To determine whether pharmacological inhibition of NMN synthesis contributes to TNF-α-induced EC apoptosis, ECs were pre-treated with DMSO or FK866 (NAMPT enzymatic inhibitor, 0.1-10 μM, 24 hrs) followed by TNF-α challenge (100 ng/ml, 24 hrs). Western blots showed that FK866 alone did not affect PARP-1 expression or cleavage and failed to alter PARP-1 cleavage in response to TNF-α (Figure 2A). Colorimetric measurements of caspase-3 activity confirmed that NAMPT- silenced ECs exhibit significantly higher levels of caspase-3 activation in response to TNF-α. In addition, FK866 failed to mimic the effect of NAMPT silencing on TNFα-induced EC apoptosis (Figure 2B) despite ~80% reductions in NAD levels compared to only ~50% reduction in NAD levels by NAMPT silencing (Figure S1). Thus, increased TNF-α-induced EC apoptosis and PARP-1 expression and cleavage observed in NAMPT-silenced EC are unlikely to be linked to NAD depletion (Figure S2).
Secreted NAMPT regulates TNF-α-mediated human lung endothelial cell (EC) apoptosis.
The divergence between intracellular NAMPT pharmacological inhibition and reductions in NAMPT expression (silencing) suggested either the involvement of secreted NAMPT in TNF-α-induced EC apoptosis or the potential for additional intracellular NAMPT functional effects beyond Sublingual NMN synthesis. To determine the role of EC-secreted eNAMPT, immunoprecipitation (IP) of eNAMPT from EC culture medium was followed by western blotting (Figure S3), studies that validated the presence of secreted eNAMPT at the same molecular size as intracellular NAMPT (iNAMPT) detected in EC lysates. EC NAMPT gene silencing reduced eNAMPT secretion (Figure 3A), as verified via a NAMPT ELISA (Figure 3B).
We next determined whether secreted eNAMPT increases pro-survival mechanisms in ECs. The effects of culture medium derived from NAMPT-silenced ECs (donor group) were evaluated for effects on EC apoptosis and compared to culture medium obtained from control-silenced-ECs (Figure 4A). The addition of culture medium from unsilenced ECs to TNF-α-challenged, NAMPT- silenced ECs significantly reduced caspase-3 activation (Figure 4B). In contrast, these reductions in TNF-α-induced caspase-3 activation by conditioned media were significantly less when culture medium from NAMPT-silenced ECs (with reduced levels of NAMPT in the conditioned media) was utilized, suggesting that a secreted factor(s) reduces EC apoptosis with NAMPT the likely candidate evoking these responses. The incubation of control non-silenced ECs with various concentration of FK866 (1-100 μM) for 24 hrs failed to affect basal NAMPT secretion (Figure S4) indicating that changes in NAD levels are not the cause of the reduced apoptosis.
To confirm that secreted eNAMPT is the effector regulating EC apoptotic responses, we next utilized a goat polyclonal antibody raised against NAMPT that abolishes TLR4 binding and NFκB activation (27). NAMPT-silenced EC incubated with control, conditioned medium supplemented with the NAMPT neutralizing antibody exhibited increased levels of TNF-α-induced caspase-3 activation compared to EC treated with conditioned medium supplemented with control IgG or conditioned medium alone (Figure 4C). To further examine the mechanisms by which secreted eNAMPT alters TNF-α-induced apoptosis, ECs were transfected with a NAMPT-HA plasmid and conditioned media containing ectopically-secreted fusion NAMPT protein sampled after 48 hrs with HA-tagged NAMPT detected in supernatant by IP and by ELISA (data not showed). In a separate set of experiments, ECs were pre-treated with LPS-RS (1 μg/ml) or with vehicle PBS for 1 hr, followed by supplementation with conditioned medium obtained from transfected ECs or with empty growth media EGM-2 for 48 hrs. After 24 hrs of exposure to conditioned media, ECs were challenged with TNF-α (100 ng/ml) for 24 hrs and assayed for caspase-3 activity. ECs exposed to conditioned media containing high levels of HA-NAMPT demonstrated reduced TNF-α-induced caspase-3 activation compared to ECs exposed to EGM-2 media alone. This protective effect of HA-NAMPT-containing conditioned media was attenuated by EC pre-treatment with the TLR4 inhibitor, LPS-RS. These studies indicate that secreted eNAMPT is a critical determinant in regulation of TNF-α-induced EC apoptosis via ligation of TLR4 receptor as we have published previously (27). The TLR4 receptor ligand, LPS, increased EC caspase-3 activation, however, pre-incubation of NAMPT-silenced cells with LPS (10 ng/ml) prior to TNF-α challenge not only failed to confer anti-apoptosis protection, but was additive in augmenting caspase-3 activation (Figure S6). These studies indicate that NAMPT exerts anti-apoptosis protection via unknown mechanisms that are distinct from other TLR4 agonists.
Recombinant human NAMPT (rhNAMPT) protects human lung EC against TNF-α-induced EC apoptosis. To further validate the role of eNAMPT in regulation of TNF-α-induced EC apoptosis, ECs were pre-treated with 100 ng/ml rhNAMPT for 24 hrs followed by TNF-α challenge (24 hrs) with assessment of levels of the immunoreactive PARP-1 cleaved fragment (DEVD214). rhNAMPT alone failed to alter basal levels of PARP-1 cleavage, however, rhNAMPT pretreatment reduced TNF-α-mediated PARP-1 cleavage compared to TNF-α-challenged EC without rhNAMPT pretreatment (Figure 5A). In addition, EC pre-treated with rhNAMPT followed by TNF-α challenge exhibited reduced DNA strand breaks (TUNEL assay, fluorescein) compared with TNF-α- challenged EC without rhNAMPT pretreatment (Figure 5B). rhNAMPT pretreatment effects on TNF-α-mediated PARP-1 cleavage were dose-dependent (5-80 ng/ml) (Figure S7). These results demonstrate that rhNAMPT confers resistance to pro-apoptotic stimuli such as TNF-α.