Nur77 Contributes to the Pathogenesis of Pulmonary Edema

Pulmonary edema occurs when the safety mechanisms of the lung are overwhelmed by either high transvascular pressure gradients or increased microvascular permeability, which makes a large amount of tissue fluid unable to be absorbed by the pulmonary lymph and pulmonary vein system in a very short period of time. The fluid seeps from the pulmonary capillary, and accumulates in the alveolus, thus causing serious impairment of pulmonary ventilation and ventilation function. Pulmonary edema can cause dyspnea, sitting upright, cyanosis, sweating, and paroxysmal cough and is one of the most common causes for admission to the ICU [1].


Introduction
Pulmonary edema occurs when the safety mechanisms of the lung are overwhelmed by either high transvascular pressure gradients or increased microvascular permeability, which makes a large amount of tissue fluid unable to be absorbed by the pulmonary lymph and pulmonary vein system in a very short period of time. The fluid seeps from the pulmonary capillary, and accumulates in the alveolus, thus causing serious impairment of pulmonary ventilation and ventilation function. Pulmonary edema can cause dyspnea, sitting upright, cyanosis, sweating, and paroxysmal cough and is one of the most common causes for admission to the ICU [1].

Pulmonary microvascular endothelial cells (ECs) are integral
to the alveoli-capillary barrier of the lung. The EC barrier integrity is disrupted in pulmonary edema [2]. The EC gaps allow for permeability of fluid, neutrophils and cytokines into the pulmonary parenchymal space [3]. The neutrophils that infiltrate the lungs express pro-inflammatory cytokines such as tumor necrosis factoralpha (TNF-α), interleukin-1 beta (IL-1β), and contribute to both the endothelial and epithelial integrity disruption of the barriers [4,5].
In this article, we use both in vitro and in vivo models to explore the potential roles of Nur77 in pulmonary edema.

Mice
The nuclear receptor Nur77 is actively involved in a variety of pulmonary diseases such as lung cancer, airway inflammation, ARDS and pulmonary artery hypertension. However, it remains unknown whether Nur77 plays a role in the pathogenesis of pulmonary edema. The objective of this study is to identify the relationship between Nur77 and pulmonary edema. By using quantitative real-time PCR (qRT-PCR), immunofluorescent staining and Western Blot, we found that Nur77 expression was markedly upregulated in endothelial cells in response to pulmonary edema. After the injection of SiNur77 endothelial cell edema reduces and restores its former structure. In conclusion, we believe that Nur77 plays a pivotal role in the pathogenesis of pulmonary edema.
Medical Center, Shanghai Jiaotong University and were performed in accordance with the standards established by Chinese government. Intraperitoneal injection of 0.1ml/10g adrenaline (1mg/ml) was administered to 8 weeks old male mice. Thirty minutes after injection, mice were anesthetized with isoflurane and the lungs were harvested.

Cell Isolation
Lung tissues were cut to 1mm3 and the filtrate was harvested.
5ul anti-CD31 antibody and 20ul magnetic beads were mixed for 20minutes at room temperature. Mix the filtrate and the mixture of antibody and beads in a ratio of 1:3 for 2 hours at room temperature.
Then pulmonary endothelial cells were harvested.

Quantitative Real Time-PCR (qRT-PCR)
Total RNA was extracted from lung tissues and pulmonary endothelial cells using Trizol reagent kit (Invitrogen). The RNA concentration was measured using Nano Drop 2000 (Thermo Fisher Scientific, Inc). Then qRT-PCR analysis was performed.
Briefly, cDNA was synthesized from total RNA using High Capacity cDNA Archive Kit (Applied Biosystem). qRT-PCR was

Statistical Analyses
Results are presented as mean±SD. Student's t test was employed to determine statistical significance. In all cases, P<0.05 was considered statistically significant.

Experimental Protocol and Establishment of Mice Pulmonary Edema Models
To define the role of Nur77 in the pathogenesis of pulmonary edema, we first set up mice pulmonary edema models with

Total Lung Nur77 Increased in Pulmonary Edema Models
The expression of NR4A1 gene increased under the condition of hypoxia (Figure 2A). Total lung Nur77 increased in pulmonary edema models compared with control ( Figure 2B). **P< 0.01.

Purification of Pulmonary Endothelial Cells
Specific marker of vascular endothelial cells Cdh5 was quantified by qRT-PCR. Pulmonary endothelial cells were stained by Immunofluorescent staining (VE-Cadherin/Dapi). Purified vascular endothelial cells were obtained by methods mentioned above ( Figure 3). **P< 0.01.
The expression of NR4A1 gene increased under the condition of hypoxia.

B.
Total lung Nur77 increased in pulmonary edema models compared with control.

Nur77 Increased in Edematous Pulmonary Endothelial Cells
We sought to explorethe impact of Nur77 on edematous pulmonary endothelial cells. Edematous pulmonary endothelial cells were harvested. Western Blot and qRT-PCR were performed.

Endothelial Edema Decreased After the Injection of SiNur77
After intraperitoneal injection of adrenaline in C57BL/6 mice,

Discussion
Pulmonary edema is caused by the disruption of the pulmonary endothelial barrier [13]. The incidence of pulmonary edema is high and the prognosis is poor. Pulmonary edema can lead to severe hypoxemia. Nur77 is induced in vascular ECs by several stimuli, such as hypoxia, TNF-α, and VEGF, and modulate EC growth, survival, and angiogenesis [11,12]. This is consistent with our research. Nur77 expression was markedly upregulated in endothelial cells in response to pulmonary edema. Vascular endothelial growth factor (VEGF) is reported to have profound effects by regulating the epithelial-endothelial barrier, vascular permeability, and inflammatory cytokines [14]. Elevated VEGF levels may be associated with pulmonary inflammation and edema [15]. In ECs exposed to VEGF, Nur77 is one of the most robustly upregulated genes [16]. VEGF-mediated upregulation of Nur77 is mediated through activation of the PKD/HDAC7/MEF2 pathway [17,18]. This suggests that Nur77 may damage the endothelial cell barrier and cause pulmonary edema by regulating VEFG.
In summary, Nur77 has emerged as an important regulator of inflammation in various diseases. However, its role in the pathogenesis of pulmonary edema is largely unknown. In this study, we document that Nur77 expression was markedly upregulated in endothelial cells in response to pulmonary edema. This is consistent with the theory that pulmonary edema is caused by the disruption of the integrity of ECs [4,5]. And after the injection of SiNur77, the edema and abscission of decreased EC decreased. However, it remains unclear which signal pathway does Nur77 work through and which cytokines have changed in pulmonary edema. These questions require further study. In summary, the data reported herein suggests that Nur77 plays a pivotal role in the pathogenesis of pulmonary edema.