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Activated Akt protects the lung from oxidant-induced injury and delays death of mice.

Lu Y, Parkyn L, Otterbein LE, Kureishi Y, Walsh K, Ray A, Ray P - J. Exp. Med. (2001)

Bottom Line: The serine-threonine kinase Akt has been implicated in inhibiting cell death induced by different stimuli including growth factor withdrawal, cell cycle discordance, DNA damage, and loss of cell adhesion in different cell types.However, the in vivo relevance of this prosurvival pathway has not been explored.This is the first demonstration of the in vivo protective function of Akt in the context of oxidant-induced lung injury.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Pulmonary and Critical Care Section, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
Oxidant-induced injury to the lung causes extensive damage to lung epithelial cells. Impaired protection and repair of the lung epithelium can result in death. The serine-threonine kinase Akt has been implicated in inhibiting cell death induced by different stimuli including growth factor withdrawal, cell cycle discordance, DNA damage, and loss of cell adhesion in different cell types. However, the in vivo relevance of this prosurvival pathway has not been explored. Here we show that a constitutively active form of Akt introduced intratracheally into the lungs of mice by adenovirus gene transfer techniques protects mice from hyperoxic pulmonary damage and delays death of mice. This is the first demonstration of the in vivo protective function of Akt in the context of oxidant-induced lung injury.

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Constitutively active Akt inhibits hyperoxic lung injury in mice. (A) C57BL/6 mice were infected intratracheally with 109 PFU of either control virus (Ad-EGFP) or Ad-myr-Akt (HA tagged) and then exposed to 100% O2. Lungs were prepared for histology by perfusing the animal through the right ventricle with PBS to remove all blood. Lungs were then inflated to constant pressure (20 cm water) with Streck Tissue fixative (Streck Labs.) instilled through a tracheostomy tube. 5-μM lung sections were mounted on slides and stained with hematoxylin and eosin according to established procedures. All of the control mice died by 72 h of O2 exposure and displayed hyaline membrane formation (white arrows), edema (black arrow), widening of alveolar septae (asterisk), and widespread inflammation and hemorrhage (left), whereas none of the mice expressing myr-Akt died at 72 h of initiation of O2 exposure and also had no signs of lung injury at this time point (middle). Mice expressing myr-Akt displayed hyaline membrane formation, patchy edema, and hemorrhage at later time points (∼120 h after hyperoxia; right). (B) Expression of constitutively active, HA epitope–tagged Akt in mouse lungs at different times of hyperoxic exposure. Lung homogenates were prepared and immunoprecipitations were carried out with anti-HA antibody coupled to agarose followed by Western blot analysis using antibody against phospho-Akt. As myristoylation of Akt causes constitutive phosphorylation of Akt, the increased level of phospho-Akt with time was a reflection of increased level of protein expression from the transgene as confirmed by stripping the blot and reprobing with anti-Akt antibody. This experiment was repeated twice with similar results.
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Figure 3: Constitutively active Akt inhibits hyperoxic lung injury in mice. (A) C57BL/6 mice were infected intratracheally with 109 PFU of either control virus (Ad-EGFP) or Ad-myr-Akt (HA tagged) and then exposed to 100% O2. Lungs were prepared for histology by perfusing the animal through the right ventricle with PBS to remove all blood. Lungs were then inflated to constant pressure (20 cm water) with Streck Tissue fixative (Streck Labs.) instilled through a tracheostomy tube. 5-μM lung sections were mounted on slides and stained with hematoxylin and eosin according to established procedures. All of the control mice died by 72 h of O2 exposure and displayed hyaline membrane formation (white arrows), edema (black arrow), widening of alveolar septae (asterisk), and widespread inflammation and hemorrhage (left), whereas none of the mice expressing myr-Akt died at 72 h of initiation of O2 exposure and also had no signs of lung injury at this time point (middle). Mice expressing myr-Akt displayed hyaline membrane formation, patchy edema, and hemorrhage at later time points (∼120 h after hyperoxia; right). (B) Expression of constitutively active, HA epitope–tagged Akt in mouse lungs at different times of hyperoxic exposure. Lung homogenates were prepared and immunoprecipitations were carried out with anti-HA antibody coupled to agarose followed by Western blot analysis using antibody against phospho-Akt. As myristoylation of Akt causes constitutive phosphorylation of Akt, the increased level of phospho-Akt with time was a reflection of increased level of protein expression from the transgene as confirmed by stripping the blot and reprobing with anti-Akt antibody. This experiment was repeated twice with similar results.

Mentions: Having confirmed the ability of myr-Akt to induce activation of downstream signaling pathways in lung epithelial cells, we investigated whether expression of Ad-myr-Akt could prevent oxidant-induced lung injury and death in animals. In these experiments, control adenovirus or adenovirus expressing myr-Akt 17 was introduced intratracheally into mice and the mice were immediately exposed to 100% O2. Although all of the control mice died within 72 h of initiation of exposure to 100% O2, all of the mice infected with Ad-myr-Akt lived longer (Fig. 2). The mice were killed at 120 h at which point they had begun to display respiratory distress. Thus, mice expressing activated Akt showed a significant increase in survival time (P < 0.0001). Histological examination of lung sections showed hyaline membrane formation, hemorrhage, inflammation, and gross pulmonary edema in the lungs of the control mice at 72 h (Fig. 3 A). In contrast, the lungs of mice infected with Ad-myr-Akt appeared normal and free of any hyaline membrane, hemorrhage, inflammation, or edema at 72 h after hyperoxia. At 120 h of exposure to 100% O2, the lungs of the myr-Akt–expressing mice displayed hyaline membrane formation, hemorrhage, inflammation, and edema. To confirm expression and activation of myr-Akt in the lungs of the infected mice, lung extracts were immunoprecipitated with anti-hemagglutinin (HA) antibody and the immunoprecipitates were analyzed by immunoblotting with antibody to phosphorylated Akt. As shown in Fig. 3 B, adenovirus-derived transgenic expression of activated Akt was evident within 24 h of infection in mice infected with Ad-myr-Akt but not in those infected with the control virus, and the expression was detectable at 96 h after infection, the last time point tested in this assay. It is well established that hyperoxic injury involves an initiation phase after exposure to hyperoxia which is followed by an inflammatory phase and a destructive phase. Collectively, our data suggest that activated Akt can significantly delay the initiation phase of acute lung injury.


Activated Akt protects the lung from oxidant-induced injury and delays death of mice.

Lu Y, Parkyn L, Otterbein LE, Kureishi Y, Walsh K, Ray A, Ray P - J. Exp. Med. (2001)

Constitutively active Akt inhibits hyperoxic lung injury in mice. (A) C57BL/6 mice were infected intratracheally with 109 PFU of either control virus (Ad-EGFP) or Ad-myr-Akt (HA tagged) and then exposed to 100% O2. Lungs were prepared for histology by perfusing the animal through the right ventricle with PBS to remove all blood. Lungs were then inflated to constant pressure (20 cm water) with Streck Tissue fixative (Streck Labs.) instilled through a tracheostomy tube. 5-μM lung sections were mounted on slides and stained with hematoxylin and eosin according to established procedures. All of the control mice died by 72 h of O2 exposure and displayed hyaline membrane formation (white arrows), edema (black arrow), widening of alveolar septae (asterisk), and widespread inflammation and hemorrhage (left), whereas none of the mice expressing myr-Akt died at 72 h of initiation of O2 exposure and also had no signs of lung injury at this time point (middle). Mice expressing myr-Akt displayed hyaline membrane formation, patchy edema, and hemorrhage at later time points (∼120 h after hyperoxia; right). (B) Expression of constitutively active, HA epitope–tagged Akt in mouse lungs at different times of hyperoxic exposure. Lung homogenates were prepared and immunoprecipitations were carried out with anti-HA antibody coupled to agarose followed by Western blot analysis using antibody against phospho-Akt. As myristoylation of Akt causes constitutive phosphorylation of Akt, the increased level of phospho-Akt with time was a reflection of increased level of protein expression from the transgene as confirmed by stripping the blot and reprobing with anti-Akt antibody. This experiment was repeated twice with similar results.
© Copyright Policy
Related In: Results  -  Collection

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Figure 3: Constitutively active Akt inhibits hyperoxic lung injury in mice. (A) C57BL/6 mice were infected intratracheally with 109 PFU of either control virus (Ad-EGFP) or Ad-myr-Akt (HA tagged) and then exposed to 100% O2. Lungs were prepared for histology by perfusing the animal through the right ventricle with PBS to remove all blood. Lungs were then inflated to constant pressure (20 cm water) with Streck Tissue fixative (Streck Labs.) instilled through a tracheostomy tube. 5-μM lung sections were mounted on slides and stained with hematoxylin and eosin according to established procedures. All of the control mice died by 72 h of O2 exposure and displayed hyaline membrane formation (white arrows), edema (black arrow), widening of alveolar septae (asterisk), and widespread inflammation and hemorrhage (left), whereas none of the mice expressing myr-Akt died at 72 h of initiation of O2 exposure and also had no signs of lung injury at this time point (middle). Mice expressing myr-Akt displayed hyaline membrane formation, patchy edema, and hemorrhage at later time points (∼120 h after hyperoxia; right). (B) Expression of constitutively active, HA epitope–tagged Akt in mouse lungs at different times of hyperoxic exposure. Lung homogenates were prepared and immunoprecipitations were carried out with anti-HA antibody coupled to agarose followed by Western blot analysis using antibody against phospho-Akt. As myristoylation of Akt causes constitutive phosphorylation of Akt, the increased level of phospho-Akt with time was a reflection of increased level of protein expression from the transgene as confirmed by stripping the blot and reprobing with anti-Akt antibody. This experiment was repeated twice with similar results.
Mentions: Having confirmed the ability of myr-Akt to induce activation of downstream signaling pathways in lung epithelial cells, we investigated whether expression of Ad-myr-Akt could prevent oxidant-induced lung injury and death in animals. In these experiments, control adenovirus or adenovirus expressing myr-Akt 17 was introduced intratracheally into mice and the mice were immediately exposed to 100% O2. Although all of the control mice died within 72 h of initiation of exposure to 100% O2, all of the mice infected with Ad-myr-Akt lived longer (Fig. 2). The mice were killed at 120 h at which point they had begun to display respiratory distress. Thus, mice expressing activated Akt showed a significant increase in survival time (P < 0.0001). Histological examination of lung sections showed hyaline membrane formation, hemorrhage, inflammation, and gross pulmonary edema in the lungs of the control mice at 72 h (Fig. 3 A). In contrast, the lungs of mice infected with Ad-myr-Akt appeared normal and free of any hyaline membrane, hemorrhage, inflammation, or edema at 72 h after hyperoxia. At 120 h of exposure to 100% O2, the lungs of the myr-Akt–expressing mice displayed hyaline membrane formation, hemorrhage, inflammation, and edema. To confirm expression and activation of myr-Akt in the lungs of the infected mice, lung extracts were immunoprecipitated with anti-hemagglutinin (HA) antibody and the immunoprecipitates were analyzed by immunoblotting with antibody to phosphorylated Akt. As shown in Fig. 3 B, adenovirus-derived transgenic expression of activated Akt was evident within 24 h of infection in mice infected with Ad-myr-Akt but not in those infected with the control virus, and the expression was detectable at 96 h after infection, the last time point tested in this assay. It is well established that hyperoxic injury involves an initiation phase after exposure to hyperoxia which is followed by an inflammatory phase and a destructive phase. Collectively, our data suggest that activated Akt can significantly delay the initiation phase of acute lung injury.

Bottom Line: The serine-threonine kinase Akt has been implicated in inhibiting cell death induced by different stimuli including growth factor withdrawal, cell cycle discordance, DNA damage, and loss of cell adhesion in different cell types.However, the in vivo relevance of this prosurvival pathway has not been explored.This is the first demonstration of the in vivo protective function of Akt in the context of oxidant-induced lung injury.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Pulmonary and Critical Care Section, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
Oxidant-induced injury to the lung causes extensive damage to lung epithelial cells. Impaired protection and repair of the lung epithelium can result in death. The serine-threonine kinase Akt has been implicated in inhibiting cell death induced by different stimuli including growth factor withdrawal, cell cycle discordance, DNA damage, and loss of cell adhesion in different cell types. However, the in vivo relevance of this prosurvival pathway has not been explored. Here we show that a constitutively active form of Akt introduced intratracheally into the lungs of mice by adenovirus gene transfer techniques protects mice from hyperoxic pulmonary damage and delays death of mice. This is the first demonstration of the in vivo protective function of Akt in the context of oxidant-induced lung injury.

Show MeSH
Related in: MedlinePlus