Angiotensin-converting enzyme 2/angiotensin-(1–7)/Mas axis prevents lipopolysaccharide–induced apoptosis of pulmonary microvascular endothelial cells by inhibiting JNK/NF–κB pathways

OPEN SUBJECT AREAS: GENETIC ENGINEERING GENE EXPRESSION ANALYSIS TRANSCRIPTION Received 8 August 2014 Angiotensin-converting enzyme 2/ angiotensin-(1–7)/Mas axis prevents lipopolysaccharide–induced apoptosis of pulmonary microvascular endothelial cells by inhibiting JNK/NF–kB pathways Accepted 13 January 2015 Yingchuan Li, Yongmei Cao, Zhen Zeng, Mengfan Liang, Ying Xue, Caihua Xi, Ming Zhou & Wei Jiang Published 3 February 2015 Department of Anesthesiology, the Sixth People’s Hospital Affiliated to Shanghai Jiaotong University, Shanghai 200233, China. Correspondence and requests for materials should be addressed to W.J. (jiangw@sjtu. edu.cn) ACE2 and Ang–(1–7) have important roles in preventing acute lung injury. However, it is not clear whether upregulation of the ACE2/Ang–(1–7)/Mas axis prevents LPS–induced injury in pulmonary microvascular endothelial cells (PMVECs) by inhibiting the MAPKs/NF–kB pathways. Primary cultured rat PMVECs were transduced with lentiviral–borne Ace2 or shRNA–Ace2, and then treated or not with Mas receptor blocker (A779) before exposure to LPS. LPS stimulation resulted in the higher levels of AngII, Ang–(1–7), cytokine secretion, and apoptosis rates, and the lower ACE2/ACE ratio. Ace2 reversed the ACE2/ACE imbalance and increased Ang–(1–7) levels, thus reducing LPS–induced apoptosis and inflammation, while inhibition of Ace2 reversed all these effects. A779 abolished these protective effects of Ace2. LPS treatment was associated with activation of the ERK, p38, JNK, and NF–kB pathways, which were aggravated by A779. Pretreatment with A779 prevented the Ace2–induced blockade of p38, JNK, and NF–kB phosphorylation. However, only JNK inhibitor markedly reduced apoptosis and cytokine secretion in PMVECs with Ace2 deletion and A779 pretreatment. These results suggest that the ACE2/Ang–(1–7)/Mas axis has a crucial role in preventing LPS–induced apoptosis and inflammation of PMVECs, by inhibiting the JNK/NF–kB pathways. A cute respiratory distress syndrome (ARDS) is an inflammatory response to both pulmonary and extra– pulmonary stimuli, characterized by acute onset of new or worsening respiratory dysfunction. Despite improvements in intensive care with optimal ventilation support and fluid balance, the mortality of patients with ARDS remains above 30%1,2. Diffuse pulmonary endothelial cell injury that results in impairment of the alveolar–capillary barrier, and increase in microvascular endothelial permeability, are considered central to the pathogenesis of ARDS3. The renin–angiotensin system (RAS) is a complex hormonal system and a pivotal regulator in maintaining homeostasis of blood pressure and electrolyte balance; RAS also has an important role in inflammation4. Abnormal activation of the RAS is involved in the pathogenesis of cardiovascular, renal, and lung diseases5–7. Angiotensin–converting enzyme (ACE) 2, a homologue of ACE, is a recently discovered component of the RAS8. In contrast to ACE which converts angiotensin (Ang) I (AngI) to generate AngII, ACE2 reduces the generation of AngII by catalyzing the conversion of AngII to Ang–(1–7), which attenuates the vasoconstrictive, proliferative, and inflammatory effects of AngII. Hence, ACE2 has a pertinent role in the anti–inflammatory RAS–ACE2– Ang–(1–7) axis, as it counteracts the pro–inflammatory effects of the ACE–AngII axis9,10. ACE2 is a membrane–associated aminopeptidase in vascular endothelia, renal and cardiovascular tissues, and epithelia of the small intestine and testes11,12. ACE2 is also broadly expressed in almost all kinds of cell types in the lung, including endothelial and smooth muscle cells of blood vessels, types I and II alveolar epithelial cells, and bronchial epithelial cells. There is also evidence that ACE2 has an important role in the development of ARDS. In fact, ACE2 levels positively correlated with severe acute respiratory syndrome (SARS) coronavirus infection of human airway epithelia13. In addition, ACE2–deficient mice suffered more aggravated lung injury compared with wild–type mice in models of ARDS, whereas therapy with recombinant ACE2 improved ARDS in Ace2–knockSCIENTIFIC REPORTS | 5 : 8209 | DOI: 10.1038/srep08209 1 www.nature.com/scientificreports out, especially in wild–type mice14. All these findings suggest that ACE2 may prevent lung injury and may be useful as a therapeutic agent targeting ARDS. Several studies have shown that mitogen–activated protein kinases (MAPKs) may have key roles in acute lung injury. For example, inhibition of p38 MAPK phosphorylation and activity protects against pulmonary infiltration of leukocytes as well as lung edema15. Activation of p38 MAPK appears to be an important upstream signaling event associated with tumor necrosis factor (TNF)–a– induced barrier failure in the pulmonary endothelial monolayer16. Furthermore, inhibition of p38 MAPK, but not extracellular signal regulated kinase (ERK), significantly attenuated TNF–a–induced increase of endothelial permeability17. The MAPK pathway also mediates regulation of Ace2 mRNA expression in rat aortic vascular smooth muscle cells18. Lipopolysaccharide (LPS), released from the gram–negative bacterial cell wall, contributes to pulmonary inflammation and sepsis that leads to ARDS19,20. Upon recognition by toll–like receptor 4 (TLR4) on the cellular surface, LPS activates nuclear factor–kB (NF–kB) and MAPKs cascades, leading to the release of pro–inflammatory cytokines such as interleukin (IL)–1, IL–6, and TNF–a21–23. TLR4–NF–kB signaling regulates the severity of acute lung injury (ALI)24. p38 MAPK, ERK, and NF–kB are activated during LPS– induced lung injury25. Inhibition of ERK prevents LPS–induced inflammation by suppressing NF–kB transcription activity26,27. Inhibition of p38 MAPK attenuates pulmonary inflammatory responses induced by LPS and reduces the activation of NF–kB28. ACE2 was found to be beneficial for both cardiac and pulmonary protection. For instance, ACE2 inhibited cardiac fibrosis through a reduction in ERK phosphorylation29. Telmisartan protects against heart failure by upregulating the ACE2/ANG–(1–7)/Mas receptor axis, by inhibiting expression of phospho–p38 MAPK, phospho–c– jun N–terminal kinases (JNK), phospho–ERK, and phospho– MAPK–activated protein kinase–230. Furthermore, upregulating ACE2 can lessen lung injury31, and ACE2 or angiotensin–(1–7) has an important role in preventing ARDS32. However, whether upregulation of the ACE2/Ang–(1–7)/Mas axis prevents LPS–induced apoptosis of pulmonary microvascular endothelial cells by inhibiting the MAPKs/NF–kB pathways remains unknown. For the present study, we investigated whether upregulation of ACE2 expression may prevent LPS–induced pulmonary inflammation and cytotoxicity by way of the MAPK/NF–kB signal pathway. Methods Reagents. LPS from Escherichia coli, (O127:B8) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Rabbit anti–ACE, anti–ACE2, and anti–p65, and mouse anti– phospho–p65 a (...truncated)