Understanding the molecular mechanism that induces pulmonary hypertension in neonates is important for the development of improved therapeutics. In this current study, we used the chronically hypoxic calf model of pulmonary hypertension to evaluate the activity of EC-SOD in a severe neonatal disease model and to better understand the role for extracellular O2.− in the upregulation of Egr-1, a redox sensitive transcription factor implicated in vascular remodeling. We tested the hypothesis that loss of EC-SOD in chronically hypoxic calves leads to extracellular O2.− -mediated upregulation of Egr-1 via activation of MAPK pathways. We tested tissue from chronically hypoxic calves to demonstrate the decrease in EC-SOD in the pulmonary artery in vivo and then treated isolated pulmonary vascular smooth muscle cells with xanthine oxidase to show that ROS generated outside the cell could upregulate Egr-1 via activation of MAPK/ ERK1/2. We used human PASMC following siRNA knock-down of EC-SOD to confirm the impact of EC-SOD expression on Egr-1 expression and SMC proliferative ability. Our data provide new insight into the importance and targets of EC-SOD and extracellular O2.− in chronic hypoxic pulmonary hypertension.
We previously reported that EC-SOD activity is impaired following exposure to chronic hypoxia, and overexpression of lung EC-SOD in two murine models of pulmonary hypertension protected against pulmonary vascular remodeling and prevented the early upregulation of the redox sensitive transcription factor, Egr-1 
. In this study, we report that total SOD activity levels in the calf lung and pulmonary artery were very low compared to the mouse lung, and extracellular SOD activity decreased in the pulmonary artery of calves exposed to 2 weeks of chronic hypoxia. The low SOD activity level measured in the calf pulmonary artery tissue was also substantially less than published activity levels reported for baboon and human pulmonary artery; though we report, similar to the baboon and human, that EC-SOD activity accounted for the majority of the total SOD activity in the pulmonary artery under normal conditions 
. The low activity levels of total SOD may be in part due to the young age of the calves, as lung antioxidant defenses are known to be low early in life and increase in the perinatal period to prepare for the relative hyperoxia of room-air breathing. Consistent with this, we previously reported in the developing rabbit that EC-SOD activity in the lung increased during the first month of life 
. Several pieces of data support the premise that the low SOD activity is associated with an increase in oxidative stress in the pulmonary artery of the chronically hypoxic calf. The ratio of GSH/GSSG was low in the calf pulmonary artery and decreased further with hypoxia. This result is similar to our published observation in mice in which the GSH/GSSG ratio decreased by 50% in response to hypoxia 
. In chronic hypoxic mice, where we were able to examine a time-course, we had reported an early increase in EC-SOD expression and activity, which may reflect an adaptive response to combat increased oxidant stress 
. Interestingly, in this study, we measured an increase in intracellular SOD (IC-SOD) activity in the calf PA at 2 weeks of hypoxia. This may also be adaptive in response to increased intracellular oxidative stress. It is possible that intracellular SOD activity would decrease over time, as we observed in the lung of chronically hypoxic mice over a 5 week period, but the large animal models are limited by cost and the longer exposures were not feasible. The loss of EC-SOD activity was even more pronounced when considered as the percentage of total SOD activity, decreasing its contribution to total SOD activity from 60% to less than 10%. We speculate that an increased susceptibility to oxidative stress due to the low total SOD activity along with a further loss of extracellular EC-SOD could contribute to the significant remodeling in the medial and adventitial layer of the pulmonary arteries observed in the neonatal calf compared to the modest vascular remodeling characteristic of chronically hypoxic mice 
To test the impact of extracellular O2.−
on Egr-1 expression in PASMC, we treated cells with XO, as an enzymatic source of O2.−
. This exogenous model has potential relevance to the in vivo
setting, as XO is upregulated in the hypoxic pulmonary circulation and can contribute to pulmonary hypertension 
. A key finding in the cell culture experiments was that exogenous generation of extracellular O2.−
by XO in smooth muscle cells strongly upregulated Egr-1 mRNA and protein expression. Furthermore, cells isolated from chronically hypoxic calves had a more marked response to XO than cells isolated from normoxic calves. Since the SMC are a major source of vascular EC-SOD and we detected less EC-SOD in the pulmonary artery of chronically hypoxic calves, we speculate that the loss of EC-SOD in these cells contributed to the enhanced upregulation of Egr-1 in response to XO. This was supported by our finding in human PASMC in which knock-down of EC-SOD increased Egr-1 mRNA and protein. In the bovine cell culture model, pretreatment with both SOD and catalase was required to fully block the XO-induced signal. This indicates that H2
, which also will increase with XO treatment due to the rapid dismutation of O2.−
or its direct generation of H2
, was capable of inducing Egr-1 expression in this system. In contrast, in the human cells, knocked down of EC-SOD expression was sufficient to increase Egr-1, consistent with our previous observation in the mouse model, that overexpression of EC-SOD in the lung blunted the hypoxic induction of Egr-1 expression
. These data demonstrate that we can use XO as a model to test how extracellular ROS can regulate Egr-1 mRNA expression; however, we must also consider the impact of exogenous vs. endogenous sources of ROS and the model when we interpret the data. Our data support the conclusion that extracellular O2.−
may upregulate Egr-1 directly or following its dismutation to H2
. Consistent with our cell culture findings, one published study reported that the exogenous administration of H2
in cardiac cells also increased Egr-1 mRNA and protein expression 
. In our study when SOD+catalase was compared to catalase pretreatment alone, there is a small difference, suggesting that O2.−
may have an effect independent of its dismutation to H2
. It is also possible that EC-SOD modulates Egr-1 expression by regulating NO bioavailability. This mechanism was not tested in this study. Overall, these data indicate that while the bovine cell culture experiments do not fully mimic the in vivo
setting, extracellular O2.−
, either directly or indirectly following its dismutation to H2
, can upregulate Egr-1 in smooth muscle cell, and provides an opportunity to further understand how O2.−
can regulate the redox sensitive transcription factor, Egr-1.
Published studies have shown enhanced induction of Egr-1 with hypoxia 
, For example, fetal bovine pulmonary artery fibroblasts exhibited an increase in Egr-1 mRNA by Northern blot analysis following four hours of 3% oxygen 
. However, we observed that exposure of PASMCs to hypoxia (1% for 1–4 hours), in addition to generating lower concentrations of extracellular O2.−
than XO, also had a much smaller impact on Egr-1 mRNA expression. This demonstrates that hypoxic induction of Egr-1 is specific to tissue or cell types, particularly given the known heterogeneity in cells within the vessel wall. The response can also vary with the hypoxic condition and the extent of hypoxic exposure. We speculate that an important source of extracellular ROS in the pulmonary circulation is that generated by recruited or resident inflammatory cells in response to hypoxia. Thus, a short bolus exposure to hypoxia in a single vascular cell type does not mimic the in vivo model system. While we focused in this study on the impact of extracellular O2.−
on Egr-1 regulation, our data with antimycin A suggest that under certain conditions, intracellular ROS production can also modulate the expression of this transcription factor. Further studies will dissect the origin and sources of extracellular ROS, distinct from cytosolic or mitochondrial sources, in the lung and pulmonary circulation in response to hypoxia. Since the use of hypoxia in cultured vascular cells was limited by the low levels of extracellular O2.−
generated and a minimal impact on Egr-1 regulation, we selected XO treatment as a model to test whether extracellular O2.−
upregulated Egr-1 via the activation of MAPK pathways.
There is extensive data implicating the MAPK/ERK1/2 pathway in the regulation of Egr-1 and in the pathogenesis of chronic hypoxic pulmonary hypertension 
. Furthermore, it is well-established that reactive oxygen species can activate MAPK through its phosphorylation 
. These two observations formed the basis of our decision to test whether ROS generated outside the cell upregulated Egr-1 via MAPK. We found that extracellular ROS, generated by XO, phosphorylated ERK1/2, while inhibition of the ERK1/2 pathway prevented the upregulation of Egr-1. This identifies the importance of MAPK/ERK pathway in the redox regulation of Egr-1 in neonatal PASMC. A recent study showed that an increased extracellular oxidation in vascular smooth muscle cells activated the EGFR membrane receptor, leading to phosphorylation of ERK1/2 and activation of downstream transcription factors 
. Our work thus provides a basis for future studies to better understand how the activity of EC-SOD may regulate extracellular redox state and further dissect its targets leading to ERK1/2 phosphorylation and regulation of Egr-1. Accordingly, targeting Egr-1 regulation may represent a novel therapeutic strategy to prevent PA remodeling.
The regulation of Egr-1 has been implicated in vascular remodeling in a number of models including the calf model of chronic hypoxic pulmonary hypertension. For example, upregulation of Egr-1 contributes to cyclin D1 expression and hypoxia-induced cell proliferation in fetal lung fibroblasts and stimulates insulin-like growth factor-1 receptor, resulting in vascular remodeling of vein grafts 
. In addition, a pro-proliferative and anti-apoptotic phenotype has been attributed to SMC in pulmonary hypertension. Consistent with these studies, we were also able to show that knock-down of EC-SOD in human PASMC upregulated cyclin D1 and ERK1/2 activation, augmented proliferation and blunted apoptosis, providing direct evidence for EC-SOD in regulating PASMC phenotype.
In summary, we report that the neonatal calf has low SOD activity levels in the pulmonary artery and EC-SOD activity decreases further in the calf with pulmonary hypertension secondary to chronic hypoxia. The loss of EC-SOD is associated with an increase in Egr-1 mRNA in the pulmonary artery. We thus tested the impact of extracellular O2.− and its product, H2O2, on Egr-1 expression in vascular smooth muscle and found that exogenous production of these ROS, via xanthine oxidase, upregulated Egr-1. Hypoxia itself had a minimal effect on either Egr-1 expression or O2.− production. Extracellular ROS together with activation of ERK1/2 by phosphorylation regulated the increase in Egr-1. Therefore, targeting Egr-1 by controlling extracellular ROS generation and thereby balancing the cellular redox status could be a potential therapeutic pathway for pulmonary artery remodeling. Our data provided new insight into the role of extracellular O2.− and EC-SOD in the pathogenesis of pulmonary hypertension.