Perinatal changes in superoxide generation in the ovine lung: Alterations associated with increased pulmonary blood flow

Abstract

Although alterations in ROS generating systems are well described in several vascular disorders, there is very limited information on the perinatal regulation of these systems in the lung both during normal development and in pulmonary hypertension. Thus, this study was undertaken to explore how the two predominant superoxide generating systems, nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) and xanthine oxidase (XO), are developmentally regulated in control lambs and in our established lamb model of increased pulmonary blood flow (Shunt) over the first 2months of life. We found that the levels of p47(phox), p67(phox), and Rac1 subunits of NADPH oxidase complex were altered. During the first two months of life there was no change in p47(phox) protein levels in either normal or Shunt lambs. However, both p67(phox) and Rac1 protein levels decreased over time. In addition, p47(phox) protein levels were significantly increased in shunt lambs at 2- and 4-, but not 8-weeks of age compared to age-matched controls while levels of the p67(phox) subunit were decreased at 8-weeks of age in the Shunts but unchanged at other time periods. Furthermore, Rac1 protein expression was significantly increased in the Shunts only at 4weeks of age. These data correlated with a significant increase in NADPH oxidase dependent superoxide generation at 2- and 4-, but not 8-weeks of age in the Shunts. During normal development XO levels significantly increased over time in normal lambs but significantly decreased in the Shunts. In addition, XO protein levels were significantly increased in the Shunt at 2- and 4-weeks of age but significantly decreased at 8-weeks. Again this correlated with a significant increase in XO dependent superoxide generation at 2- and 4-, but not 8-weeks of age in the Shunts. Collectively, our findings suggest that NADPH oxidase and XO are major contributors to superoxide generation both during the normal development and during the development of pulmonary hypertension.

Copyright 2010 Elsevier Inc. All rights reserved.

Figures

Figure 1. Developmental changes in p47phox subunit levels in peripheral lung in Control and Shunt lambs at 2-, 4-, and 8-weeks of age

Protein extracts (50µg), prepared from peripheral lung of Control (A) and Shunt (B) lambs at 2-, 4-, and 8-weeks of age, were separated on a 4–20% Tris-SDS-HEPES gel, electrophoretically transferred to a PVDF membrane and analyzed using a specific antiserum raised against p47phox protein. p47phox protein levels were normalized for loading using β-actin. A representative blot is shown in each panel. Developmentally there are no significant differences in the p47phox protein levels in either Control or Shunt lambs. Values are mean ± SD; n=6 control and n=6 shunt at each age. The protein extracts (50µg), prepared from peripheral lung of 2-(C), 4-(D), and 8-week (E) Control and Shunt animals, were also analyzed to determine changes in p47protein levels between Control and Shunt lambs. Again p47phox protein levels were normalized for loading using β-actin. Each panel contains a representative blot. There is a significant increase in p47phox protein levels in Shunt lambs at 2-and 4-weeks of age but not at 8-weeks of age. Values are mean ± SD; n=6 control and n=6 shunt at each age; *P<0.05 vs. control. In vivo localization of p47phox protein in pulmonary vasculature of 4-week old Control and Shunt lung tissues is shown (F–G). eNOS was used as an endothelial cell marker and Caldesmon as marker of smooth muscle cells. A stack of images was taken every 2µm using confocal microscopy to identify the actual co-localization. Magnification is ×20.

Figure 1. Developmental changes in p47phox subunit levels in peripheral lung in Control and Shunt lambs at 2-, 4-, and 8-weeks of age

Protein extracts (50µg), prepared from peripheral lung of Control (A) and Shunt (B) lambs at 2-, 4-, and 8-weeks of age, were separated on a 4–20% Tris-SDS-HEPES gel, electrophoretically transferred to a PVDF membrane and analyzed using a specific antiserum raised against p47phox protein. p47phox protein levels were normalized for loading using β-actin. A representative blot is shown in each panel. Developmentally there are no significant differences in the p47phox protein levels in either Control or Shunt lambs. Values are mean ± SD; n=6 control and n=6 shunt at each age. The protein extracts (50µg), prepared from peripheral lung of 2-(C), 4-(D), and 8-week (E) Control and Shunt animals, were also analyzed to determine changes in p47protein levels between Control and Shunt lambs. Again p47phox protein levels were normalized for loading using β-actin. Each panel contains a representative blot. There is a significant increase in p47phox protein levels in Shunt lambs at 2-and 4-weeks of age but not at 8-weeks of age. Values are mean ± SD; n=6 control and n=6 shunt at each age; *P<0.05 vs. control. In vivo localization of p47phox protein in pulmonary vasculature of 4-week old Control and Shunt lung tissues is shown (F–G). eNOS was used as an endothelial cell marker and Caldesmon as marker of smooth muscle cells. A stack of images was taken every 2µm using confocal microscopy to identify the actual co-localization. Magnification is ×20.

Figure 1. Developmental changes in p47phox subunit levels in peripheral lung in Control and Shunt lambs at 2-, 4-, and 8-weeks of age

Protein extracts (50µg), prepared from peripheral lung of Control (A) and Shunt (B) lambs at 2-, 4-, and 8-weeks of age, were separated on a 4–20% Tris-SDS-HEPES gel, electrophoretically transferred to a PVDF membrane and analyzed using a specific antiserum raised against p47phox protein. p47phox protein levels were normalized for loading using β-actin. A representative blot is shown in each panel. Developmentally there are no significant differences in the p47phox protein levels in either Control or Shunt lambs. Values are mean ± SD; n=6 control and n=6 shunt at each age. The protein extracts (50µg), prepared from peripheral lung of 2-(C), 4-(D), and 8-week (E) Control and Shunt animals, were also analyzed to determine changes in p47protein levels between Control and Shunt lambs. Again p47phox protein levels were normalized for loading using β-actin. Each panel contains a representative blot. There is a significant increase in p47phox protein levels in Shunt lambs at 2-and 4-weeks of age but not at 8-weeks of age. Values are mean ± SD; n=6 control and n=6 shunt at each age; *P<0.05 vs. control. In vivo localization of p47phox protein in pulmonary vasculature of 4-week old Control and Shunt lung tissues is shown (F–G). eNOS was used as an endothelial cell marker and Caldesmon as marker of smooth muscle cells. A stack of images was taken every 2µm using confocal microscopy to identify the actual co-localization. Magnification is ×20.

Figure 2. Developmental changes in p67phox subunit levels in peripheral lung in Control and Shunt lambs at 2-, 4-, and 8-weeks of age

Protein extracts (50µg), prepared from peripheral lung of Control (A) and Shunt (B) lambs at 2-, 4-, and 8-weeks of age, were separated on a 4–20% Tris-SDS-HEPES gel, electrophoretically transferred to a PVDF membrane and analyzed using a specific antiserum raised against p67phox protein. P67phox protein levels were normalized for loading using β-actin. A representative blot is shown in each panel. Developmentally there was a significant decrease in p67phox protein levels in both Control (A) and Shunt (B) lambs at 4-and 8-weeks compared to 2-weeks of age. Values are mean ± SD; n=6 control and n=6 shunt at each age. *P<0.05 vs. 2-weeks of age. The protein extracts (50µg), prepared from peripheral lung of 2-(C), 4-(D), and 8-week (E) Control and Shunt animals, were also analyzed to determine changes in p67phox protein levels between Control and Shunt lambs. Again p67phox protein levels were normalized for loading using β-actin. Each panel contains a representative blot. There was a significant decrease in p67phox protein levels in Shunt lambs at 8-weeks but no changes at 2-or 4weeks of age. Values are mean ± SD; n=6 control and n=6 shunt at each age; *P <0.05 vs. control. In vivo localization of p67phox protein in pulmonary vasculature of 8-week old Control and Shunt lung tissues is shown (F–G). eNOS was used as an endothelial cell marker and Caldesmon as marker of smooth muscle cells. A stack of images was taken every 2µm using confocal microscopy to identify actual co-localization. Magnification is ×20.

Figure 2. Developmental changes in p67phox subunit levels in peripheral lung in Control and Shunt lambs at 2-, 4-, and 8-weeks of age

Protein extracts (50µg), prepared from peripheral lung of Control (A) and Shunt (B) lambs at 2-, 4-, and 8-weeks of age, were separated on a 4–20% Tris-SDS-HEPES gel, electrophoretically transferred to a PVDF membrane and analyzed using a specific antiserum raised against p67phox protein. P67phox protein levels were normalized for loading using β-actin. A representative blot is shown in each panel. Developmentally there was a significant decrease in p67phox protein levels in both Control (A) and Shunt (B) lambs at 4-and 8-weeks compared to 2-weeks of age. Values are mean ± SD; n=6 control and n=6 shunt at each age. *P<0.05 vs. 2-weeks of age. The protein extracts (50µg), prepared from peripheral lung of 2-(C), 4-(D), and 8-week (E) Control and Shunt animals, were also analyzed to determine changes in p67phox protein levels between Control and Shunt lambs. Again p67phox protein levels were normalized for loading using β-actin. Each panel contains a representative blot. There was a significant decrease in p67phox protein levels in Shunt lambs at 8-weeks but no changes at 2-or 4weeks of age. Values are mean ± SD; n=6 control and n=6 shunt at each age; *P <0.05 vs. control. In vivo localization of p67phox protein in pulmonary vasculature of 8-week old Control and Shunt lung tissues is shown (F–G). eNOS was used as an endothelial cell marker and Caldesmon as marker of smooth muscle cells. A stack of images was taken every 2µm using confocal microscopy to identify actual co-localization. Magnification is ×20.

Figure 2. Developmental changes in p67phox subunit levels in peripheral lung in Control and Shunt lambs at 2-, 4-, and 8-weeks of age

Protein extracts (50µg), prepared from peripheral lung of Control (A) and Shunt (B) lambs at 2-, 4-, and 8-weeks of age, were separated on a 4–20% Tris-SDS-HEPES gel, electrophoretically transferred to a PVDF membrane and analyzed using a specific antiserum raised against p67phox protein. P67phox protein levels were normalized for loading using β-actin. A representative blot is shown in each panel. Developmentally there was a significant decrease in p67phox protein levels in both Control (A) and Shunt (B) lambs at 4-and 8-weeks compared to 2-weeks of age. Values are mean ± SD; n=6 control and n=6 shunt at each age. *P<0.05 vs. 2-weeks of age. The protein extracts (50µg), prepared from peripheral lung of 2-(C), 4-(D), and 8-week (E) Control and Shunt animals, were also analyzed to determine changes in p67phox protein levels between Control and Shunt lambs. Again p67phox protein levels were normalized for loading using β-actin. Each panel contains a representative blot. There was a significant decrease in p67phox protein levels in Shunt lambs at 8-weeks but no changes at 2-or 4weeks of age. Values are mean ± SD; n=6 control and n=6 shunt at each age; *P <0.05 vs. control. In vivo localization of p67phox protein in pulmonary vasculature of 8-week old Control and Shunt lung tissues is shown (F–G). eNOS was used as an endothelial cell marker and Caldesmon as marker of smooth muscle cells. A stack of images was taken every 2µm using confocal microscopy to identify actual co-localization. Magnification is ×20.

Figure 3. Developmental changes in Rac1 levels in peripheral lung in Control and Shunt lambs at 2-, 4-, and 8-weeks of age

Protein extracts (50 µg), prepared from peripheral lung of Control (A) and Shunt (B) lambs at 2-, 4-, and 8-weeks of age, were separated on a 4–20% Tris-SDS-HEPES gel, electrophoretically transferred to a PVDF membrane and analyzed using a specific antiserum raised against Rac1. Rac1 protein levels were normalized for loading using β-actin. A representative blot is shown in each panel. Developmentally there was a significant decrease in Rac1 protein levels in both Control (A) and Shunt (B) lambs at 4- and 8-weeks compared to 2-weeks of age. Values are mean ± SD; n=6 control and n=6 shunt at each age. *PIn vivo localization of Rac1 protein in pulmonary vasculature of 4-week old Control and Shunt lung tissues is shown (F–G). eNOS was used as an endothelial cell marker and Caldesmon as marker of smooth muscle cells. A stack of images was taken every 2µm using confocal microscopy to identify actual co-localization. Magnification is ×20.

Figure 3. Developmental changes in Rac1 levels in peripheral lung in Control and Shunt lambs at 2-, 4-, and 8-weeks of age

Protein extracts (50 µg), prepared from peripheral lung of Control (A) and Shunt (B) lambs at 2-, 4-, and 8-weeks of age, were separated on a 4–20% Tris-SDS-HEPES gel, electrophoretically transferred to a PVDF membrane and analyzed using a specific antiserum raised against Rac1. Rac1 protein levels were normalized for loading using β-actin. A representative blot is shown in each panel. Developmentally there was a significant decrease in Rac1 protein levels in both Control (A) and Shunt (B) lambs at 4- and 8-weeks compared to 2-weeks of age. Values are mean ± SD; n=6 control and n=6 shunt at each age. *PIn vivo localization of Rac1 protein in pulmonary vasculature of 4-week old Control and Shunt lung tissues is shown (F–G). eNOS was used as an endothelial cell marker and Caldesmon as marker of smooth muscle cells. A stack of images was taken every 2µm using confocal microscopy to identify actual co-localization. Magnification is ×20.

Figure 3. Developmental changes in Rac1 levels in peripheral lung in Control and Shunt lambs at 2-, 4-, and 8-weeks of age

Protein extracts (50 µg), prepared from peripheral lung of Control (A) and Shunt (B) lambs at 2-, 4-, and 8-weeks of age, were separated on a 4–20% Tris-SDS-HEPES gel, electrophoretically transferred to a PVDF membrane and analyzed using a specific antiserum raised against Rac1. Rac1 protein levels were normalized for loading using β-actin. A representative blot is shown in each panel. Developmentally there was a significant decrease in Rac1 protein levels in both Control (A) and Shunt (B) lambs at 4- and 8-weeks compared to 2-weeks of age. Values are mean ± SD; n=6 control and n=6 shunt at each age. *PIn vivo localization of Rac1 protein in pulmonary vasculature of 4-week old Control and Shunt lung tissues is shown (F–G). eNOS was used as an endothelial cell marker and Caldesmon as marker of smooth muscle cells. A stack of images was taken every 2µm using confocal microscopy to identify actual co-localization. Magnification is ×20.

Figure 4. NOX2 and NOX4 mRNA levels in peripheral lung in Control and Shunt lambs at 2-, 4-, and 8-weeks of age

NOX2 and NOX4 mRNA levels were quantified by qRT-PCR analysis. There was a significant decrease in NOX2 mRNA levels in 2-week old Shunt lambs compared to age matched Controls (A). Whereas, NOX2 mRNA levels were unchanged 4- and 8- week old Shunt lambs (B–C). There were no detectable differences in NOX4 mRNA levels in 2-, 4-, 8- week Shunt lambs compared to age matched Controls (D–F). Values are mean ± SD; n=6 control and n=6 shunt at each age; *P

Figure 4. NOX2 and NOX4 mRNA levels in peripheral lung in Control and Shunt lambs at 2-, 4-, and 8-weeks of age

NOX2 and NOX4 mRNA levels were quantified by qRT-PCR analysis. There was a significant decrease in NOX2 mRNA levels in 2-week old Shunt lambs compared to age matched Controls (A). Whereas, NOX2 mRNA levels were unchanged 4- and 8- week old Shunt lambs (B–C). There were no detectable differences in NOX4 mRNA levels in 2-, 4-, 8- week Shunt lambs compared to age matched Controls (D–F). Values are mean ± SD; n=6 control and n=6 shunt at each age; *P

Figure 5. NADPH oxidase-dependent superoxide levels in the peripheral lung of Control and Shunt lambs

NADPH oxidase-dependent Superoxide levels in peripheral lung tissue of Control and Shunt lambs at 2-, 4-, and 8-weeks of age were estimated using electron paramagnetic resonance (EPR). Developmentally there is a developmental increase in superoxide production in both the Control-(A) and Shunt-lambs (B). *P

Figure 6. Developmental changes in xanthine oxidase (XO) levels in peripheral lung in Control and Shunt lambs at 2-, 4-, and 8-weeks of age

Protein extracts (50µg), prepared from peripheral lung of Control (A) and Shunt (B) lambs at 2-, 4-, and 8-weeks of age, were separated on a 4–20% Tris-SDS-HEPES gel, electrophoretically transferred to a PVDF membrane and analyzed using a specific antiserum raised against XO. XO protein levels were normalized for loading using β-actin. A representative blot is shown in each panel. Developmentally there was a significant increase in XO protein levels in the Control lambs (A) at 8-weeks of age compared to 2-weeks of age. In addition there was a significant decrease in XO protein levels in Shunt (B) lambs at both 4-and 8-weeks compared to 2-weeks of age. Values are mean ± SD; n=6 control and n=6 shunt at each age. *P<0.05 vs. control.In vivo localization of XO protein in pulmonary vasculature of 4 week old Control and Shunt lung tissues is shown (F–G). eNOS was used as an endothelial cell marker and Caldesmon as marker of smooth muscle cells. A stack of images was taken every 2µm using confocal microscopy to identify actual co-localization. Magnification is ×20.

Figure 6. Developmental changes in xanthine oxidase (XO) levels in peripheral lung in Control and Shunt lambs at 2-, 4-, and 8-weeks of age

Protein extracts (50µg), prepared from peripheral lung of Control (A) and Shunt (B) lambs at 2-, 4-, and 8-weeks of age, were separated on a 4–20% Tris-SDS-HEPES gel, electrophoretically transferred to a PVDF membrane and analyzed using a specific antiserum raised against XO. XO protein levels were normalized for loading using β-actin. A representative blot is shown in each panel. Developmentally there was a significant increase in XO protein levels in the Control lambs (A) at 8-weeks of age compared to 2-weeks of age. In addition there was a significant decrease in XO protein levels in Shunt (B) lambs at both 4-and 8-weeks compared to 2-weeks of age. Values are mean ± SD; n=6 control and n=6 shunt at each age. *P<0.05 vs. control.In vivo localization of XO protein in pulmonary vasculature of 4 week old Control and Shunt lung tissues is shown (F–G). eNOS was used as an endothelial cell marker and Caldesmon as marker of smooth muscle cells. A stack of images was taken every 2µm using confocal microscopy to identify actual co-localization. Magnification is ×20.

Figure 6. Developmental changes in xanthine oxidase (XO) levels in peripheral lung in Control and Shunt lambs at 2-, 4-, and 8-weeks of age

Protein extracts (50µg), prepared from peripheral lung of Control (A) and Shunt (B) lambs at 2-, 4-, and 8-weeks of age, were separated on a 4–20% Tris-SDS-HEPES gel, electrophoretically transferred to a PVDF membrane and analyzed using a specific antiserum raised against XO. XO protein levels were normalized for loading using β-actin. A representative blot is shown in each panel. Developmentally there was a significant increase in XO protein levels in the Control lambs (A) at 8-weeks of age compared to 2-weeks of age. In addition there was a significant decrease in XO protein levels in Shunt (B) lambs at both 4-and 8-weeks compared to 2-weeks of age. Values are mean ± SD; n=6 control and n=6 shunt at each age. *P<0.05 vs. control.In vivo localization of XO protein in pulmonary vasculature of 4 week old Control and Shunt lung tissues is shown (F–G). eNOS was used as an endothelial cell marker and Caldesmon as marker of smooth muscle cells. A stack of images was taken every 2µm using confocal microscopy to identify actual co-localization. Magnification is ×20.

Figure 7. Xanthine oxidase (XO)-dependent superoxide levels in the peripheral lung of Control and Shunt lambs

XO-dependent superoxide levels were determined in the peripheral lung tissues from control and shunt lambs using EPR. Developmentally there were no significant differences in the XO dependent superoxide generation in either the Control (A) or Shunt lambs (B). But there was a significant increase in XO dependent superoxide generation in Shunt-compared to Control-lambs at 2-and 4--weeks (C & D) but not at 8-weeks of age (E). Values are mean ± SD; n=6 control and n=6 shunt at each age. *P

Figure 7. Xanthine oxidase (XO)-dependent superoxide levels in the peripheral lung of Control and Shunt lambs

XO-dependent superoxide levels were determined in the peripheral lung tissues from control and shunt lambs using EPR. Developmentally there were no significant differences in the XO dependent superoxide generation in either the Control (A) or Shunt lambs (B). But there was a significant increase in XO dependent superoxide generation in Shunt-compared to Control-lambs at 2-and 4--weeks (C & D) but not at 8-weeks of age (E). Values are mean ± SD; n=6 control and n=6 shunt at each age. *P