Command, Key Laboratory of Tropical Zone Trauma Care and Tissue Repair of PLA, Guangzhou, P.R. China.
authors.forename: Zhifeng
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Department of intensive care unit, Guangzhou General Hospital of Guangzhou Military Command, Key Laboratory of Tropical Zone Trauma Care and Tissue Repair of PLA, Guangzhou, P.R. China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Geng
authors.affiliation: Department of intensive care unit, Guangzhou General Hospital of Guangzhou Military Command, Key Laboratory of Tropical Zone Trauma Care and Tissue Repair of PLA, Guangzhou, P.R. China.
authors.forename: Yunsong
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of intensive care unit, Guangzhou General Hospital of Guangzhou Military Command, Key Laboratory of Tropical Zone Trauma Care and Tissue Repair of PLA, Guangzhou, P.R. China.
authors.forename: Huasheng
authors.initials: H
authors.lastname: Tong
authors.affiliation: Department of intensive care unit, Guangzhou General Hospital of Guangzhou Military Command, Key Laboratory of Tropical Zone Trauma Care and Tissue Repair of PLA, Guangzhou, P.R. China.
authors.forename: Youqing
authors.initials: Y
authors.lastname: Tang
authors.affiliation: Department of intensive care unit, Guangzhou General Hospital of Guangzhou Military Command, Key Laboratory of Tropical Zone Trauma Care and Tissue Repair of PLA, Guangzhou, P.R. China.
authors.forename: Junmin
authors.initials: J
authors.lastname: Qiu
authors.affiliation: Department of intensive care unit, Guangzhou General Hospital of Guangzhou Military Command, Key Laboratory of Tropical Zone Trauma Care and Tissue Repair of PLA, Guangzhou, P.R. China.
authors.forename: Lei
authors.initials: L
authors.lastname: Su
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000502:physiology; D018869:Heat-Shock Response / Q000502:physiology*; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D017382:Reactive Oxygen Species; D002118:Calcium