Figure 1.
Heat stress reduces cell viability and increased cytotoxicity in HUVEC cells.
Cells were exposed to the indicated temperature for 2 h, and were further incubated at 37°C for 6 h. The percentages of viability and cell death were assessed by WST-1 (a) and LDH release assays (b). Percent viability is expressed relative to control cells cultured at 37°C. The data shown represent the mean ±SD of at least three independent experiments, peformed in triplicate. *P<0.05, statistically significant relative to control.
Figure 2.
Intense heat stress induces apoptotic DNA fragmentation and activates caspase-3 and PARP in HUVEC cells.
Cells underwent intense heat stress (43°C) for 2 h, and were further incubated at 37°C for different times as indicated (0 h, 1 h, 3 h, 6 h, or 9 h). (a) DNA fragmentation using DNA electrophoresis and fluorescent staining. (b) Western blot analysis of cleaved PARP. (c) Enzymatic activity of caspase-3 was measured in cell lysates using the fluorogenic substrate Ac-DEVD-AMC. Each value is the mean ± SD of at least three separate experiments, *P<0.05, **P<0.01, compared with control group (37°C).
Figure 3.
Intense heat stress induces the UPR in HUVEC cells.
Cells were exposed to intense heat stress (43°C) for 2 h, and were further incubated at 37°C for 0 h, 1 h, 3 h, 6 h, or 9 h. (a) and (c) Expression of P-PERK, P-elF2a, ATF4, ATF6, XBP1-s, and GRP78 were determined by Western Blot. (b) RT-qPCR analysis of XBP-1s target gene expression (EDEM). (d) Quantification of western blots for PERK phosphorylation, eIF2a phosphorylation, ATF4, ATF6, XBP1-s and GRP78 after heat stress. Graphs represent mean±S.D. of at least three independent experiments. *P<0.05, compared with control group (37°C).
Figure 4.
Intense heat stress induces apoptosis by triggering the mitochondrial pathway in HUVEC cells.
Cells udnerwent intense heat stress (43°C) for 2 h, and were further incubated at 37°C for the indicated times (0 h, 1 h, 3 h, 6 h, or 9 h). (a) and (f) Expression of GADD153, Apaf-1 and cleaved PARP were determined by Western blot. (b–e) Enzymatic activity of caspase-9, -3, -8 and -4 were measured in cell lysates using the fluorogenic substrates Ac-LEHD-AFC, Ac-DEVD-AMC, Ac-ATAD-AFC and Ac-IETD-pNA, respectively, and was expressed relative to the control at 37°C. Data are shown as the mean ± SD of at least three independent experiments, *P<0.05, **P<0.01, compared with control group (37°C).
Figure 5.
Intense heat stress-induced apoptosis in HUVEC cells over-expressing Bcl-xl.
HUVEC cells stably overexpressing the anti-apoptotic protein Bcl-xl or vector control were cultured at 43°C for 2 h, and further incubated for 6 h. (a) Western blot analysis of bcl-2 protein expression (cropped) in transfected cells. β-actin served as an internal control. (b) and (d) Enzymatic activity of caspase-9, and -3 was measured in cell lysates using the fluorogenic substrates Ac-LEHD-AFC and Ac-DEVD-AMC, respectively, and was expressed relative to the control at 37°C. (c) Expression of Apaf-1 and cleaved PARP were determined by Western blot. (e) Nucleosomal DNA fragmentation was evaluated using DNA electrophoresis and fluorescent staining. Each value is the mean ± SD of at least three separate experiments, *P<0.05, compared with heat stress group (43°C).
Figure 6.
Calcium elevation induced by intense heat stress activates the mitochondrial apoptotic pathway.
(a) Cells underwent intense heat stress (43°C) for 2 h, and were further incubated at 37°C for the indicated times (0 h, 0.5 h, 1 h, 1.5 h, 2 h). The effect of heat stress on calcium overload was determined using flow cytometric analysis with the fluorescent probe Fluo-3/AM. (b) Cells underwent heat stress as described above, and were further incubated at 37°C for the indicated times (0 h, 1 h, 3 h, 6 h, or 9 h). (c) Cells were pretreated with 20 M BAPTA-AM, then exposed to intense heat stress (43°C) for 2 h, and incubated at 37°C for 6 h. Expression of Apaf-1 and cleaved PARP were determined by Western blot analysis. Enzymatic activity of caspase-9 and -3 was measured in cell lysates using the fluorogenic substrates Ac-LEHD-AFC and Ac-DEVD-AMC, respectively, and activity was expressed relative to the control at 37°C. DNA fragmentation using DNA electrophoresis and fluorescent staining. Each value represents the mean ± SD of at least three separate experiments, *P<0.05, **P<0.01, compared to control group (37°C), #P<0.05, compared to heat stress group (43°C).
Figure 7.
Effect of intense heat stress on the expression of Ca2+channel release related protein (IP3R).
(a) Cells were exposed to intense heat (43°C) for 2 h, and were further incubated at 37°C for 0 h, 1 h, 3 h, 6 h, or 9 h. Expression of phospho-IP3R, RYR, and SERCA were determined by Western blot. (b) and (c) Cells were treated with 2.5 µM Xestospongin B(XeB) prior to heat stress, and further incubated at 37°C for 1 h; phosphorylation of IP3R was determined by Western blot analysis. The effect of heat stress on calcium overload was determined by flow cytometric analysis of cells labeled with the fluorescent probe Fluo-3/AM. Each value represents the mean ± SD of at least three independent experiments; *P<0.05, compared to the heat stress group (43°C).
Figure 8.
ROS is involved in induction of the calcium-mediated mitochondrial apoptotic pathway induced by intense heat stress in HUVEC cells.
(a)–(b) Cells were exposed to intense heat (43°C) for 2 h, then incubated at 37°C for 0 h, 0.5 h, 1 h, 1.5 h, or 2 h. Cells were labeled with DHE (red fluorescence), DHR (green fluorescence), or DAF-FMDA (green fluorescence) for detection of superoxide, H2O2, or nitric oxide, respectively. X/XO (0.05 mM X+0.01 U XO), H2O2 (25 µM) and SNP (0.01 mol/L) were used as a positive control for O2−, H2O2 and NO, respectively. (a) Confocal laser scanning microscopy images of fluorescently labeled cells. Representative images of the best of three independent image acquisitions are shown. (b) Flow cytometry analysis heat stress-induced ROS (O2−, H2O2, NO). (c)–(d) Cells were pretreated with MnTBAP prior to 2 hr of heat stress (43°C), then further incubated at 37°C for 1 h. (c) Flow cytometric analysis of ROS generation (O2−). (d) The effect of heat stress on calcium overload was analyzed by flow cytometry using the fluorescent probe Fluo-3/AM. (e)–(h) Cells were pretreated with MnTBAP prior to heat stress (43°C) for 2 h, and further incubated at 37°C for 6 h. Cell death was assessed by the WST-1 (e) and LDH release assays (f). (g) Expression of Apaf-1 and cleaved PARP were determined by Western blot analysis. Enzymatic activity of caspase-9 and -3 was measured in cell lysates using the fluorogenic substrates Ac-LEHD-AFC and Ac-DEVD-AMC, respectively; caspase activity was expressed relative to the control at 37°C. (h) Nucleosomal DNA fragmentation using DNA electrophoresis and fluorescent staining. (i) Cells were pretreated with 20 M BAPTA-AM, exposed to intense heat (43°C) for 2 h, and incubated at 37°C for 1 h. Flow cytometric analysis of ROS (O2−) was performed as in (c). Each value represents the mean ± SD of at least three independent experiments; *P<0.05, compared with heat stress group (43°C).