In this study, we successfully established a rat model of PAH with LIRI and demonstrated that CYP2J2 overexpression and EETs could reduce inflammation, oxidative stress and apoptosis caused by PAH with LIRI, and also reduce pulmonary artery pressure and improve vascular remodeling in rats with PAH with LIRI.
Inflammatory response, oxidative stress and apoptosis play an important role during LIRI. Although LIRI could be reduced by anti-inflammatory, antioxidant and anti-apoptotic action21, the development and treatment of LIRI are also affected by its comorbidities, especially when combined with PAH. PAH is characterized by pulmonary vasoconstriction and pulmonary vascular remodeling, narrowing and occlusion of small pulmonary arteries, and increased vascular resistance22, which affect the reperfusion and aggravate the inflammatory response and lung injury. The endothelial cell dysfunction and in situ thrombosis caused by PAH increase thrombosis during lung ischemia10,23, which also reduces reperfusion and aggravates the inflammatory response and lung injury. Therefore, it is particularly important to improve endothelial cell injury and vascular remodeling in PAH when treating LIRI in combination with PAH. In this study, we demonstrated that CYP2J2 overexpression and EETs could alleviate LIRI, when combined with PAH, through anti-inflammation, anti-oxidative stress and anti-apoptosis while improving PAH vascular remodeling.
In the inflammatory cascade caused by lung ischemia and reperfusion and subsequent activation of inflammatory cells, the activation of alveolar macrophages releases many inflammatory mediators which damage the vascular endothelium and alveolar epithelium. In the present study, we tested IL-1β and IL-6, which directly involved in the initiation of lung injury by inducing early inflammatory responses, releasing toxic products and increasing lung vascular permeability as proinflammatory factors24,25. Moreover, TNF-α stimulates the activation and aggregation of neutrophils26, which also plays an essential role in the acute lung injury. However, IL-10 is an anti-inflammatory factor that inhibits the inflammatory response of LIRI27. In this study, CYP2J2 overexpression significantly reduced the levels of IL-1β, IL-6, TNF-a and increased the level of IL-10 in rats with PAH combined with LIRI while improving lung injury and endothelial cell injury in rats in the PAH + IR group. In addition, CYP2J2 overexpression and EETs in the study increased PPARγ levels in vitro, suggesting that the anti-inflammatory effects of CYP2J2 overexpression and EETs may be related to the PPARγ pathway. PPARγ is considered to be the "gatekeeper" of extracellular matrix and vascular cell homeostasis, which helps maintain endothelial cell homeostasis and suppresses the inflammatory responses28. The activation of PPARγ can inhibit NF-κB downstream of PPARγ, regulate pro-inflammatory factors and cell adhesion molecules, suppress inflammatory responses and inhibit inflammatory cell adhesion to the vessel wall. Therefore, PPARγ can reduce LIRI by decreasing inflammatory factor levels, inhibiting apoptosis and alleviating oxidative stress, and reducing vascular endothelial cell injury and vascular remodeling29. These data suggest that CYP2J2 overexpression and EETs may exert anti-inflammatory effects via PPARγ activation to attenuate PAH with LIRI.
The development of LIRI is also affected by oxidative stress and apoptosis. In the present study, we found that PAH with lung ischemia-reperfusion leads to ROS synthesis and further triggers mitochondria-associated events and apoptosis. ROS play an important role in oxidative stress injury by causing structural damage to cells through protein inactivation, lipid peroxidation, and DNA damage30. In LIRI, the lack of oxygen supply during ischemia terminates ATP synthesis. Meanwhile, rapid ATP depletion leads to ATP-dependent ion pump dysfunction, decreased mitochondrial membrane potential, increased ROS synthesis, and triggered apoptosis31,32. In lung ischemia, rapid ATP depletion leads to inactivation of ATP-sensitive potassium channels and free entry of sodium, calcium and water into the cell, causing endothelial cell membrane depolarization and abnormal endothelial cell function, accompanied by NADPH oxidase activation and consequently increased ROS synthesis and apoptosis. Simultaneously, we also found that CYP2J2-EETs could attenuate apoptosis caused by PAH with LIRI, and this mechanism is at least partialy associated with the activation of the PI3K/ Akt pathway. The PI3K/ Akt pathway is an important anti-apoptotic pathway. Activated Akt activates or inhibits several downstream apoptosis-related protein families (e.g., Bcl-2 family, BAX, etc.), thereby inhibiting apoptosis33, but this protective effect could be inhibited by LY294002 (Sigma), a selective inhibitor of PI3K34. The above results suggest that CYP2J2 overexpression and EETs can activate PI3K/Akt signaling pathway, attenuate apoptosis in pulmonary artery endothelial cells, and protect against PAH with LIRI.
In addition, EETs, a sort of endothelium-derived hyperpolarizing factor (eEDHF), can maintain normal endothelial cell and vascular function and can relax vascular smooth muscle cells by activating Ca2+-sensitive K+ channels35. However, Strielkov et al. demonstrated that EETs relaxed pulmonary arteries in normoxia but constricted in anoxia36. In this study, CYP2J2 decreased the pulmonary artery pressure of the rat with PAH in combined with LIRI, which might be due to the vasoprotective effects of CYP2J2 and EETs on pulmonary arteries before IR, including relaxing pulmonary arteries and decreasing vascular remodeling. Furthermore, CYP2J2 and EETs can reduce the upregulation of cytokine-induced adhesion molecules, inhibite inflammatory cell adhesion to the vascular wall and suppress the migration of rat aortic smooth muscle cells17. In the present study, CYP2J2 gene transfection also effectively improved pulmonary artery pressure in rats with PAH combined with LIRI, and exogenous EETs improved endothelial cell injury treated with TNF-α and anoxia reoxygenation through the sequence. These results suggested that CYP2J2 overexpression and EETs could inhibit pulmonary vascular endothelial cell injury and reduce pulmonary hypertension.
Our study also has some limitations. Firstly, it is currently believed that LIRI has multiple pathogenic mechanisms, such as microvascular dysfunction, platelet activation, intracellular calcium overload, etc. Whether CYP2J2 and EETs act through other pathways needs further study. Secondly, the effects and mechanisms by which CYP2J2 and EETs act may vary depending on the concentration, but the control of CYP2J2 protein concentration could hardly be achieved by CYP2J2 gene transfection in this study, which also needs further study. In addition, the long-term effects of CYP2J2 and EETs in PAH with LIRI also need to be further investigated. Finally, the development of both PAH and LIRI is complicated. When PAH combined with LIRI, there is a possible interaction between the them, still, their roles and mechanisms have not been fully clarified, and further studies are needed to clarify this interrelationship.