Targeting NOX4 alleviates sepsis-induced acute lung injury via attenuation of redox-sensitive activation of CaMKII/ERK1/2/MLCK and endothelial cell barrier dysfunction

Abstract

Increased pulmonary vascular permeability due to endothelial cell (EC) barrier dysfunction is a major pathological feature of acute respiratory distress syndrome/acute lung injury (ARDS/ALI), which is a devastating critical illness with high incidence and excessive mortality. Activation of NADPH oxidase (NOX) induces EC dysfunction via production of reactive oxygen species (ROS). However, the role(s) of NOX isoform(s), and their downstream signaling events, in the development of ARDS/ALI have remained unclear. Cecal Ligation Puncture (CLP) was used to induce preclinical septic ALI in wild-type mice and mice deficient in NOX2 or p47phox, or mice transfected of control siRNA, NOX1 or NOX4 siRNA in vivo. The survival rate of the CLP group at 24 h (26.6%, control siRNA treated) was substantially improved by NOX4 knockdown (52.9%). Mice lacking NOX2 or p47phox, however, had worse outcomes after CLP (survival rates at 0% and 8.3% respectively), whereas NOX1-silenced mice had similar survival rate (30%). NOX4 knockdown attenuated lung ROS production in septic mice, whereas NOX1 knockdown, NOX2 knockout, or p47phox knockout in mice had no effects. In addition, NOX4 knockdown attenuated redox-sensitive activation of the CaMKII/ERK1/2/MLCK pathway, and restored expression of EC tight junction proteins ZO-1 and Occludin to maintain EC barrier integrity. Correspondingly, NOX4 knockdown in cultured human lung microvascular ECs also reduced LPS-induced ROS production, CaMKII/ERK1/2/MLCK activation and EC barrier dysfunction. Scavenging superoxide in vitro and in vivo with TEMPO, or inhibiting CaMKII activation with KN93, had similar effects as NOX4 knockdown in preserving EC barrier dysfunction. In summary, we have identified a novel, selective and causal role of NOX4 (versus other NOX isoforms) in inducing lung EC barrier dysfunction and injury/mortality in a preclinical CLP-induced septic model, which involves redox-sensitive activation of CaMKII/ERK1/2/MLCK pathway. Targeting NOX4 may therefore prove to an innovative therapeutic option that is markedly effective in treating ALI/ARDS.

Keywords: Acute lung injury (ALI); Acute respiratory distress syndrome (ARDS); Endothelial barrier dysfunction; Endothelial cell (EC); Endothelial permeability; NADPH oxidase (NOX); NOX1; NOX2; NOX4; Occludin; Reactive oxygen species (ROS); Tight junction; ZO-1; p22phox; p47phox.

Fig. 1

NOX4 knockdown markedly improves survival and attenuates acute lung injury in CLP-induced septic mice. Sepsis was induced by cecal ligation and puncture (CLP) in wild-type (WT) mice and mice deficient in NOX2 or p47phox, or mice transfected of control siRNA, NOX1 or NOX4 siRNA. Natural death time was recorded by observation every 30 min starting 12 h after CLP, and the death time was presented as a Kaplan-Meier plot using the Prism software, and log-rank test was used to compare survival between groups. Other mice were harvested at 16 h post CLP and lung tissues harvested for analyses of histomorphology, Evans Blue index and wet-to-dry weight ratio were used to assess severity of acute lung injury. (A) Survival curves in sham group, CLP groups treated with control siRNA, NOX1 or NOX4 siRNA, CLP WT group and CLP groups made in NOX2 or p47phox knockout mice. **p < 0.01, ^##p < 0.01. (B) Representative images of H&E staining of lung tissue sections and semiquantitative histological scores of lung injury in experimental groups described in panel A. H&E stainings indicate signs of inflammation (red arrows), edema (blue arrows), hemorrhage (black arrows), and alveolar septal thickening (green arrows) from the cross sections of the lung. Bar = 100 μm. Data are presented as Mean±SEM, n = 5. **p < 0.01 vs. CLP group. (C–D) Lung wet-to-dry weight ratio and Evans Blue index were determined in experimental groups as described in panel A. Data are presented as Mean±SEM, n = 5. *p < 0.05, **p < 0.01 vs. control siRNA transfected CLP group. Data indicate NOX4 knockdown in CLP mice is selectively and robustly effective in improving survival and attenuating acute lung injury. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

NOX4 knockdown decreases ROS production in CLP-induced septic mice. Sepsis was induced by CLP in wild-type (WT) mice and mice deficient in NOX2 or p47phox, or mice transfected of control siRNA, NOX1 or NOX4 siRNA. Mouse lung tissues were freshly harvested at 16 h post CLP for DHE fluorescent imaging, or at 1, 2, 4, 6 h post CLP for Western blotting analyses. (A): Representative images of DHE fluorescent imaging of lung tissue sections in sham group, CLP groups treated with control siRNA, NOX1 or NOX4 siRNA, CLP WT group and CLP groups made in NOX2 or p47phox knockout mice. Freshly prepared lung OCT sections were incubated with DHE (2 μmol/L) in the dark for 30 min, and fluorescent images were captured using a Nikon A1R Confocal Microscope. Photographs were taken at ×200 magnification. Data are presented as Mean±SEM. n = 10. **p < 0.01 vs. CLP group. (B) Representative Western blots and quantitative data of NOX1, NOX2, NOX4 and p22phox protein expression in mice at 1, 2, 4, 6 h post CLP. Data are presented as Mean±SEM; n = 3. *p < 0.05, **p < 0.01 vs. 0 h group. (C) Representative Western blots and quantitative data of NOX1, NOX2, NOX4, and p22phox expression in lung tissues of control siRNA or NOX1 siRNA treated mice with or without CLP for 2 h, indicating that NOX1 siRNA attenuated the expression of NOX1, while having no effects on the expression of NOX2, NOX4, and p22phox. Data are presented as Mean±SEM; n = 3. **p < 0.01. (D) Representative Western blots and quantitative data of NOX1, NOX2, NOX4, and p22phox expression in lung tissues of WT and NOX2 knockout mice with or without CLP for 2 h, indicating that NOX2 knockout had absence of NOX2 but upregulated expression of NOX4 and p22phox. Data are presented as Mean±SEM; n = 3. **p < 0.01. (E) Representative Western blots and quantitative data of NOX1, NOX2, NOX4, and p22phox in lung tissues of control siRNA or NOX4 siRNA treated mice with or without CLP for 2 h, indicating that NOX4 siRNA attenuated the expression of NOX4 and p22phox, while having no effect on the protein expression of NOX1 and NOX2. Data are presented as Mean±SEM; n = 3. **p < 0.01. (F) Representative Western blots and quantitative data of p47phox and p22phox in lung tissues of WT or p47phox knockout mice with or without CLP for 2 h, indicating that p47phox knockout had absence of p47phox but upregulated expression of p22phox. Data are presented as Mean±SEM; n = 3. *p < 0.05.

Fig. 3

NOX4 knockdown prevents CLP-induced pulmonary hyperpermeability via CaMKII/ERK1/2/MLCK pathway inactivation. Sepsis was induced by CLP in mice transfected of control siRNA or NOX4 siRNA. Mouse lung tissues were harvested at 1, 2, 4, 6 h post CLP for Western blotting analyses. In parallel experiments, septic mice were pretreated with either KN93 (0.05 mg/day/mouse) or KN92 (0.05 mg/day/mouse) for 1 week before exposure to CLP for 16 h to examine effects of KN93 on pulmonary hyperpermeability in CLP-treated mice. (A): Representative Western blots and quantitative data of CaMKII phosphorylation in mouse lung tissues at 1, 2, 4, 6 h post CLP. Data are presented as Mean±SEM; n = 3. *p < 0.05, **p < 0.01 vs. 0 h group. (B): Representative Western blots and quantitative data of CaMKII, ERK1/2, and MLCK phosphorylation in sham mice or CLP treated mice transfected of control siRNA or NOX4 siRNA. Results indicate NOX4 knowdown markedly attenuated phosphorylation of CaMKII, ERK1/2 and MLCK. Data are presented as Mean±SEM; n = 3. **p < 0.01 vs. Control-CLP group. (C): Representative fluorescent images of DHE detection of ROS production in CLP mice with saline or KN92/KN93 treatment, indicating the upstream production of superoxide was not affected by CaMKII inhibition KN93. Data are presented as Mean±SEM; n = 3. (D): Representative Western blots and quantitative data of p-CaMKII expression in sham mice, or CLP treated mice pretreated with KN92 or KN93. Results indicate KN93 inhibition of CaMKII phosphorylation. Data are presented as Mean±SEM; n = 3. **p < 0.01. (E–F): Lung wet-to-dry weight ratio and Evans Blue index were determined in PBS or KN92/KN93 treated CLP mice, indicating attenuation of pulmonary hyperpermeability by KN93 treatment in vivo. Data are presented as Mean±SEM, n = 5. **p < 0.01 vs. CLP group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

NOX4 knockdown preserves expression of endothelial tight junction proteins ZO-1 and Occludin in CLP-induced septic mice. Sepsis was induced by CLP in mice transfected of control siRNA or NOX4 siRNA. Mouse lung tissues were harvested at 16 h post CLP for Western blotting, immunohistochemical or immunofluorescent analyses. (A): Representative Western blots and quantitative data of Occludin and ZO-1 protein expression in sham mice or CLP treated mice transfected of control siRNA or NOX4 siRNA. Data are presented as Mean±SEM; n = 3; **p < 0.01 vs. CLP group. (B): Representative immunohistochemical staining images of Occludin and ZO-1 in lung paraffin sections. Dark brown dots indicate positively stained cells. Morphometric analysis of immunostained area for each protein in relation to total area was performed quantitatively using Image-Pro Plus software. Scale bars: 100 μm. Data are presented as Mean±SEM. n = 3. *p < 0.05, **p < 0.01 vs. control siRNA transfected CLP group. (C): Representative immunofluorescent staining images of Occludin and ZO-1 in lung OCT sections. Cryosections of lung were stained for Occludin (green), ZO-1 (red) and DAPI (blue). The fluorescent images were captured using a Nikon A1R confocal microscope. Densities of Occludin and ZO-1 fluorescence were quantified using the Image J software. Scale bars: 50 μm. Data are presented as Mean±SEM; n = 3. *p < 0.05, **p < 0.01 vs. Control siRNA transfected CLP group. Results from A-C indicate that NOX4 knockdown markedly reversed CLP-induced deficiencies in ZO-1 and Occludin expression. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Pharmacological scavenging of ROS by TEMPO displayed similar effects with NOX4 knockdown in protection against pulmonary hyperpermeability in CLP-induced septic mice. Mice were treated with TEMPO (superoxide scavenger; 100 mg/kg) or PBS for 1 h prior to induction of sepsis with CLP in mice transfected of control siRNA or NOX4 siRNA. Mice were harvested at 16 h post CLP and lung tissues harvested for DHE fluorescent imaging, lung wet-to-dry weight ratio and Evans Blue index to assess effects of TEMPO on severity of acute lung injury. (A): Representative DHE fluorescent images of superoxide production in CLP mice with or without TEMPO treatment/ Data indicate that TEMPO substantially attenuated CLP induced increase in ROS production. Photographs were taken at ×200 magnification. Data are presented as Mean±SEM; n = 10. **p < 0.01 vs. CLP group. (B–C): Lung wet-to-dry weight ratio and Evans Blue index were determined in PBS or TEMPO treated CLP mice Data indicate that TEMPO treatment markedly attenuated acute lung injury in CLP mice in vivo. Data are presented as Mean±SEM, n = 5. **p < 0.01 vs. CLP group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

NOX4 knockdown attenuates LPS-induced endothelial permeability via CaMKII/ERK1/2/MLCK pathway inactivation. Sepsis was mimicked in vitro by LPS (1 μg/ml) stimulation in HPMECs pre-transfected with control siRNA or NOX4 siRNA. (A): Representative Western blots and quantitative data of NOX4 protein expression in HPMECs transfected with control siRNA or NOX4 siRNA, indicating that NOX4 siRNA transfection effectively attenuated NOX4 protein abundance. Data are presented as Mean±SEM. n = 3. **p < 0.01. (B): Changes in TEER values in real time following LPS treatment up to 8 h. Data indicate that NOX4 knockdown substantially alleviated LPS induced decrease in TEER values in HPMECs. Data are presented as Mean±SEM. n = 6–9, **p < 0.01 vs. control siRNA transfected LPS group. (C): Representative images of DHE staining of HPMECs in control group or LPS treated groups transfected with control siRNA or NOX4 siRNA, indicating that NOX4 knockdown completely abrogated ROS production in response to LPS treatment. Cells were incubated with DHE (2 μM) in the dark for 30 min, and fluorescent images were captured using a Nikon A1R Confocal Microscope. Photographs were taken at ×200 magnification. Data are presented as Mean±SEM. n = 3. **p < 0.01 vs. control siRNA transfected LPS group. (D): Representative Western blots and qualitative data of CaMKII phosphorylation in HPMECs following LPS challenge, indicating a time-dependent increase. Data are presented as Mean±SEM. n = 3. **p < 0.01 vs. 0 h group. (E): Representative Western blots and quantitative data of CaMKII, ERK1/2, and MLCK phosphorylation in control or LPS (1 μg/ml) treated HPMECs transfected of control siRNA or NOX4 siRNA. Results indicate that NOX4 knockdown inactivated CaMKII/ERK1/2/MLCK pathway in LPS-treated HPMECs. Data are presented as Mean±SEM; n = 3. *p < 0.05, **p < 0.01 vs. control siRNA transfected LPS group. Results in F–H indicate that pharmacological inhibition of CaMKII is protective endothelial barrier dysfunction in LPS treated HPMECs. Cells were incubated with either KN93 (1 μmol/L) or KN92 (1 μmol/L) for 24 h and then exposed to LPS (1 μg/ml) for 2 h. (F): Representative fluorescent images of DHE detection of ROS production in LPS treated HPMECs with KN92 or KN93 treatment. Data indicate that the upstream production of superoxide was not affected by CaMKII inhibition KN93. Data are presented as Mean±SEM; n = 3. (G): Representative Western blots and quantitative data of p-CaMKII expression in control HPMECs, or LPS stimulated HPMECs pre-treated with KN92 or KN93. KN93 attenuated CaMKII phosphorylation while KN92 had no effects. Data are presented as Mean±SEM; n = 3. **p < 0.01 vs. LPS group. (H): Changes in TEER values in real time in LPS-stimulated HPMECs pre-treated with KN92 or KN93. Data indicate that KN93 treatment markedly preserved TEER values in LPS-treated HMPECs. Data are presented as Mean±SEM. n = 6–9, **p < 0.01 vs. LPS group. (I): Representative Western blots and quantitative data of Occludin and ZO-1 expression in control HPMECs or LPS (1 μg/ml) stimulated HPMECs transfected with control siRNA or NOX4 siRNA. Data are presented as Means±SEM. n = 3. **p < 0.01 vs. LPS group. (J): Representative immunofluorescent analysis of ZO-1 expression in control HPMECs or LPS (1 μg/ml) stimulated HPMECs transfected with control siRNA or NOX4 siRNA. Cells were grown on a glass chamber slid to confluence and then challenged with LPS (1 μg/ml) for 6 h. Immunofluorescent staining of ZO-1 (red), and nuclei (blue) was imaged by a Nikon A1R Confocal microscope. Scale bars: 100 μm. Data are presented as Means±SEM. n = 3. **p < 0.01 vs. LPS group. Results in (I–J) indicate that NOX4 knockdown preserved protein abundance of Occudin and ZO-1 following LPS stimulation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

NOX4 knockdown attenuates LPS-induced endothelial permeability via CaMKII/ERK1/2/MLCK pathway inactivation. Sepsis was mimicked in vitro by LPS (1 μg/ml) stimulation in HPMECs pre-transfected with control siRNA or NOX4 siRNA. (A): Representative Western blots and quantitative data of NOX4 protein expression in HPMECs transfected with control siRNA or NOX4 siRNA, indicating that NOX4 siRNA transfection effectively attenuated NOX4 protein abundance. Data are presented as Mean±SEM. n = 3. **p < 0.01. (B): Changes in TEER values in real time following LPS treatment up to 8 h. Data indicate that NOX4 knockdown substantially alleviated LPS induced decrease in TEER values in HPMECs. Data are presented as Mean±SEM. n = 6–9, **p < 0.01 vs. control siRNA transfected LPS group. (C): Representative images of DHE staining of HPMECs in control group or LPS treated groups transfected with control siRNA or NOX4 siRNA, indicating that NOX4 knockdown completely abrogated ROS production in response to LPS treatment. Cells were incubated with DHE (2 μM) in the dark for 30 min, and fluorescent images were captured using a Nikon A1R Confocal Microscope. Photographs were taken at ×200 magnification. Data are presented as Mean±SEM. n = 3. **p < 0.01 vs. control siRNA transfected LPS group. (D): Representative Western blots and qualitative data of CaMKII phosphorylation in HPMECs following LPS challenge, indicating a time-dependent increase. Data are presented as Mean±SEM. n = 3. **p < 0.01 vs. 0 h group. (E): Representative Western blots and quantitative data of CaMKII, ERK1/2, and MLCK phosphorylation in control or LPS (1 μg/ml) treated HPMECs transfected of control siRNA or NOX4 siRNA. Results indicate that NOX4 knockdown inactivated CaMKII/ERK1/2/MLCK pathway in LPS-treated HPMECs. Data are presented as Mean±SEM; n = 3. *p < 0.05, **p < 0.01 vs. control siRNA transfected LPS group. Results in F–H indicate that pharmacological inhibition of CaMKII is protective endothelial barrier dysfunction in LPS treated HPMECs. Cells were incubated with either KN93 (1 μmol/L) or KN92 (1 μmol/L) for 24 h and then exposed to LPS (1 μg/ml) for 2 h. (F): Representative fluorescent images of DHE detection of ROS production in LPS treated HPMECs with KN92 or KN93 treatment. Data indicate that the upstream production of superoxide was not affected by CaMKII inhibition KN93. Data are presented as Mean±SEM; n = 3. (G): Representative Western blots and quantitative data of p-CaMKII expression in control HPMECs, or LPS stimulated HPMECs pre-treated with KN92 or KN93. KN93 attenuated CaMKII phosphorylation while KN92 had no effects. Data are presented as Mean±SEM; n = 3. **p < 0.01 vs. LPS group. (H): Changes in TEER values in real time in LPS-stimulated HPMECs pre-treated with KN92 or KN93. Data indicate that KN93 treatment markedly preserved TEER values in LPS-treated HMPECs. Data are presented as Mean±SEM. n = 6–9, **p < 0.01 vs. LPS group. (I): Representative Western blots and quantitative data of Occludin and ZO-1 expression in control HPMECs or LPS (1 μg/ml) stimulated HPMECs transfected with control siRNA or NOX4 siRNA. Data are presented as Means±SEM. n = 3. **p < 0.01 vs. LPS group. (J): Representative immunofluorescent analysis of ZO-1 expression in control HPMECs or LPS (1 μg/ml) stimulated HPMECs transfected with control siRNA or NOX4 siRNA. Cells were grown on a glass chamber slid to confluence and then challenged with LPS (1 μg/ml) for 6 h. Immunofluorescent staining of ZO-1 (red), and nuclei (blue) was imaged by a Nikon A1R Confocal microscope. Scale bars: 100 μm. Data are presented as Means±SEM. n = 3. **p < 0.01 vs. LPS group. Results in (I–J) indicate that NOX4 knockdown preserved protein abundance of Occudin and ZO-1 following LPS stimulation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Pharmacological scavenging of ROS by TEMPO displayed similar effects with NOX4 knockdown in protection against LPS-induced barrier dysfunction in HPMECs. HPMECs were transfected with control siRNA or NOX4 siRNA, and then treated with TEMPO (0.1 mmo/L) for 1 h prior to LPS (1 μg/ml) stimulation. Cells were incubated in ECIS for real time monitoring of changes in TEER values up to 8 h, or harvested at 2 h post LPS for DHE fluorescent imaging of ROS production. (A): Representative DHE fluorescent images of superoxide production in LPS treated HPMECs with or without TEMPO treatment. Data indicate that TEMPO substantially attenuated LPS induced ROS production. Photographs were taken at ×200 magnification. Data are presented as Mean±SEM; n = 5. **p < 0.01 vs. LPS group. (B): Changes in TEER values after TEMPO treatment in LPS exposed HPMECs. Data indicate that TEMPO treatment markedly abrogated LPS induced decline in TEER values. Data are presented as Means±SEM. n = 6–9. **p < 0.01 vs. LPS group.

Fig. 8

Targeting NOX4 alleviates sepsis-induced acute lung injury via attenuation of redox-sensitive activation of CaMKII/ERK1/2/MLCK and endothelial cell barrier dysfunction. During ALI/ARDS, activation of NOX4, rather than other NOX isoforms, produces ROS and redox-sensitively activates CaMKII/ERK1/2/MLCK pathway to result in endothelial cell tight junction deficiency and barrier dysfunction, leading to acute lung injury and death in CLP-induced septic mice. RNAi inhibition of NOX4, scavenging superoxide radicals, or pharmacological inhibition of CaMKII can block this pathway and relieve endothelial cell barrier dysfunction and lung injuries in CLP mice. Targeting NOX4 may therefore prove to an innovative therapeutic option that is markedly effective in treating ALI/ARDS.