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Mechanisms of Susceptibility to Hyperoxia-induced Lung Injury in Adults and Neonates

Environmental Genetics

Reactive oxygen species (ROS) have been implicated in the pathogenesis of many acute and chronic clinical disorders such as ALI and ARDS, bronchopulmonary dysplasia (BPD), ischemia-reperfusion injury, atherosclerosis, neurodegenerative diseases, and cancer. The lung is particularly at risk to the toxic effects of ROS because it interfaces with various oxidants such as environmental pollutants and cigarette smoke. ROS cause cellular damage in the lung by oxidizing nucleic acids, proteins, and membrane lipids. Hyperoxia (80-100 percent O2) causes inflammation and extensive death of capillary endothelial and alveolar epithelial cells that result in noncardiogenic pulmonary edema and severe impairment of respiratory functions.

 

Inter-strain differences in hyperoxic lung injury indicate that genetic background is an important risk factor. Furthermore, evidence supports genetic predisposition in some individuals to ALI and BPD, both of which have an etiology associated with oxidant stress. Previous studies in our lab used positional cloning of susceptibility to ALI phenotypes in adult mice to identify the candidate susceptibility gene NF-E2 related factor 2 (Nrf2), a transcription factor for antioxidant response element (ARE)-mediated gene expression and regulation ( Cho et al, 2002  ). Proof of concept investigations validated an important role for Nrf2 in this ( Cho et al, 2002  ) and other lung disease models (e.g. Cho et al, 2004  ; (Cho et al, 2009  ).

 

BPD is a chronic lung disease and common outcome that develops in ~20% of very low birth weight infants each year in the U.S. Lung injury in BPD is thought to result from early developmental arrest which is associated with prenatal exposure and genetic factors ('new' BPD) or from structural damage of relatively immature lungs at saccular phase ('old' BPD) that receive respiratory support with mechanical ventilation and prolonged oxygenation. Major pathologic features of BPD are impaired alveolarization leading to simplified air space, inflammation, and respiratory distress. Angiogenesis proteins including vascular endothelial growth factor (VEGF), keratinocyte growth factor, and matrix metalloproteases (MMPs) including MMP-9 are essential in lung development to protect against BPD pathogenesis. In contrast, cathepsin S, transforming growth factor (TGF)-β and cytokines such as IL-1β contribute to lung injury in experimental BPD. Heritability estimates for BPD are as high as 80 percent, but specific genetic mechanisms are unclear.

 

The overall objective of this project is to identify mechanisms that modify susceptibility to oxidant stress-induced lung injury in adults and neonates. The specific aims of this project are to:

 

  1. determine molecular mechanism(s) of cardiopulmonary responses to hyperoxia in adult mice
  2. determine mechanisms of susceptibility to hyperoxia-induced BPD phenotypes in neonatal mice
  3. determine whether functional SNPs in candidate susceptibility genes associate with risk of ALI/ARDS and BPD

 

Ongoing Projects in the Laboratory:

Mechanisms of cardiopulmonary responses to hyperoxia in adult mice

 

We previously completed global lung gene expression analysis of Nrf2+/+ and Nrf2-/- mice and found putative downstream molecular mechanisms of O2 toxicity and Nrf2-mediated ALI protection ( Cho et al, 2005  ). The Nrf2-dependent novel genes regulated during the development of hyperoxia-induced ALI included Pparg (or Nr1C3) which encodes peroxisome proliferator activated receptor gamma (PPARγ). Subsequently, we characterized the mechanisms through which Nrf2 regulates PPARγ, and determined the function of PPARγ in the pathogenesis of hyperoxia-induced lung injury ( Cho et al, 2010  Cho et al, 2010). Results demonstrated an essential protective role for Nrf2-driven PPARγ against ALI in mice (Figure 1) and new insights into therapeutic potential of PPARγ and its agonist in oxidative and inflammatory disorders.

 

Suggested mechanisms for Nrf2-mediated protection of ALI through induction of PPARγ

Figure 1. Suggested mechanisms for Nrf2-mediated protection of ALI through induction of PPARγ.


 

The pulmonary and cardiovascular systems are known to be regulated in 'concert' indicating that alterations in cardiovascular function may play a critical role in development of pulmonary morbidity and mortality caused by hyperoxia. Understanding the cardiovascular system response to pulmonary oxidant challenge may have important clinical implications. Previously, we identified significant QTLs for HR/HRV parameters in quiescent inbred and RI strains ( Howden et al, 2008  ). In this study, radio-telemetry transmitters were surgically implanted in mice that are susceptible [B6 and A/J] and resistant [C3 and DBA/2J (D2)] to hyperoxic lung injury and mice were exposed to hyperoxia (>95 percent O2) or air. We found reduced minute ventilation (VE), HR, and total power (TP) of HRV [composed of low frequency (LF) and high frequency (HF)] in all strains during O2.

 

Interestingly, the lag time before these changes began was strain dependent (Figure 2). Notably, changes in VE began ~10 h prior to onset of severe lung injury in B6 mice, suggesting that HR reduction could be used as a predictive tool for risk of lung injury development. We then performed linkage analyses of HRV responses to hyperoxia in recombinant inbred strains and identified significant QTLs on chromosomes 3, 5, and 9, each of which has candidate susceptibility genes. This is the first study to describe cardiopulmonary physiology in real time to continuously inhaled O2 in laboratory animals. Changes in HR/HRV are useful to predict adverse pulmonary outcomes in response to hyperoxia, and have important implications for prognosis of severe ALI/ARDS. Furthermore, QTLs and candidate genes for differential susceptibility in HR and HRV changes by O2 provide insight into genetic mechanisms associated with cardiac responses to hyperoxia.

 

Differential minute ventilation (A) and heart rate (B) responses to hyperoxia in 4 inbred strains of mice

Figure 2. Differential minute ventilation (A) and heart rate (B) responses to hyperoxia in 4 inbred strains of mice. Values are means from 20 min of recording of pulmonary function data or ECG per h ± SEM (n=8/group).


 

Mechanisms of hyperoxia-induced BPD pathogenesis in neonatal mice

 

This new project is an integrated, translational program focused on mechanisms of development of BPD phenotypes in mice and a prospective cohort of very low birthweight children and their parents recruited in Buenos Aires, Argentina in collaboration with Fernando Polack, Ph.D., (Vanderbilt).

 

Extensive lung development takes place in preterm newborns with body weight < 1000g (2.5 lb) born at 24 to 36 wk of gestation. Critical morphologic processes in this saccular phase include widening of distal airways to prepare subsequent formation of alveoli (the alveolar phase), differentiation of type 1 and 2 pneumocytes, and thinning of the air-blood barrier. Therapeutically administered hyperoxia is considered to be a contributing factor in development of BPD, and ROS are implicated in its pathogenesis. Because previous studies identified a protective role for Nrf2 in oxidant-induced injury in adult mice, we hypothesized that Nrf2 is also important in pathogenesis of BPD phenotypes in neonates. Hyperoxia enhanced mortality and growth retardation (65% vs. 85% b.w. relative to air controls) in Nrf2-/- relative to Nrf2+/+ mice. Nrf2-/- neonates also developed significantly greater inflammation, protein edema, and cell death (lysis and apoptosis) after 3 d O2. Hyperoxia caused exudative-phase diffuse alveolar damage in 50% of neonatal Nrf2-/- lungs examined, while no lungs from Nrf2+/+ mice had this severe pathology. Alveolar development in Nrf2+/+ mice after 3 d O2 was comparable to air controls with branched septum and alveoli present in 67% of mice. Importantly, fewer Nrf2-/- neonates had developing multilobular alveoli and branched septi after 3 d O2. Genome-wide mRNA expression analyses also identified a number of major functional categories that were Nrf2-dependent during lung development and during hyperoxia exposure. Collectively, these results suggest an important role for Nrf2 in normal lung development as well as during BPD pathogenesis (Figure 3).

 

Proposed role for Nrf2 in saccular to alveolar stage transition and in BPD pathogenesis learned from mice

Figure 3. Proposed role for Nrf2 in saccular to alveolar stage transition and in BPD pathogenesis learned from mice.



Association of functional polymorphisms in susceptibility genes with ALI/ARDS

 

An important attribute of inbred mice for modeling environmental lung diseases is the homology between human and mouse genomes. Therefore, identification of the chromosomal location of susceptibility genes in the mouse provides the basis for potentially localizing a homologous gene in the human, and may lead to a means to identify susceptible individuals and/or design strategies to protect against oxidant injury. We have taken advantage of this homology to translate our findings in the hyperoxia susceptibility model to human acute lung injury disease.

 

To test whether human Nrf2 is an important determinant of ALI/ARDS, we initially re-sequenced Nrf2 in four different ethnic populations and identified three new Nrf2 promoter polymorphisms at positions -617 (C/A), -651 (G/A) and -653 (A/G) ( Marzec et al, 2007  ). Variations at -653/-651 and -617 are predicted to alter the consensus recognition sequences for myeloid zinc finger-1 (MZF-1) and Nrf2, respectively, perhaps affecting Nrf2 transcription. We then found that promoter SNPs C–617A or G–651A affect basal level expression of Nrf2, thereby attenuating ARE-mediated gene transcription. Importantly, with Dr. Jason Christie (U Penn) we tested for association of functional SNPs (-617, -651) with risk for ALI in a blunt force trauma cohort. Significantly higher risk for developing ALI after major trauma was found in patients with -617 A SNP [OR 6.44; 95% CI 1.34, 30.8; p = 0.021] relative to patients with wild type (-617 CC).

 

A major ARE-responsive effector antioxidant/phase II gene in the lung is NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 catalyzes two-electron reduction of a variety of quinone compounds, which prevents generation of free radicals and ROS, and protects cells from oxidative damage. NQO1 is highly inducible by oxidant stress, xenobiotics including aromatic hydrocarbons and certain industrial acrylates, and antioxidants such as sulforaphane. We identified three novel NQO1 promoter SNPs (-1103, -1221, -1293) and found that the A-1221C SNP decreased in vitro transcription of NQO1 basally and after exposure to oxidant stressors ( Reddy et al, 2009  ). The -1221 SNP is predicted to disrupt binding of the transcription factor sterol regulatory element-binding protein 1 (SREBP-1), but confirmation is necessary. In the same cohort described above, A−1221C significantly associated with lower incidence of ALI after major trauma [unadjusted OR 0.48 relative to AA genotype (95% CI 0.25, 0.93; p=0.029)].

 

These translational investigations (Figure 4) provided novel insight to mechanisms of susceptibility to ALI, and may help to identify patients who are predisposed to develop ALI under at-risk conditions, such as trauma and sepsis. Furthermore, these findings may have important implications in other human diseases.

 

Schematic of the translational study design that identified Nrf2 (and subsequently NQO1) as a candidate gene for acute lung injury (ALI) in mouse and human populations
Figure 4. Schematic of the translational study design that identified Nrf2 (and subsequently NQO1) as a candidate gene for acute lung injury (ALI) in mouse and human populations ( Marzec et al, 2007  ).

 

Additional Investigations:

 

  • Genetic and genomic mechanisms of BPD pathogenesis in neonatal mice
  • Neonatal exposure to hyperoxia and effects on adult responses to environmental exposures
  • Prevention of murine ALI and BPD phenotypes by Nrf2 activating phytochemical antioxidants
  • Effect of BPD phenotypes no adult disease and a role for Nrf2
  • Evaluate the role of additional candidate genes in ALI/ARDS and high-risk BPD populations

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