cell biochemistry and function Cell Biochem Funct 2015; 33: 44–49. Published online 16 December 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cbf.3085
The role of placenta growth factor in the hyperoxia-induced acute lung injury in an animal model Liang Zhang1, Li-Jie Yuan2, Shuang Zhao3, Yu Shan1, Hong-Min Wu1 and Xin-Dong Xue4* 1
Department of Department of 3 Department of 4 Department of 2
Neonatology, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning, China Biochemistry and Molecular Biology, Harbin Medical University Daqing Campus, Daqing, China Pediatrics, The Fourth People Hospital of Shenyang, Shenyang, Liaoning, China Pediatrics, Shengjing Hospital of China Medical University, Shenyang, Liaoning, China
Prolonged exposure to hyperoxia leads to acute lung injury. Alveolar type II cells are main target of hyperoxia-induced lung injury. However, the cellular and molecular mechanisms remain unknown. Here, we aimed to investigate the role of placental growth factor (PLGF) in hyperoxia-induced lung injury. Using experimental hyperoxia-induced lung injury model of neonatal rat and mouse lung epithelial type II cells (MLE-12), we examined the levels of PLGF in bronchoalveolar lavage fluid and in the supernatants of MLE-12 cells. Our results revealed that exogenous PLGF induced hyperoxia-induced lung injury. Furthermore, PLGF triggered a shift of vinculin from insoluble to soluble cell fraction, similar to the observation under hyperoxia stimulation. Moreover, we observed significantly reduced phosphorylation of focal adhesion kinase and increased permeability in MLE-12 cells treated with PLGF. These results suggest that PLGF triggers focal adhesion disassembly in alveolar type II cells via inhibiting the activation of focal adhesion kinase. Our findings reveal a novel role of PLGF in hyperoxia-induced lung injury and provide a potential target for the management of hyperoxia-induced acute lung injury. Copyright © 2014 John Wiley & Sons, Ltd. key words—placental growth factor; focal adhesion; hyperoxia; focal adhesion kinase; alveolar type II cells; neonates; acute lung injury
INTRODUCTION Mechanical ventilation with hyperoxia is necessary to treat patients with respiratory failure and distress. In particular, mechanical ventilation with hyperoxia is most widely used in neonatal intensive care units to increase the survival of preterm and term infants, who are in urgent need of life support procedure. However, mechanical ventilation is known to cause acute lung injury (ALI) by inducing proinflammatory cytokines.1 In addition, prolonged exposure to hyperoxia leads to ALI as well.2 Hyperoxia-induced ALI often progresses to acute respiratory distress syndrome, which is characterized by alveolar damage with inflammatory cell infiltration, alveolar oedema and haemorrhage, perhaps as a result of increased production of a variety of inflammatory cytokines.3,4 Therefore, understanding the effects of inflammatory cytokines on alveolar epithelial cells under hyperoxia is important for supplemental oxygen therapy. Alveolar type II cells act as stem cells for alveolar epithelial restoration after lung injuries and during normal tissue recovery.5 Alveolar type II cells are main target of
*Correspondence to: Xin-Dong Xue, Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang, Liaoning, China. E-mail: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
hyperoxia-induced lung injury.6 Several reports indicated that inflammatory cytokines induced the apoptosis of alveolar type II cells, contributing to hyperoxia-induced lung injuries.7–9 Placental growth factor (PLGF) is a homodimeric protein that shares 53% homology to vascular endothelial growth factor (VEGF).10 Recent studies suggest that VEGF may worsen pulmonary oedema during ALI.11 PLGF plays an important role in pathological conditions related to angiogenesis, vascular leakage and inflammation.12 However, the effects of PLGF on hyperoxiainduced lung injury remain unknown. Here, we provided new evidence to demonstrate that PLGF induces the disassembly of focal adhesion in alveolar type II cells, which contributes to hyperpermeability during hyperoxia-induced lung injury.
MATERIALS AND METHODS Cell lines and reagents Mouse lung epithelial type II cells (MLE-12) were purchased from ATCC (Manassas, VA, USA). Recombinant mouse PLGF was obtained from R&D Systems. Horseradish peroxidase (HRP; type IV) and o-phenylenediamine were from Sigma-Aldrich (St. Louis, MO, USA). Antibodies Received 30 September 2014 Revised 8 November 2014 Accepted 10 November 2014
plgf promotes ali specific for focal adhesion kinase (FAK) and phospho-FAK were from Cell Signalling (Boston, MA, USA). Animals Within 12h of birth, pups were divided into two group, group A is hyperoxia-exposed group and group B is air-exposed group. Group A were pooled in Plexiglas chambers (Biosperix, NY, USA) into which oxygen was continuously delivered to keep a constant level of 90% oxygen and CO2 concentration < 0.5%. The oxygen concentration was continuously monitored using Proox110-O2 and the CO2 level was monitored with Proox-CO2 (Biospherix, NY, USA). The chamber contained soda lime in a container for the removal of excess CO2. Temperature and relative humidity were maintained at 22°C∼27°C and 50%∼70% respectively. For a daily 30min period, the chamber was opened to allow provision of fresh food and water, change the rat litter and exchange the surrogate mothers between the two groups to avoid oxygen toxicity in the mothers and to eliminate maternal effects between groups. 8 rats of each group were sacrificed on day l,3,5,7 and 14. The lungs of mice were surgically removed from anesthetized animals. After general anaesthesia with pentobarbital by injection intraperitoneally, a tracheostomy was placed with a 22-gauge catheter. One millilitre normal saline was infused and removed gently with a tuberculin syringe. Lavage fluid was centrifuged at 300 g for 10 min at 4 °C to sediment cells and cell debris, and the supernatant was recovered. The protocols were approved by the Institutional Animal Care and Use committee of China Medical University. The concentration of PLGF in cellfree supernatant was determined using a mouse PLGF ELISA quantification kit according to the manufacturer’s instruction. Quantitative real-time PCR Total RNA was isolated with Trizol (Invitrogen) according to the manufacturer’s instructions, and the reverse transcriptase reaction was performed with 1 μg of total RNA using a PrimeScript RT Master Mix Kit (Takara, Tokyo, Japan) with random primers. Relative real-time PCR was performed on an ABI PRISM 7500HT Sequence Detection System (Applied Biosystems) using SYBR Premix Ex Taq kit (Takara, Tokyo, Japan) according to the manufacturer’s protocols. The relative expression levels of indicated genes were normalized to glyceraldehyde-3-phosphate dehydrogenase and analyzed by the 2(-delta delta C(T)) method. Horseradish peroxidase (HRP) flux measurement MLE-12 cells were cultured on the Transwell inserts (0.4 μm pore size, Costar, Cambridge, MA, USA) and incubated with 100 ng/ml recombinant mouse PLGF for the indicated time or incubated with different concentrations of PLGF for 2 h, along with 0.4 mg/ml HRP, simultaneously. At the end of experiments, the media from the lower chamber were collected and HRP content of the samples were assayed colorimetrically. HRP flux was expressed as nanogram passed per cm2 surface area per hour. Copyright © 2014 John Wiley & Sons, Ltd.
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Transepithelial electrical resistance (TEER) measurement TEER was measured using a Millicell-ERS (Milipore, Billerica, MA, USA) according to the manufacturer’s protocol and then was calculated in ohms cm2 by multiplying the value by the surface area of the monolayer. Western blot analysis Confluent MLE-12 cells were washed and extracted in Triton X-100 lysis buffer (25 mM HEPES, 150 mM NaCl, 4 mM EDTA, 1% Triton X-100) and centrifuged to collect the soluble fraction. The pellets were dissolved in sodium dodecyl sulfate (SDS) lysis buffer (25 mM HEPES, 4 mM EDTA, 1% SDS) to obtain the insoluble fraction. Protein concentrations were determined using bicinchoninic acid protein assay reagent kit (Pierce, Indianapolis, IN, USA). Equal portions of the soluble and insoluble fractions were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred electrophoretically to polyvinylidene difluoride membrane (Millipore, Billerica, MA) and processed for immunoblotting with specific antibodies. Protein bands were visualized using Amersham™ ECL Plus Western Blotting Detection Reagents (GE Healthcare, Piscataway, NJ, USA). Immunofluorescence MLE-12 cell monolayers grown on glass coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking with 5% BSA in PBS, the cells were incubated with vinculin antibodies. The glass slides were analyzed using immunofluorescence microscopy (Olympus, Japan). Statistical analysis The values were expressed as mean ± SD. Statistical significance of the differences was analyzed by Student’s t test for comparisons between two groups and by a one-way ANOVA for comparisons among more than three groups. P values
The role of placenta growth factor in the hyperoxia-induced acute lung injury in an animal model Liang Zhang1, Li-Jie Yuan2, Shuang Zhao3, Yu Shan1, Hong-Min Wu1 and Xin-Dong Xue4* 1
Department of Department of 3 Department of 4 Department of 2
Neonatology, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning, China Biochemistry and Molecular Biology, Harbin Medical University Daqing Campus, Daqing, China Pediatrics, The Fourth People Hospital of Shenyang, Shenyang, Liaoning, China Pediatrics, Shengjing Hospital of China Medical University, Shenyang, Liaoning, China
Prolonged exposure to hyperoxia leads to acute lung injury. Alveolar type II cells are main target of hyperoxia-induced lung injury. However, the cellular and molecular mechanisms remain unknown. Here, we aimed to investigate the role of placental growth factor (PLGF) in hyperoxia-induced lung injury. Using experimental hyperoxia-induced lung injury model of neonatal rat and mouse lung epithelial type II cells (MLE-12), we examined the levels of PLGF in bronchoalveolar lavage fluid and in the supernatants of MLE-12 cells. Our results revealed that exogenous PLGF induced hyperoxia-induced lung injury. Furthermore, PLGF triggered a shift of vinculin from insoluble to soluble cell fraction, similar to the observation under hyperoxia stimulation. Moreover, we observed significantly reduced phosphorylation of focal adhesion kinase and increased permeability in MLE-12 cells treated with PLGF. These results suggest that PLGF triggers focal adhesion disassembly in alveolar type II cells via inhibiting the activation of focal adhesion kinase. Our findings reveal a novel role of PLGF in hyperoxia-induced lung injury and provide a potential target for the management of hyperoxia-induced acute lung injury. Copyright © 2014 John Wiley & Sons, Ltd. key words—placental growth factor; focal adhesion; hyperoxia; focal adhesion kinase; alveolar type II cells; neonates; acute lung injury
INTRODUCTION Mechanical ventilation with hyperoxia is necessary to treat patients with respiratory failure and distress. In particular, mechanical ventilation with hyperoxia is most widely used in neonatal intensive care units to increase the survival of preterm and term infants, who are in urgent need of life support procedure. However, mechanical ventilation is known to cause acute lung injury (ALI) by inducing proinflammatory cytokines.1 In addition, prolonged exposure to hyperoxia leads to ALI as well.2 Hyperoxia-induced ALI often progresses to acute respiratory distress syndrome, which is characterized by alveolar damage with inflammatory cell infiltration, alveolar oedema and haemorrhage, perhaps as a result of increased production of a variety of inflammatory cytokines.3,4 Therefore, understanding the effects of inflammatory cytokines on alveolar epithelial cells under hyperoxia is important for supplemental oxygen therapy. Alveolar type II cells act as stem cells for alveolar epithelial restoration after lung injuries and during normal tissue recovery.5 Alveolar type II cells are main target of
*Correspondence to: Xin-Dong Xue, Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang, Liaoning, China. E-mail: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
hyperoxia-induced lung injury.6 Several reports indicated that inflammatory cytokines induced the apoptosis of alveolar type II cells, contributing to hyperoxia-induced lung injuries.7–9 Placental growth factor (PLGF) is a homodimeric protein that shares 53% homology to vascular endothelial growth factor (VEGF).10 Recent studies suggest that VEGF may worsen pulmonary oedema during ALI.11 PLGF plays an important role in pathological conditions related to angiogenesis, vascular leakage and inflammation.12 However, the effects of PLGF on hyperoxiainduced lung injury remain unknown. Here, we provided new evidence to demonstrate that PLGF induces the disassembly of focal adhesion in alveolar type II cells, which contributes to hyperpermeability during hyperoxia-induced lung injury.
MATERIALS AND METHODS Cell lines and reagents Mouse lung epithelial type II cells (MLE-12) were purchased from ATCC (Manassas, VA, USA). Recombinant mouse PLGF was obtained from R&D Systems. Horseradish peroxidase (HRP; type IV) and o-phenylenediamine were from Sigma-Aldrich (St. Louis, MO, USA). Antibodies Received 30 September 2014 Revised 8 November 2014 Accepted 10 November 2014
plgf promotes ali specific for focal adhesion kinase (FAK) and phospho-FAK were from Cell Signalling (Boston, MA, USA). Animals Within 12h of birth, pups were divided into two group, group A is hyperoxia-exposed group and group B is air-exposed group. Group A were pooled in Plexiglas chambers (Biosperix, NY, USA) into which oxygen was continuously delivered to keep a constant level of 90% oxygen and CO2 concentration < 0.5%. The oxygen concentration was continuously monitored using Proox110-O2 and the CO2 level was monitored with Proox-CO2 (Biospherix, NY, USA). The chamber contained soda lime in a container for the removal of excess CO2. Temperature and relative humidity were maintained at 22°C∼27°C and 50%∼70% respectively. For a daily 30min period, the chamber was opened to allow provision of fresh food and water, change the rat litter and exchange the surrogate mothers between the two groups to avoid oxygen toxicity in the mothers and to eliminate maternal effects between groups. 8 rats of each group were sacrificed on day l,3,5,7 and 14. The lungs of mice were surgically removed from anesthetized animals. After general anaesthesia with pentobarbital by injection intraperitoneally, a tracheostomy was placed with a 22-gauge catheter. One millilitre normal saline was infused and removed gently with a tuberculin syringe. Lavage fluid was centrifuged at 300 g for 10 min at 4 °C to sediment cells and cell debris, and the supernatant was recovered. The protocols were approved by the Institutional Animal Care and Use committee of China Medical University. The concentration of PLGF in cellfree supernatant was determined using a mouse PLGF ELISA quantification kit according to the manufacturer’s instruction. Quantitative real-time PCR Total RNA was isolated with Trizol (Invitrogen) according to the manufacturer’s instructions, and the reverse transcriptase reaction was performed with 1 μg of total RNA using a PrimeScript RT Master Mix Kit (Takara, Tokyo, Japan) with random primers. Relative real-time PCR was performed on an ABI PRISM 7500HT Sequence Detection System (Applied Biosystems) using SYBR Premix Ex Taq kit (Takara, Tokyo, Japan) according to the manufacturer’s protocols. The relative expression levels of indicated genes were normalized to glyceraldehyde-3-phosphate dehydrogenase and analyzed by the 2(-delta delta C(T)) method. Horseradish peroxidase (HRP) flux measurement MLE-12 cells were cultured on the Transwell inserts (0.4 μm pore size, Costar, Cambridge, MA, USA) and incubated with 100 ng/ml recombinant mouse PLGF for the indicated time or incubated with different concentrations of PLGF for 2 h, along with 0.4 mg/ml HRP, simultaneously. At the end of experiments, the media from the lower chamber were collected and HRP content of the samples were assayed colorimetrically. HRP flux was expressed as nanogram passed per cm2 surface area per hour. Copyright © 2014 John Wiley & Sons, Ltd.
45
Transepithelial electrical resistance (TEER) measurement TEER was measured using a Millicell-ERS (Milipore, Billerica, MA, USA) according to the manufacturer’s protocol and then was calculated in ohms cm2 by multiplying the value by the surface area of the monolayer. Western blot analysis Confluent MLE-12 cells were washed and extracted in Triton X-100 lysis buffer (25 mM HEPES, 150 mM NaCl, 4 mM EDTA, 1% Triton X-100) and centrifuged to collect the soluble fraction. The pellets were dissolved in sodium dodecyl sulfate (SDS) lysis buffer (25 mM HEPES, 4 mM EDTA, 1% SDS) to obtain the insoluble fraction. Protein concentrations were determined using bicinchoninic acid protein assay reagent kit (Pierce, Indianapolis, IN, USA). Equal portions of the soluble and insoluble fractions were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred electrophoretically to polyvinylidene difluoride membrane (Millipore, Billerica, MA) and processed for immunoblotting with specific antibodies. Protein bands were visualized using Amersham™ ECL Plus Western Blotting Detection Reagents (GE Healthcare, Piscataway, NJ, USA). Immunofluorescence MLE-12 cell monolayers grown on glass coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking with 5% BSA in PBS, the cells were incubated with vinculin antibodies. The glass slides were analyzed using immunofluorescence microscopy (Olympus, Japan). Statistical analysis The values were expressed as mean ± SD. Statistical significance of the differences was analyzed by Student’s t test for comparisons between two groups and by a one-way ANOVA for comparisons among more than three groups. P values
