Alda-1 attenuates hyperoxia-induced mitochondrial dysfunction in lung vascular endothelial cells.
Patil Sahebgowda Sidramagowda,Hernández-Cuervo Helena,Fukumoto Jutaro,Narala Venkata Ramireddy,Saji Smita,Borra Monica,Alleyn Matthew,Lin Muling,Soundararajan Ramani,Lockey Richard,Kolliputi Narasaiah,Galam Lakshmi
Acute lung injury (ALI) is a major cause of morbidity and mortality worldwide, especially in aged populations. Mitochondrial damage is one of the key features of ALI. Hyperoxia-induced lung injury model in mice has been widely used for ALI study because it features many ALI phenotypes including, but not limited to, mitochondrial and vascular endothelial cell damage. Recently, accumulating evidence has shown that mitochondrial aldehyde dehydrogenase 2 (ALDH2) has a protective effect against oxidative stress mediated cell damage in epithelial cells. However, it is not known whether ALDH2 protects against oxidative stress in vascular endothelial cells. In this current study, we attempted to find the capacity of Alda-1 [(N-(1,3benzodioxol-5-ylmethyl)-2,6- dichloro-benzamide), an ALDH2 activator] to protect against oxidative stress in human microvascular endothelial cells (HMVEC). HMVEC pretreated with Alda-1 prior to hyperoxic exposure vs non-treated controls showed i) lower 4-hydroxynonenal (4-HNE) levels, ii) significantly decreased expressions of Bax and Cytochrome C, iii) partially restored activity and expression of ALDH2 and iv) significantly improved mitochondrial membrane potential. These results suggest that ALDH2 protein in lung vascular endothelial cells is a promising therapeutic target for the treatment of ALI and that Alda-1 is a potential treatment option.
Bcl-2 overexpression in type II epithelial cells does not prevent hyperoxia-induced acute lung injury in mice.
Métrailler-Ruchonnet Isabelle,Pagano Alessandra,Carnesecchi Stéphanie,Khatib Karim,Herrera Pedro,Donati Yves,Bron Camille,Barazzone Constance
American journal of physiology. Lung cellular and molecular physiology
Bcl-2 is an anti-apoptotic molecule preventing oxidative stress damage and cell death. We have previously shown that Bcl-2 is able to prevent hyperoxia-induced cell death when overexpressed in a murine fibrosarcoma cell line L929. We hypothesized that its specific overexpression in pulmonary epithelial type II cells could prevent hyperoxia-induced lung injury by protecting the epithelial side of the alveolo-capillary barrier. In the present work, we first showed that in vitro Bcl-2 can rescue murine pulmonary epithelial cells (MLE12) from oxygen-induced cell apoptosis, as shown by analysis of LDH release, annexin V/propidium staining, and caspase-3 activity. We then generated transgenic mice overexpressing specifically Bcl-2 in lung epithelial type II cells under surfactant protein C (SP-C) promoter (Tg-Bcl-2) and exposed them to hyperoxia. Bcl-2 did not hinder hyperoxia-induced mitochondria and DNA oxidative damage of type II cell in vivo. Accordingly, lung damage was identical in both Tg-Bcl-2 and littermate mice strains, as measured by lung weight, bronchoalveolar lavage, and protein content. Nevertheless, we observed a significant lower number of TUNEL-positive cells in type II cells isolated from Tg-Bcl-2 mice exposed to hyperoxia compared with cells isolated from littermate mice. In summary, these results show that although Bcl-2 overexpression is able to prevent hyperoxia-induced cell death at single cell level in vitro and ex vivo, it is not sufficient to prevent cell death of parenchymal cells and to protect the lung from acute damage in mice.
Hyperoxia decreases glycolytic capacity, glycolytic reserve and oxidative phosphorylation in MLE-12 cells and inhibits complex I and II function, but not complex IV in isolated mouse lung mitochondria.
Das Kumuda C
High levels of oxygen (hyperoxia) are frequently used in critical care units and in conditions of respiratory insufficiencies in adults, as well as in infants. However, hyperoxia has been implicated in a number of pulmonary disorders including bronchopulmonary dysplasia (BPD) and adult respiratory distress syndrome (ARDS). Hyperoxia increases the generation of reactive oxygen species (ROS) in the mitochondria that could impair the function of the mitochondrial electron transport chain. We analyzed lung mitochondrial function in hyperoxia using the XF24 analyzer (extracellular flux) and optimized the assay for lung epithelial cells and mitochondria isolated from lungs of mice. Our data show that hyperoxia decreases basal oxygen consumption rate (OCR), spare respiratory capacity, maximal respiration and ATP turnover in MLE-12 cells. There was significant decrease in glycolytic capacity and glycolytic reserve in MLE-12 cells exposed to hyperoxia. Using mitochondria isolated from lungs of mice exposed to hyperoxia or normoxia we have shown that hyperoxia decreased the basal, state 3 and state3 μ (respiration in an uncoupled state) respirations. Further, using substrate or inhibitor of a specific complex we show that the OCR via complex I and II, but not complex IV was decreased, demonstrating that complexes I and II are specific targets of hyperoxia. Further, the activities of complex I (NADH dehydrogenase, NADH-DH) and complex II (succinate dehydrogenase, SDH) were decreased in hyperoxia, but the activity of complex IV (cytochrome oxidase, COX) remains unchanged. Taken together, our study show that hyperoxia impairs glycolytic and mitochondrial energy metabolism in in tact cells, as well as in lungs of mice by selectively inactivating components of electron transport system.
WISP-3/CCN6 inhibits apoptosis by regulating caspase pathway after hyperoxia in lung epithelial cells.
Wei Shuquan,Wang Kangwei,Zhao Zhuxiang,Huang Xiaomei,Tang Wanna,Zhao Ziwen
Cell death is a normal phenomenon in the course of biological development, moreover, which is also a prominent feature in lung exposed to hyperoxia. Severe hypoxia occurs in ALI/ARDS patients, who generally require high concentration oxygen therapy assisted by mechanical ventilation. Nevertheless, high oxygen can cause excessive reactive oxygen species (ROS), leading to apoptosis in lung epithelial cells, which has been reported in our previous study. Herein, the correlation between increments of ROS and CCN6 expression was negative in CCN6-mediated the mitochondria dependent, intrinsic apoptotic pathway. Our latest research explained that CCN6 can inhibit caspase-8 mediated extrinsic apoptotic pathway to protect cells from hyperoxia-induced apoptosis. As demonstrated by Western Blot Analysis, Caspase 8 cleavage and Caspase 3 cleavage in CCN6-depleted cells exceeded the control group treated with high oxygen (48 h). And deletion of CCN6 enhanced caspase-8 activation after hyperoxia shown by Flow Cytometry. Although, it is unclear how CCN6 participated in the regulation of apoptotic pathways, the future targeted therapy drugs inhibiting CCN6 may be useful in the treatment of ALI/ARDS.
Mouse lung development and NOX1 induction during hyperoxia are developmentally regulated and mitochondrial ROS dependent.
Datta Ankur,Kim Gina A,Taylor Joann M,Gugino Sylvia F,Farrow Kathryn N,Schumacker Paul T,Berkelhamer Sara K
American journal of physiology. Lung cellular and molecular physiology
Animal models demonstrate that exposure to supraphysiological oxygen during the neonatal period compromises both lung and pulmonary vascular development, resulting in a phenotype comparable to bronchopulmonary dysplasia (BPD). Our prior work in murine models identified postnatal maturation of antioxidant enzyme capacities as well as developmental regulation of mitochondrial oxidative stress in hyperoxia. We hypothesize that consequences of hyperoxia may also be developmentally regulated and mitochondrial reactive oxygen species (ROS) dependent. To determine whether age of exposure impacts the effect of hyperoxia, neonatal mice were placed in 75% oxygen for 72 h at either postnatal day 0 (early postnatal) or day 4 (late postnatal). Mice exposed to early, but not late, postnatal hyperoxia demonstrated decreased alveolarization and septation, increased muscularization of resistance pulmonary arteries, and right ventricular hypertrophy (RVH) compared with normoxic controls. Treatment with a mitochondria-specific antioxidant, (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mitoTEMPO), during early postnatal hyperoxia protected against compromised alveolarization and RVH. In addition, early, but not late, postnatal hyperoxia resulted in induction of NOX1 expression that was mitochondrial ROS dependent. Because early, but not late, exposure resulted in compromised lung and cardiovascular development, we conclude that the consequences of hyperoxia are developmentally regulated and decrease with age. Attenuated disease in mitoTEMPO-treated mice implicates mitochondrial ROS in the pathophysiology of neonatal hyperoxic lung injury, with potential for amplification of ROS signaling through NOX1 induction. Furthermore, it suggests a potential role for targeted antioxidant therapy in the prevention or treatment of BPD.
Hyperoxia Causes Mitochondrial Fragmentation in Pulmonary Endothelial Cells by Increasing Expression of Pro-Fission Proteins.
Ma Cui,Beyer Andreas M,Durand Matthew,Clough Anne V,Zhu Daling,Norwood Toro Laura,Terashvili Maia,Ebben Johnathan D,Hill R Blake,Audi Said H,Medhora Meetha,Jacobs Elizabeth R
Arteriosclerosis, thrombosis, and vascular biology
OBJECTIVE:We explored mechanisms that alter mitochondrial structure and function in pulmonary endothelial cells (PEC) function after hyperoxia. APPROACH AND RESULTS:Mitochondrial structures of PECs exposed to hyperoxia or normoxia were visualized and mitochondrial fragmentation quantified. Expression of pro-fission or fusion proteins or autophagy-related proteins were assessed by Western blot. Mitochondrial oxidative state was determined using mito-roGFP. Tetramethylrhodamine methyl ester estimated mitochondrial polarization in treatment groups. The role of mitochondrially derived reactive oxygen species in mt-fragmentation was investigated with mito-TEMPOL and mitochondrial DNA (mtDNA) damage studied by using ENDO III (mt-tat-endonuclease III), a protein that repairs mDNA damage. Drp-1 (dynamin-related protein 1) was overexpressed or silenced to test the role of this protein in cell survival or transwell resistance. Hyperoxia increased fragmentation of PEC mitochondria in a time-dependent manner through 48 hours of exposure. Hyperoxic PECs exhibited increased phosphorylation of Drp-1 (serine 616), decreases in Mfn1 (mitofusion protein 1), but increases in OPA-1 (optic atrophy 1). Pro-autophagy proteins p62 (LC3 adapter-binding protein SQSTM1/p62), PINK-1 (PTEN-induced putative kinase 1), and LC3B (microtubule-associated protein 1A/1B-light chain 3) were increased. Returning cells to normoxia for 24 hours reversed the increased mt-fragmentation and changes in expression of pro-fission proteins. Hyperoxia-induced changes in mitochondrial structure or cell survival were mitigated by antioxidants mito-TEMPOL, Drp-1 silencing, or inhibition or protection by the mitochondrial endonuclease ENDO III. Hyperoxia induced oxidation and mitochondrial depolarization and impaired transwell resistance. Decrease in resistance was mitigated by mito-TEMPOL or ENDO III and reproduced by overexpression of Drp-1. CONCLUSIONS:Because hyperoxia evoked mt-fragmentation, cell survival and transwell resistance are prevented by ENDO III and mito-TEMPOL and Drp-1 silencing, and these data link hyperoxia-induced mt-DNA damage, Drp-1 expression, mt-fragmentation, and PEC dysfunction.
Reactive oxygen and nitrogen species induce cell apoptosis via a mitochondria-dependent pathway in hyperoxia lung injury.
Zou Dongmei,Li Jing,Fan Qianqian,Zheng Xuemei,Deng Jian,Wang Shaohua
Journal of cellular biochemistry
Hyperoxia-induced lung injury limits the application of mechanical ventilation on rescuing the lives of premature infants and seriously ill and respiratory failure patients, and its mechanisms are not completely understood. In this article, we focused on the relationship between hyperoxia-induced lung injury and reactive oxygen species (ROS), reactive nitrogen species (RNS), mitochondria damage, as well as apoptosis in the pulmonary epithelial II cell line RLE-6TN. After exposure to hyperoxia, the cell viability was significantly decreased, accompanied by the increase in ROS, nitric oxide (NO), inflammatory cytokines, and cell death. Furthermore, hyperoxia triggered the loss of mitochondrial membrane potential (▵Ψm), thereby promoting cytochrome c to release from mitochondria to cytoplasm. Further studies conclusively showed that the Bax/Bcl-2 ratio was enlarged to activate the mitochondria-dependent apoptotic pathway after hyperoxia treatment. Intriguingly, the effects of hyperoxia on the level of ROS, NO and inflammation, mitochondrial damage, as well as cell death were reversed by free radical scavengers N-acetylcysteine and hemoglobin. In addition, a hyperoxia model of neonatal Sprague-Dawley (SD) rats presented the obvious characteristics of lung injury, such as a decrease in alveolar numbers, alveolar mass edema, and disorganized pulmonary structure. The effects of hyperoxia on ROS, RNS, inflammatory cytokines, and apoptosis-related proteins in lung injury tissues of neonatal SD rats were similar to that in RLE-6TN cells. In conclusion, mitochondria are a primary target of hyperoxia-induced free radical, whereas ROS and RNS are the key mediators of hyperoxia-induced cell apoptosis via the mitochondria-dependent pathway in RLE-6TN cells.
[Effects of heme oxygenase-1/carbon monoxide pathway on the mitochondrial fusion in rat alveolar epithelial type II cells stimulated by lipopolysaccharide].
Jia Haojuan,Shi Jia,Dong Shu'an,Zhang Yuan,Yu Jianbo
Zhonghua wei zhong bing ji jiu yi xue
OBJECTIVE:To investigate the effects of heme oxygenase-1/carbon monoxide (HO-1/CO) pathway on mitochondrial fusion in rat alveolar epithelial type II cells (AEC II) stimulated by lipopolysaccharide (LPS). METHODS:Once the cultured in vitro rat AEC II cells line RLE-6TN reached confluency of 85%, they were subcultured and randomly divided into seven groups (n = 5 each). RLE-6TN cells were routinely cultured in control group. The cells in LPS group was stimulated with 10 mg/L LPS to reproduce the model of endotoxin challenge in AECII cells. The cells in carbon monoxide-releasing molecule-2 (CORM-2, in vitro CO release agent) + LPS group (CL group) and Hemin (HO-1 inducer) + LPS group (HL group) were pretreated with 100 μmol/L CORM-2 or 20 μmol/L Hemin for 1 hour, respectively, followed by 10 mg/L LPS stimulation. The cells in zinc protoporphyrin-IX (ZnPP-IX, HO-1 inhibitor) + LPS group (ZL group) was pretreated with 10 μmol/L ZnPP-IX for 0.5 hour followed by 10 mg/L LPS stimulation. The cells in CORM-2 + ZnPP-IX + LPS group (CZL group) and Hemin + ZnPP-IX + LPS group (HZL group) were pretreated with 100 μmol/L CORM-2 or 20 μmol/L Hemin respectively for 1 hour, and other treatments were similar to those previously described in ZL group. At 24 hours after LPS stimulation, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the supernatant were determined by enzyme linked immunosorbent assay (ELISA), the protein expressions of HO-1, mitochondrial fusion related proteins 1 and 2 (Mfn1, Mfn2) and optic atrophy 1 (OPA1) were determined by Western Blot. RESULTS:Compared with control group, IL-6 and TNF-α contents in the supernatant were increased, HO-1 protein expression was up-regulated, Mfn1, Mfn2 and OPA1 protein expressions were down-regulated in all treatment groups. Compared with LPS group, IL-6 and TNF-α contents were significantly decreased after CORM-2 or Hemin pretreatment [IL-6 (ng/L): 48.6±3.7, 48.4±3.1 vs. 58.7±2.5; TNF-α (ng/L): 40.7±5.3, 39.4±4.3 vs. 51.8±5.1], the protein expressions of HO-1, Mfn1, Mfn2 and OPA1 were significantly up-regulated (HO-1 protein: 0.873±0.051, 0.839±0.061 vs. 0.671±0.044; Mfn1 protein: 0.673±0.037, 0.654±0.025 vs. 0.568±0.021; Mfn2 protein: 0.676±0.044, 0.683±0.035 vs. 0.571±0.043; OPA1 protein: 0.648±0.031, 0.632±0.031 vs. 0.554±0.032; all P < 0.05); while opposite effects were found after ZnPP-IX preincubation, and there were significant differences in IL-6 and TNF-α contents and protein expressions of HO-1, Mfn1, Mfn2 and OPA1 as compared with those of LPS group [IL-6 (ng/L): 69.8±5.1 vs. 58.7±2.5, TNF-α (ng/L): 61.9±3.3 vs. 51.8±5.1, HO-1 protein: 0.545±0.023 vs. 0.671±0.044, Mfn1 protein: 0.406±0.051 vs. 0.568±0.021, Mfn2 protein: 0.393±0.051 vs. 0.571±0.043, OPA1 protein: 0.372±0.050 vs. 0.554±0.032; all P < 0.05]. There were no significant differences in the parameters mentioned above between HL group and CL group, as well as among LPS, CZL and HZL groups. CONCLUSIONS:HO-1/CO pathway promotes mitochondrial fusion and alleviates inflammatory response in LPS-induced rat AEC II cells.