Human umbilical vein endothelial cells (HUVECs) were obtained from Cell Applications (San Diego, CA, USA) and Taiwan Medical Cell and Bioresource Collection and Research Center (BCRC, Taiwan). Cells at passages 3-5 were used for the experiments with ALDH2-silencing RNA (siALDH2, Santa Cruz, sc-60147) and Alda-1 (Adooq Bioscience, A15805, 20 mM in DMSO as stock solution, 1:1,000 dilution (20μM) for the in vitro experiments). According to previous published literatures and our own experiences (24–26, 38), we believe that DMSO in such concentration would not exert obvious toxic or protective effects on endothelial cells. HUVECs were cultured in M200 medium supplemented with endothelial growth factor (Gibco, Medium 200 and LSGS) and maintained in a humidified atmosphere at 37°C and 5% CO₂. The control cells were maintained in an incubator at 37°C. For heat stress induction, cells were subjected to 42°C for 2 h and then at 37°C overnight, as previously described (11, 15).

Protein lysates from the cells and lung tissues were subjected to SDS-PAGE followed by electroblotting onto PVDF membranes. The membranes were probed with monoclonal antibodies against ALDH2 (Abcam, ab108306), cytochrome c (Santa Cruz, sc-7159), caspase 3 (CST, #9662), 4-HNE (Abcam, ab46545), p-p38 (CST, #9215), p38 (CST, #9212), p-p65 (CST, #3033), p65 (CST, #3034), NOX1 (GeneTex, GTX103888), NOX4 (GeneTex, GTX121929) and GAPDH (Santa Cruz, sc-32233). Bands were visualized by chemiluminescence detection reagents, and densitometric analysis was conducted with imaging processing software (Multi Gauge, Fujifilm). These data are expressed as the fold changes relative to the controls.

ALDH2 activity was measured using an ALDH2 activity assay kit according to the manufacturer’s protocol (Abcam, ab115348, Cambridge, UK). In brief, the activity was estimated by measuring the conversion of oxidized nicotinamide adenine dinucleotide (NAD⁺) to reduced nicotinamide adenine dinucleotide (NADH) at an absorbance of 450 nm every 5 minutes for 2 hours period in the lung tissues and every 30 minutes for a 6 hours period (39).

ROS measurement was performed according to the manufacturer’s recommendations (OxiSelecte in vitro ROS/reactive nitrogen species (RNS) assay kit, Green Fluorescence; Catalog #STA-347, Cell Biolabs, Inc., San Diego, CA, USA). This in vitro assay measured total ROS/RNS free radical activity. Unknown ROS or RNS samples or standards were added to the wells with a catalyst that helps accelerate the oxidative reaction. Samples were measured fluorometrically against hydrogen peroxide. The free radical content in the samples was determined by comparison with a hydrogen peroxide standard curve. In brief, the cell lysates were stained with 2’,7’-dichlorofluorescein diacetate (DCFH-DA), which is oxidized by ROS to form fluorescent 2’,7’-dichlorofluorescein and were measured at an excitation wavelength of 488 nm and an emission wavelength of 535 nm. In addition, the dihydroethidium (DHE) method was also used to detect superoxide production at an excitation wavelength of 518 nm and an emission wavelength of 606 nm. The samples were loaded onto black 96-well plates and incubated for 30 min at 37°C, and the relative fluorescence units (RFUs) were determined by a fluorescence microplate reader (BMG Labtech, Ortenberg, Germany).

Knock-in mice on a C57BL/6J background with an inactivating point mutation in ALDH2 (ALDH2*2) were generated by homologous recombination, as previously described (34). There were no significant phenotypic changes in ALDH2*2 KI mice. Homozygous ALDH2*2 KI mice were used for the experiments.

C57BL/6J mice and homozygous ALDH2*2 KI mice with a C57BL/6J background were used for the animal experiments. The mice were directly exposed to whole-body heating (WBH) at 42°C for 1 h at 80% relative humidity using a temperature-controlled environmental chamber from room temperature and then returned to room temperature for 6-hour recovery period. Rectal temperature was measured using a copper-constantan thermocouple probe inserted into the rectum and connected to a thermometer. After the 1-h heating period, the mice were returned to their home cages and given food and water ad libitum (40). These mice were treated with either vehicle control (20% DMSO and 20% PEG 400 in 100μl PBS) or Alda-1 [16 mg/kg in 100 μl in PBS with 20% DMSO and 20% PEG 400 (Sigma, 06855)] intraperitoneally 30 minutes prior to WBH as previously described with some modification (34, 41). Previous literatures had showed that there were no obvious toxic or protective effects of this DMSO preparation on animal experiments (34, 42). Mice were considered adequately anesthetized when no attempt to withdraw the limb after pressure was observed. At the end of the study, the mice were euthanized by exsanguination under anesthesia. Bronchioalveolar lavage fluid (BALF) was collected at the end of the experiment by slowly irrigating the right lung with two separate 0.7-ml aliquots of PBS, of which 1.2 ml could be retrieved consistently. To avoid overdistention, the pressure should be kept less than 20 mmH₂O. All experimental protocols and procedures were approved by the Institutional Animal Care Committee of the National Defense Medical Center (Taipei, Taiwan) (43).

Lungs were collected after the mice were sacrificed following WBH. The wet weights of the organs were measured, and their dry weights were determined after the tissues were fully dried in an oven at 105°C. The water content was calculated as a percentage according to the following formula: 100×(wet weight-dry weight)/wet weight. One BALF aliquot was used immediately to measure the total cell counts. Erythrocytes were lysed using erythrocyte lysis buffer (Sigma, 1814389001), and the BALF was centrifuged at 400 g for 5 min and the supernatant was discarded. The pelleted cells were resuspended in 1.0 ml of PBS for the total leukocyte count by using a hemocytometer as previously described (44). The protein concentration in the supernatant was determined using bicinchoninic acid (BCA) method (Pierce, Rockford, IL, USA).

Lung injury was evaluated by histological analysis as described previously. In brief, lung tissue was fixed in 10% formalin solution for 24 h and stained with hematoxylin and eosin (H&E). Lung injury was scored based on (1) the infiltration or accumulation of neutrophils in the airspace or vessel wall and on (2) the thickness of the alveolar wall. These two observations were scored from 0 (normal) to 5 (most severe injury or greatest thickness, respectively) (45).

Cell proliferation was analyzed by the MTT assay (Sigma, #11465007001) in accordance with the manufacturer’s protocols. Briefly, 5,000 cells/well were grown in 96-well plates, exposed to heat stress at 42°C for 2 h and recovered at 37°C overnight. Then, the cells were incubated with MTT medium in a 5% CO₂ incubator at 37°C. After 4 h, the absorbance was measured at 570 nm using a Clariostar microplate reader (BMG Labtech, Ortenberg, Germany).

Cell apoptosis was evaluated by a TUNEL assay using a fluorescein direct in situ apoptosis detection kit (Millipore, S7110) according to the manufacturer’s recommended protocol. Apoptosis was determined as the percentage of positive cells per 1,000 DAPI-stained nuclei, and the cells were visualized under a fluorescence microscope (Nikon Eclipse 50i) at a magnification of 100×.

Cellular aging was assessed with a senescence cell staining kit according to the manufacturer’s instructions (Sigma, CS0030). Cultured HUVECs were fixed and then incubated with fresh X-gal staining solution (1 mg/ml, 5 mmol/l potassium ferrocyanide, 5 mmol/l potassium ferricyanide, and 2 mmol/l MgCl₂; pH 6). After the cells were stained, the numbers of blue-stained and total cells were determined, and the percentage of β-galactosidase-positive cells was calculated.

The mitochondrial membrane potential (ΔΨm) has been used as a parameter of mitochondrial function (46). To assess mitochondrial function in HUVECs after HS, JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’- tetraethylbenzimidazolcarbocyanine iodide) staining (BD Biosciences, 551302) was performed and assessed by fluorescence microscopy (Nikon Eclipse 50i) at a magnification of 200×. The images represent the merging of red and green channels. In addition, the fluorescence intensity of the cells was measured by a fluorescence microplate reader (BMG Labtech, Ortenberg, Germany). The data are expressed as the ratio of red fluorescence to green fluorescence intensity.

The serum levels of creatine kinase (CK), aspartate transferase (AST), and blood urea nitrogen (BUN) were measured by a FUJI Dri-chem slide on a FUJIDRI-CHEM 4000i instrument.

All experiments were performed independently at least 3 times, and all continuous variables are presented as the mean ± standard deviation (SD). The F test for equal variance was performed before the differences among groups were analyzed. Comparisons between two groups were analyzed using Student’s t test. For multiple groups, the data were analyzed using one-way ANOVA. For post hoc analysis, the Tukey test was used to correct for multiple comparisons, and the Fisher Least Significant Difference test was used for planned comparisons. Target protein expression measured by immunoblotting was analyzed by densitometry and is expressed as percent changes relative to an internal control or as the phosphorylated protein level relative to the total protein expression. Statistical significance was defined as a P value less than 0.05. Analyses were performed using a statistical software package (SPSS version 16.0 for Windows; SPS, Inc; Chicago, IL, USA) and GraphPad software.

There were no significant differences in body temperature between wild-type (WT) and ALDH2*2 KI mice after WBH ( Supplementary Figure 1 ). Compared to WT mice, ALDH2*2 KI mice were more vulnerable to WBH with increased mortality rates ( Figure 1A ). ALDH2*2 mice were susceptible to WBH-induced ALI, there was a significant increase in the wet/dry ratio of lung tissue in WBH-induced mice ( Figure 1B ). WBH significantly induced pathological fluid accumulation and inflammatory cell infiltration in the lung ( Figure 1C ). Increased inflammatory cells, protein levels and ROS production were observed in the BALF of ALDH2*2 KI mice subjected to WBH compared with WT mice ( Figures 1D –F ). In lung homogenates, the ROS production was increased in ALDH2*2 KI mice subjected to WBH compared with that in WT mice ( Figure 1G ). ALDH2 activity was decreased in the livers of ALDH2*2 KI mice compared with WT mice (Figure 1H). WBH significantly induced the accumulation of 4-HNE, NOX1, the phosphorylated-p65:p65 ratio and the phosphorylated-p38:p38 ratio ( Figure 1I ). There were no significant differences in NOX4 expression between the groups. In addition, there were significantly elevated serum levels of AST, CK and BUN in ALDH2*2 KI mice subjected to WBH compared with WT mice ( Supplementary Figure 2 ). Taken together, these results indicate that ALDH2*2 KI mice are susceptible to WBH-induced ALI.

To further confirm the effect of ALDH2 on heat stress in vitro, silencing ALDH2 was used in HUVECs with or without heat stress. As expected, the protein and activity of ALDH2 were decreased by silencing ALDH2 ( Figures 2A , B ). There was increased ROS accumulation in HUVECs subjected to 42°C for 2 h ( Figures 2C , D ). Silencing ALDH2 reduced the viability and exaggerated apoptosis and senescence of HUVECs subjected to heat stress ( Figures 2E –G ). Consistent with the results of previous studies (5, 47), heat stress induced mitochondrial dysfunction and apoptosis. We found silencing ALDH2 attenuated the level of ΔΨm ( Figure 2H ) and increased the expression of cytochrome c and cleaved caspase 3 relative to the HS in HUVECs ( Figure 2I ). Meanwhile, the phosphorylated-p65:p65 ratio, the phosphorylated-p38:p38 ratio, the expression of NOX1 and the toxic 4-HNE were accumulated in the silencing ALDH2 subjected to HS in HUVECs ( Figure 2I ). NOX4 expression was not significantly different between the group. These results suggest that reduced ALDH2 exacerbates heat stress induced NF-kB inflammatory pathways, ROS production, mitochondrial dysfunction and reduces the viability of HUVECs in vitro.

To understand the effect of ALDH2 activation on HS, Alda-1 was pretreated in HS induced HUVECs. Consistent with previous studies (29, 31, 48), Alda-1 (20μM) significantly augmented ALDH2 activity ( Figure 3A ) and reduced heat stress-induced ROS accumulation ( Figures 3B, C ). Alda-1 ameliorated the heat stress-induced cell death ( Figure 3D ) and reduced heat stress-induced apoptosis ( Figure 3E ) and senescence ( Figure 3F ) in HUVECs. Alda-1 reversed the heat stress-reduced ΔΨm ( Figure 3G ). Alda-1 attenuated the heat stress-induced phosphorylated-p65:p65 ratio, phosphorylated-p38:p38 ratio, cytochrome c, cleaved caspase 3, NOX1 and 4-HNE accumulation ( Figure 3H ). There were no significant differences regarding NOX4 expression. These results suggest that activation of ALDH2 by Alda-1 reduces heat stress induced mitochondrial dysfunction and ROS accumulation of HUVECs in vitro.

We then tested the protective effects of Alda-1 on WBH-induced ALI in vivo. Pretreatment with Alda-1 significantly increased HS-related survival rates by 25% ( Figure 4A ). Alda-1 ameliorated WBH-induced ALI, there was a significant decrease in wet/dry ratio of lung tissue in mice that received WBH exposure with Alda-1 pretreatment ( Figure 4B ). In addition, Alda-1 significantly reduced pathological fluid accumulation and inflammatory cell infiltration in the lung ( Figure 4C ). There were decreased inflammatory cell protein levels and ROS production in the BALF of Alda-1-pretreated mice subjected to WBH ( Figures 4D –F ). In lung homogenates, mice subjected to WBH increased ROS accumulation by 2.8-fold relative to the control, whereas pretreatment with Alda-1 reduced the level by 2.9-fold ( Figure 4G ). As expected, Alda-1-treated mice had significantly elevated ALDH2 activity ( Figure 4H ) in liver homogenates. Alda-1 significantly decreased the WBH-induced accumulation of 4-HNE, NOX1 expression, the phosphorylated-p65:p65 ratio, the phosphorylated-p38:p38 ratio, cytochrome c expression and cleaved caspase 3 expression ( Figure 4I ). NOX4 expression was not significantly different between the groups. Alda-1 attenuated HS-induced elevations in serum levels of AST, CK, and BUN ( Supplementary Figure 2 ). These results suggest that pretreatment with Alda-1 ameliorates WBH-induced ALI in vivo through reduced activation of NF-kB and apoptotic pathways and ROS accumulation.

We are aware that the development of HS could be multifactorial in nature, including other cell types, mediators and pathways. Infiltrated immune cells in BALF could be determined to further elucidate the roles of ALDH2 on HS. The roles of ALDH2 in other inflammatory cells as well HS-induced changes in pulmonary hemodynamics, lung endothelial permeability and the alveolar fluid resorption should be further investigated. Heat shock response systems, including the heat shock factor-1 (HSF-1) and heat shock protein (HSP) stress systems, provide protection against thermal insult by regulating the transcription of several HSPs and promoting chaperone activities to alleviate proteotoxic stresses in eukaryotic cells. The interplay among HSF-1, HSPs and ALDH2 should be further explored. HSF-1 can upregulate the expression of ALDH2 via protein kinase C (82). 4-HNE targets and impairs the function of HSP70 and the endoplasmic reticulum homolog of HSP70, glucose-regulated protein 78 (83, 84).