Background
Intracellular nicotinamide phosphoribosyltransferase (NAMPT), also called visfatin or pre-B cell colony-enhancing factor, is the rate-limiting enzyme in the salvage pathway for nicotinamide adenine dinucleotide (NAD) biosynthesis. It influences the function of NAD-dependent enzymes such as sirtuins and poly (ADP-ribose) polymerases, which regulate cellular signaling, insulin resistance, apoptosis, oxidative stress response, and inflammation [
1]. All tissues and cells contain NAMPT. The ubiquitous expression of NAMPT implies the pleiotropic actions of proteins in cellular events [
1]. Moreover, NAMPT can also be found as an extracellular secreted form under inflammatory conditions; it is a potent extracellular proinflammatory inducer of the NF-kB pathway, toll-like receptor signaling, apoptosis, and leukocyte extravasation signaling [
2]. Because of the upregulation of NAMPT expression in a variety of inflammatory responses, it is implicated in the pathogenesis of various inflammatory disorders, such as atherosclerosis, psoriasis, inflammatory bowel disease, rheumatoid arthritis, and myocardial failure [
1]. Recent reports indicate that a low molecular-weight inhibitor of NAMPT, FK866, improves inflammation-related diseases in an animal model, including spinal cord injury, lipopolysaccharide (LPS)-induced myocardial impairment, myocardial infarction, inflammatory arthritis, endotoxic shock, and autoimmune encephalitis [
3‐
6]. These findings suggest that NAMPT could be a novel therapeutic target in various disorders, but a better understanding of its mechanisms of action is a prerequisite for the use of a NAMPT inhibitor as a therapeutic option in relevant diseases.
Despite advances in research in acute lung injury/acute respiratory distress syndrome (ALI/ARDS), the fundamental basis for ischemia-reperfusion (I/R)-evoked pathophysiology remains unclear. Recently, NAMPT was demonstrated as a potential novel biomarker in ALI/ARDS via genomic and genetic studies [
7,
8]. The expression of NAMPT in the lungs is markedly increased in human and animal models of ALI, and NAMPT levels are significantly increased in serum and bronchoalveolar lavage fluid (BALF) [
7]. Variations in
NAMPT polymorphisms were also significantly associated with susceptibility to sepsis and ALI [
7,
8]. Furthermore, heterozygous
NAMPT
+/- mice were significantly protected from the development and severity of ventilator-induced lung injury (VILI) [
9]. Moreno-Vinasco et al. recently reported that FK866, an inhibitor of NAMPT enzymatic function, had beneficial effects in VILI and LPS-induced lung injury [
10]. Matsuda et al. demonstrated that FK866 protected against intestinal I/R-associated ALI in mice. The protective effect of FK866 occurred via modulation of the NF-
κB pathway [
11]. These investigations strongly support a potentially important role for NAMPT in the inflammatory processes observed in ALI/ARDS.
I/R in the lungs can lead to ALI that was obviously different from the study of Matsuda et al. that ALI was associated with I/R at distant, nonpulmonary sites [
11]. I/R-induced ALI is the major cause of primary graft dysfunction in the early stages after lung transplantation. The morbidity and mortality associated with I/R-induced ALI is still high [
12]. Therefore, it is important to explore the molecular mechanisms of I/R-induced ALI and develop an effective therapy. In this study, we further determined the role of NAMPT enzymatic activity in the pathogenesis of I/R-induced acute lung injury using an inhibitor of NAMPT enzymatic function, FK-866.
Discussion
The results of our study demonstrated that FK866, an inhibitor of NAMPT enzymatic function, significantly ameliorated multiple indices of I/R-induced acute lung injury, including vascular barrier dysfunction, PAP, pulmonary neutrophil influx, BALF TNF-α, CINC-1, and IL-6 production, oxidative stress, apoptosis, and tissue damage compared with control animals. In addition, FK866 inhibited I/R-induced MAPK and Akt activation, IκB-α degradation, and nuclear translocation of NF-κB. Moreover, FK866 treatment had a similar beneficial effect on A549 epithelial cells subjected to H/R, corroborating the observations in the rat lung tissues. This indicates that FK-866 exerts its protective effects through multiple signaling cascades. Our experiments revealed the importance of NAMPT in the pathophysiology of I/R-induced lung injury.
After I/R injury, there was a significant increase in the NAMPT protein expression in the lung tissue, similar to the results of previous studies showing that NAMPT protein was highly upregulated in VILI and intestinal ischemia-reperfusion [
7,
11]. This could be due to infiltration of activated inflammatory cells with upregulated NAMPT expression into injured lung tissue [
9]. Upregulation of NAMPT expression is also observed in cytokine, LPS, mechanical stress–challenged human lung endothelial cells and human alveolar epithelial cells, and LPS-stimulated RAW264.7 cells [
7,
11,
18]. These results strongly support a significant role for NAMPT in the inflammatory events observed in ALI/ARDS. Moreover, we showed that NAMPT inhibition by FK-866 attenuated I/R-induced increases in NAMPT protein. This attenuation may block various I/R-associated inflammatory responses, thereby leading to a significant reduction of lung damage. This finding is consistent with a previous study showing that FK866 decreased the degree of positive staining of NAMPT in the spinal cord of mice subjected to spinal cord injury [
19].
Vast evidence has demonstrated that oxidative stress has a significant contribution in the pathogenesis of ALI/ARDS [
20]. In addition, neutrophil-derived oxygen radicals disrupt endothelial barrier function and integrity, and increase plasma leakage and lung tissue edema. Our data demonstrated that FK-866 suppressed oxidative stress as reflected by attenuating the protein carbonylation and peroxidation of membrane lipids in I/R lung tissue. In addition, FK-866 attenuated I/R-evoked increased neutrophil infiltration in the lung tissue, as evidenced by diminishing numbers of neutrophils and MPO-positive cells. This attenuation blocks the interaction between neutrophils and the endothelium, and reduces the production of proinflammatory cytokines and free radicals by activated neutrophils. Therefore, the anti-oxidative and anti-inflammatory effects of FK-866 appeared to attenuate lung edema as shown by the lower W/D and LW/BW ratios, reduced K
f, and decreased protein concentration in the BALF. These results agreed with those of other investigations showing that NAMPT inhibitors have the ability to attenuate vascular permeability and neutrophil infiltration in VILI, intestinal I/R, and LPS-induced lung injuries [
10,
11].
Previous investigations have implicated a complex network of inflammatory cytokines and chemokines in mediating, amplifying, and perpetuating the lung injury process [
12]. Our experiment showed that FK-866 significantly attenuated the increased levels of inflammatory mediators such as proinflammatory TNF-α, CINC-1 and IL-6 in the BALF after I/R-induced lung injury. Our findings were also comparable with those in previous investigations showing that FK866 alleviated TNF-α production, thereby reducing inflammation, and preventing the I/R lung injury [
11,
19]. In parallel with these results, FK-866 significantly suppressed caspase-3 activation and increased anti-apoptotic expression of Bcl-2 after I/R lung injury. The observations were also similar to those seen in intestinal I/R induced lung injury and spinal cord injury [
11,
19]. FK866 has been employed as an antitumor agent through NAD and subsequent ATP depletion, resulting in apoptosis in many malignant cell lines [
21]. TNF-
α, one of the major inflammatory mediators in ALI/ARDS, can initiate the apoptotic cascade through the death receptor/caspase pathway [
22]. Because FK866 inhibits TNF-
α production, it is reasonable to speculate that FK866 inhibits apoptosis in I/R-induced ALI, at least partly, through an indirect pathway [
11]. Furthermore, this discrepancy suggests that FK866 has different effector mechanisms for apoptosis of tumor cells and suppression of inflammatory reactions in inflammatory cells [
4].
NF-kB is a master regulator of inflammatory responses because it activates the transcription of a cascade of proinflammatory cytokines and chemokines. The activity of NF-κB is regulated primarily by the IκB family of inhibitory proteins, which are conjugated with NF-κB in the cytoplasm [
23]. Inappropriate activation of NF-κB is implicated in the pathogenesis of ALI/ARDS. Akt also participates in signaling pathways that lead to NF-κB activation and increased NF-κB-dependent transcription [
24]. Moreover, Akt-dependent events contribute to the development and perpetuation of ALI [
25]. Our prior investigations revealed that I/R-induced lung damage caused IκB degradation and NF-κB activation [
15,
26]. Hong et al. demonstrated that
NAMPT
+/− mice exhibited a dramatic attenuation of the VILI-mediated NF-κB pathway in the lungs [
9]. In the present experiment, FK866 suppressed the activation of the NF-κB signaling pathway in the rat lungs exposed to I/R by inhibiting Akt phosphorylation, degradation of IκBα and nuclear translocation of NF-κB. The inhibition of NF-κB activity resulted in decreased production of proinflammatory cytokines such as TNF-α, CINC-1, and IL-6. Furthermore, we conducted in vitro cell culture studies by using the A549 epithelial cell line to elucidate the direct effects of FK866 on alveolar epithelial cells. In line with the findings in the rat lungs, FK866 significantly inhibited IκBα degradation and, consequently, NF-κB p65 phosphorylation, and the production of IL-8 in A549 cells exposed to H/R. This finding was also consistent with an investigation by Matsuda et al. demonstrating that FK866 inhibited NF-κB activation in mouse lungs subjected to intestinal I/R, and LPS-stimulated RAW264.7 cells [
11]. However, in an experimental compression model of spinal cord injury, FK866 treatment prevented the activation of NF-κB but not IκBα degradation [
19]. Therefore, the precise molecular mechanisms by which NAMPT inhibition exerts its effect in the NF-κB signaling pathway need clarification.
The activation of MAPKs such as p38, ERK, and JNK is implicated in the inflammatory process of ALI/ARDS. The inhibition of p38, ERK, and JNK MAPK, effectively diminishes LPS and peritonitis-induced lung inflammation [
27‐
29]. The MAPK signaling pathways are regulated by opposing regulatory repressors from MKP-1 [
30]. MKP-1 knockout mice had increased inflammatory responses with higher levels of inflammatory mediators and more episodes of multiple organ failure after LPS challenge [
31]. In our previous study, I/R induced phosphorylation of p38, ERK, and JNK; it also decreased the level of MKP-1 protein in lung tissue [
17]. In contrast, FK-866 treatment activated MKP-1 expression and interrupted I/R-induced activation of MAPK. This may consequently restrain widespread inflammation in I/R-induced lung injury. Extracellular NAMPT is reported to trigger p38, ERK, and JNK phosphorylation and stimulates diverse biological processes in various types of cells [
1,
32]. However, whether intracellular NAMPT triggers the same intracellular pathways remains unknown.
The function of NAMPT in various diseases is not completely recognized. One reason for this lack of clarity could be the inability to discriminate between the intracellular and extracellular actions of NAMPT. NAMPT is enzymatically active both intracellularly and extracellularly [
32]. Extracellular NAMPT may also act in a non-enzymatic way to regulate activation of inflammatory cells by increasing surface expression of costimulatory molecules and inducing IL-1β, IL-6, and TNF-α production through a currently unidentified membrane receptor [
32]. The fundamental pathological event of ischemic stroke is the loss of blood supply and subsequent oxygen/nutrition shortage, which are similar in key pathophysiological processes to I/R lung injury. Accumulating evidence from in vitro and in vivo experiments reveals that NAMPT provides cerebral protection in ischemic stroke [
3]. NAMPT inhibition exacerbated brain infarction in a rat model of ischemic stroke, whereas local NAMPT overexpression in the brain and NAMPT enzymatic action protected against ischemia-induced cerebral strokes [
3]. Therefore, further investigations are needed to explore these contradictory findings.
Acknowledgements
Not applicable.