A growing body of data indicates

A growing body of data indicates that endothelial NOS (eNOS) is a rate-limiting enzyme for the synthesis of nitric oxide (NO), its downstream Tenovin-1 receptor molecule. High pathological concentrations of NO produced from inducible NO synthase (iNOS) induce apoptosis, whereas a reduction in the concentrations of NO by eNOS attenuates apoptosis [21,22]. Further research has shown that TNF-α induces apoptosis via concentration-dependent iNOS expression and NO production in neonatal mouse cardiomyocytes [23]. Several recent studies have demonstrated that hormones such as estrogen cause eNOS phosphorylation and result in eNOS release through the PI3K/Akt-dependent pathway [24]. Dimmeler showed that eNOS is continuously expressed in cardiomyocytes and is activated by calcium, calmodulin or Akt [[25], [26], [27]]. Akt is downstream of PI3K and is phosphorylated and activated by a number of growth factors, cytokines and hormones. Furthermore, we found that calpain decreases Akt activity by shearing the heat shock protein Hsp90 in septic rats/mice [13,28]. However, the role of Akt in sepsis is debatable. Many researches indicated that PI3K/Akt pathway was activated in the sepsis and cardiac dysfunction [29,30,31]. The results of the Akt inhibitor wortmanner in septic rat were inconsistent with the inhibitors alone in mice as we once reported [13] and were not as we expected, suggesting the existence of compensatory mechanisms or other signaling pathways. The involved mechanism need to be further explored. A recent study also demonstrated that eNOS/NO depressed inflammation and inflammatory factors (TNF-α, COX-2, IL-1, etc.) [32,33]. The decrease in eNOS in the vascular endothelium is often accompanied by an increase in TNF-α in sepsis mice [34], but there have been inconsistent reports [35]. Considering the importance of the Akt/eNOS/NO pathway, in particular, the activation of Akt and eNOS, we specifically investigated the relationship among p-Akt/Akt, p-eNOS/eNOS and iNOS in myocardial tissue under treatment with LPS, LPS plus wortmannin or LPS plus L-NAME. We observed that a decrease in p-Akt protein by pretreatment with wortmannin in addition to a decrease in p-eNOS and iNOS protein by pretreatment with L-NAME in septic rats. In addition, NO production is dependent on iNOS levels in septic rats, the result is consistent with a previous report [21,22]. Therefore, these findings support the view that calpain activates NF-κB and further excites TNF-α and leads to cardiac dysfunction in sepsis.
Conflict of interest
Authors\' contributions
Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (No. 81470521).
Introduction Endothelial dysfunction (ED) is an early indicator of numerous vascular diseases, as well as a key contributor to the pathophysiology of ischemia–reperfusion (I/R) injury [1]. Nitric oxide (NO) is a key endogenous substance to maintain normal endothelial function, and endothelial NO synthase (eNOS) is the key protease to regulate NO production [2]. Kruppel-like factor 2 (KLF2) is a laminar flow-inducible transcription factor primarily expressed by endothelial cells that play an important role in the regulation of endothelial function [3,4]. Studies have shown that KLF2 induces eNOS expression and increases NO production in cultured human umbilical vein endothelial cells (HUVECs) and could also activate eNOS and OH-1 in liver I/R injury. Studies have also shown that KLF2 expression is mediated by the phosphorylation of extracellular signal-regulated kinase 5 (ERK5), and KLF2 subsequently activates eNOS. However, the mechanism by which KLF2 activates eNOS remains to be elucidated [[5], [6], [7]]. It is well known that vascular NO is produced by eNOS by converting l-arginine to l-citrulline in the presence of molecular oxygen [8]. Tetrahydrobiopterin (BH4) is an essential cofactor of eNOS that aids in the coupling of l-arginine and the heme group within the oxygenase domain of eNOS. In this coupled state, eNOS produces NO to facilitate normal blood flow and to maintain an anti-inflammatory and anti-thrombotic vascular endothelial surface. A lack of BH4 can lead to eNOS uncoupling. In this regard, eNOS uncoupling may not only induce NO insufficiency but also contribute to oxidative stress under various pathological conditions, such as I/R [9,10]. In addition, increased oxidized glutathione (GSSG) levels caused by oxidative stress can induce dose-dependent eNOS S-glutathionylation (yielding eNOS-SG), aggravating eNOS uncoupling in I/R-induced ED [11]. Phosphorylation of eNOS also influences the regulation of NO levels. The phosphatidylinositol-3-kinase (PI3K)/Akt/eNOS pathway is involved in the protective effect of ischemic postconditioning. Akt, which is activated via phosphorylation by activated PI3K, phosphorylates eNOS on serine 1177 (yielding p-eNOS), thereby activating this enzyme to produce NO [12]. However, no reports on the relationship between KLF2, BH4, the PI3K pathway, and subsequent effects on eNOS activity are available. Therefore, it remains to be elucidated how KLF2 regulates eNOS activity and exerts its vascular protective function. The aim of our study was to clarify the relationship between KLF2, eNOS uncoupling, and the Akt pathway.