Direct Extracellular NAMPT Involvement in Pulmonary Hypertension and Vascular Remodeling. Transcriptional Regulation by SOX and HIF-2α

Abstract

We previously demonstrated involvement of NAMPT (nicotinamide phosphoribosyltransferase) in pulmonary arterial hypertension (PAH) and now examine NAMPT regulation and extracellular NAMPT’s (eNAMPT’s) role in PAH vascular remodeling. NAMPT transcription and protein expression in human lung endothelial cells were assessed in response to PAH-relevant stimuli (PDGF [platelet-derived growth factor], VEGF [vascular endothelial growth factor], TGF-β1 [transforming growth factor-β1], and hypoxia). Endothelial-to-mesenchymal transition was detected by SNAI1 (snail family transcriptional repressor 1) and PECAM1 (platelet endothelial cell adhesion molecule 1) immunofluorescence. An eNAMPT-neutralizing polyclonal antibody was tested in a PAH model of monocrotaline challenge in rats. Plasma eNAMPT concentrations, significantly increased in patients with idiopathic pulmonary arterial hypertension, were highly correlated with indices of PAH severity. eNAMPT increased endothelial-to-mesenchymal transition, and each PAH stimulus significantly increased endothelial cell NAMPT promoter activity involving transcription factors STAT5 (signal transducer and activator of transcription 5), SOX18 (SRY-box transcription factor 18), and SOX17 (SRY-box transcription factor 17), a PAH candidate gene newly defined by genome-wide association study. The hypoxia-induced transcription factor HIF-2α (hypoxia-inducible factor-2α) also potently regulated NAMPT promoter activity, and HIF-2α binding sites were identified between −628 bp and −328 bp. The PHD2 (prolyl hydroxylase domain-containing protein 2) inhibitor FG-4592 significantly increased NAMPT promoter activity and protein expression in an HIF-2α–dependent manner. Finally, the eNAMPT-neutralizing polyclonal antibody significantly reduced monocrotaline-induced vascular remodeling, PAH hemodynamic alterations, and NF-κB activation. eNAMPT is a novel and attractive therapeutic target essential to PAH vascular remodeling.

Clinical Relevance

Extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a proinflammatory cytokine, is increased in pulmonary arterial hypertension (PAH) and linked to PAH indices. NAMPT is transcriptionally regulated by PAH-relevant stimuli via HIF-2α (hypoxia-inducible factor-2α), SOX, and others and promotes endothelial-to-mesenchymal transition and pulmonary vascular remodeling, which is significantly attenuated by eNAMPT-neutralizing antibodies, in vivo and in vitro. Blocking extracellular NAMPT by eNAMPT antibody is a potential novel therapeutic strategy for PAH.

Pulmonary arterial hypertension (PAH) is a progressive disease affecting the pulmonary precapillary vasculature, leading to severe pulmonary vascular remodeling, persistently increased pulmonary vascular resistance (PVR), and right heart failure (1). Despite recent progress, the underlying mechanistic basis for pulmonary vascular remodeling in PAH remains unclear. Postulated contributors to PAH pathogenesis include a focus on genetic susceptibility (2), the role of inflammation (3–7), and metabolic shifts in vascular cells (8). Additional insights into the advanced vascular remodeling in PAH and identification of novel PAH targets and biomarkers are desperately needed.

We previously used genomics-intensive approaches to explore candidate genes influenced by mechanical stress (9, 10) and identified the nicotinamide phosphoribosyltransferase gene, NAMPT, as a novel candidate gene and biomarker in acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (11–14). NAMPT, also termed “visfatin” or “pre–B-cell colony-enhancing factor,” was originally characterized as a B-cell maturation factor with intracellular enzymatic activity (iNAMPT) that is the rate-limiting component of mammalian nicotinamide adenine dinucleotide biosynthesis (11, 15). NAMPT is also linked to cellular adaptation to stress responses, including resistance to senescence and apoptosis, increased cell proliferation, and regulation of the cellular redox state (16).

We recently highlighted the role of extracellular NAMPT (eNAMPT) in innate immunity as a proinflammatory cytokine that increases inflammatory cell survival and inflammatory cytokine production via ligation of TLR4 (Toll-like receptor 4) and subsequent NF-κB activation (17). In the vasculature, eNAMPT promotes endothelial cell (EC) survival (18) and angiogenic activity as well as smooth muscle cell survival (15). We previously reported a potential role for NAMPT in PAH, demonstrating that both heterozygous Nampt^+/− mice and the iNAMPT enzymatic inhibitor, FK866, reduced hypoxia-mediated PAH (19). However, the role of eNAMPT was not directly assessed.

In the present study, we focused on the mechanistic participation of eNAMPT in human PAH and in preclinical models of PAH. Subjects with PAH exhibited significant increases in plasma eNAMPT concentrations that were linked to clinical PAH indices, including diastolic ventricular stiffness. PAH-relevant stimuli significantly increase EC NAMPT promoter activity and NAMPT protein expression via STAT5 (signal transducer and activator of transcription 5), SOX18 (SRY-box transcription factor 18), SOX17 (SRY-box transcription factor 17), and HIF-2α (hypoxia-inducible factor-2α) transcription factors. eNAMPT directly increased endothelial-to-mesenchymal transition (EndMT), a cellular process that contributes to PAH vascular remodeling, and an eNAMPT-neutralizing polyclonal antibody (pAb) dramatically reduced PAH severity and vascular remodeling in vivo in a preclinical rat model of monocrotaline (MCT)-induced PAH (histologic and hemodynamic data, NF-κB activation). These studies are very consistent with an essential role for eNAMPT as a highly novel therapeutic target in amelioration of the inflammatory cascade activation, vascular remodeling, and right ventricular (RV) dysfunction essential to PAH pathobiology.

Methods

Assessment of Patients with and without Idiopathic Pulmonary Arterial Hypertension

Idiopathic pulmonary arterial hypertension (IPAH) was defined according to the 2015 European Society of Cardiology/European Respiratory Society guidelines. All subjects were prospectively recruited at the University of Arizona and provided written consent to participate with the approval of the University of Arizona Institutional Review Board. Plasma samples were obtained from 68 subjects with group 1 IPAH who had undergone cardiac catherization, 13 subjects without IPAH (systemic lupus, scleroderma, or coccidioidomycosis) who also underwent cardiac catherization, and 70 healthy subjects. Pulmonary hemodynamic indices were assessed as previously described (20). Plasma NAMPT concentrations were detected via in-house ELISA as we previously reported (18).

Immunohistochemical Assays

Paraffin-embedded tissue samples from control and PAH lungs were used for detection of NAMPT proteins with primary rabbit anti-NAMPT antibody (IHC-00011; Bethyl Laboratories) and a 3,3′-diaminobenzidine kit (VECTOR Laboratories). See the data supplement for additional details.

Cell Culture, Plasmids, and siRNA Transfection

Human pulmonary artery endothelial cells (HPAECs) were cultured as described previously (21). The plasmids or siRNAs for indicated genes were cotransfected with NAMPT-pGL3-luc plasmids with FuGENE HD transfection reagent (Promega) and transfected into HPAECs.

Gene Cloning, Mutagenesis, and 5′-Deletion Mutations

Gene cloning, mutagenesis, and luciferase activity assays were performed as previously described (22). See the data supplement for further details.

Luciferase Reporter Gene Assays

The constructs were transfected in cultured HPAECs as previously described (23, 24). Briefly, plasmid constructs were transfected into HPAECs with plasmid for Renilla luciferase reporter for herpes simplex virus thymidine kinase promoter (pRL-TK) cotransfection as a control. Luciferase activity was measured by using dual-luciferase assay kits.

Immunocytochemical Assays

HPAECs were grown on 12-mm-diameter coverslips and challenged with 1 μg/ml recombinant human NAMPT (rhNAMPT; PeproTech Inc.) or vehicle for 24 hours and then fixed in 4% paraformaldehyde (25). See the data supplement for further details.

Preclinical Rat PAH Model and Hemodynamic Measurements

A rat model of MCT-induced PAH, approved by the University of Arizona Ethics and Animal Care Committee (19, 25), was used as we previously described. MCT (60 mg/kg body weight) or vehicle was injected subcutaneously, followed by injection of saline or goat anti-NAMPT pAb (1 mg/kg/day, i.p.) for 2 weeks. Right ventricular systolic pressure (RVSP) was determined by right-sided heart catheterization with a Millar pressure transducer catheter (26). The lung vascular medial thickness in small (50 μm) and intermediate (100 μm) arteries was measured by quantitative immunohistochemistry (25). See the data supplement for further details.

Statistical Analysis

Results are expressed as mean ± SEM, except when noted otherwise, from at least three experiments. Student’s t test and ANOVA were used for comparison of luciferase activities among different constructs. The Mann-Whitney U test was used for the comparison of eNAMPT plasma concentrations between control subjects and subjects with IPAH. The correlation between eNAMPT plasma concentrations and indices of pulmonary hemodynamics and RV function was assessed with Pearson correlations. Correlations were also adjusted for age and sex. Statistical significance was defined as P < 0.05 in all tests. Linear regression was used for the multivariable analysis of eNAMPT plasma concentrations. Known potential confounders in pulmonary hypertension were included in the multivariable models. All models were adjusted for age, sex (male or female), ethnicity (non-Hispanic white or Hispanic), and body mass index (in kg/m²) (Table E2 in the data supplement).

Results

Plasma eNAMPT Concentrations and Lung NAMPT Expression Are Significantly Increased in Subjects with IPAH and Correlate with PAH Severity

The characteristics of subjects with IPAH and those with other non-PAH lung diseases are reported in Table E1. Compared with patients without IPAH with other lung diseases, patients with IPAH exhibit increased mean pulmonary arterial pressure (PAP), PVR, and arterial elastance (P < 0.05) with preserved cardiac index and stroke volume. RV systolic function was preserved in patients with IPAH with a ratio of end-systolic elastance to effective arterial elastance of approximately 1. However, subjects with IPAH exhibited reduced RV diastolic function as measured by increased RV end-diastolic elastance (P < 0.05). Plasma eNAMPT concentrations were significantly elevated in subjects with IPAH (n = 68; median [interquartile range], 51.39 [36.68–74.60]) compared with in healthy control subjects (n = 70; median [interquartile range], 12.53 [4.10–23.35]; P < 0.01) (Figure 1A). eNAMPT plasma concentrations were also increased in non-PAH lung disease cases (systemic lupus, scleroderma, or coccidioidomycosis) (P < 0.05 vs. healthy control subjects). Associations with plasma eNAMPT concentrations trended toward significance for PVR and mean PAP and were significantly correlated with the indicator for RV diastolic stiffness, end-diastolic elastance (P = 0.02; adjusted P = 0.04) (Table E2) (Figure 1B), even after adjustment for age, sex, ethnicity, and body mass index (see data supplement for adjustment strategy details). In healthy subjects, a small amount of NAMPT was expressed in pulmonary arteries, spreading through the intima, media, and externa. In patients with IPAH, in addition to increased circulating concentrations of eNAMPT, immunohistochemistry confirmed increased NAMPT expression within distal pulmonary arteries, primarily in ECs and in neointimal cells of obliterated pulmonary arteries with IPAH plexiform lesions (black arrows in Figures 1C, E1, and E2).

Figure 1.

Plasma and lung extracellular nicotinamide phosphoribosyltransferase (eNAMPT) protein concentrations are significantly increased in patients with idiopathic pulmonary arterial hypertension (IPAH). (A) Plasma concentrations of eNAMPT measured by ELISA (18) were significantly elevated in patients with IPAH (n = 68) compared with subjects without PAH who had lung disease (n = 13) and healthy control subjects (n = 70) (error bars mean ± SD) (P < 0.001). PH = pulmonary hypertension. (B) Correlation between pulmonary hemodynamics and plasma eNAMPT concentrations revealed significant linkage with the indicator for diastolic ventricular stiffness, end-diastolic elastance (Eed) (P = 0.02 and adjusted P = 0.04). adj = adjusted; mPAP = pulmonary artery pressure; PVR = pulmonary vascular resistance; PVRi = PVR index. (C) Immunohistochemical studies of NAMPT expression in paraffin-embedded lung sections from patients with IPAH and subjects without IPAH were performed with rabbit anti-NAMPT and a 3,3′-diaminobenzidine kit. NAMPT (brown) expression localizes in the distal pulmonary arteries (PAs) and shows high NAMPT expression in endothelial and neointimal cells of obliterated PAs in patients with IPAH (arrows), not in healthy subjects. Scale bars: 50 μm.

PAH-Relevant Stimuli Increase NAMPT Promoter Activity and Protein Expression in Human Lung ECs

Blood concentrations of PDGF-BB (platelet-derived growth factor BB), VEGF (vascular endothelial growth factor), and TGF-β1 (transforming growth factor-β1) are recognized to be elevated in patients with PAH compared with healthy control individuals (27). The impact of these PAH-relevant stimuli on EC transcriptional regulation of NAMPT was assessed using a 3-kb NAMPT luciferase reporter promoter assay. PDGF-BB (10 ng/ml for 0–24 h) significantly increased NAMPT promoter activity in a time- and dose-dependent manner beginning at 1 hour, peaking at 8 hours (8.2-fold) (P < 0.01 vs. 0 h), and remaining elevated at 24 hours (2.3- to 8.2-fold increase) (Figure 2A). NAMPT promoter activity was also increased across a range of PDGF-BB concentrations (0.1–50 ng/ml, 4 h, 1.8- to 8.5-fold increases) compared with in control subjects (Figure 2B). Similar to PDGF, both VEGF (100 ng/ml) and TGF-β1 (2 ng/ml) significantly increased NAMPT promoter activity in a time-dependent manner (three- to ninefold; P < 0.01) (Figure 2C), indicating rapid upregulation of NAMPT expression in response to PAH-relevant growth factor stimulation. Consistent with NAMPT promoter activation, Figures 2A–2D depict the increases in NAMPT protein in EC lysates (via Western blotting) after exposure to PDGF, VEGF, and TGF-β1 (24 h).

Figure 2.

PAH stimuli and vascular transcription factors regulate NAMPT promoter activity and protein expression. The plasmids containing luciferase reporter and 3-kb NAMPT promoter (NAMPT-pGL3-luc) were transfected into human pulmonary artery endothelial cells (HPAECs). Cells were next exposed to either PAH-relevant stimuli or vehicle for the indicated number of hours, then lysed, and luciferase activity was measured using the dual-luciferase assay kit. (A) PDGF-BB (platelet-derived growth factor BB) 10 ng/ml stimulation resulted in significantly increased NAMPT promoter activity from 1 hour to 24 hours (*P < 0.01 vs. 0 h). (B) Exposure to PDGF-BB (from 0.1 to 50 ng/ml) significantly increased NAMPT promoter activity at 4 hours (*P < 0.01 vs. 0 ng/ml). (C) Both VEGF (vascular endothelial growth factor; 100 ng/ml) and TGF-β1 (transforming growth factor-β1; 2 ng/ml) significantly increased NAMPT promoter activity in a time-dependent fashion at 4 hours and 24 hours (error bars mean ± SEM) (^#P < 0.01 vs. control at 4 h and *P < 0.01 vs. control 24 h). n = 6. (D) To confirm the results of the effect of NAMPT promoter activity on NAMPT expression, we assessed protein expression of NAMPT after HPAECs were exposed to 10 ng/ml PDGF-BB, 100 ng/ml VEGF, and 2 ng/ml TGF-β1 for 24 hours by immunoblotting. These results showed increased protein expression consistent with that shown in C as well as in our prior report of significantly increased NAMPT protein expression in preclinical PAH models (14). (E) NAMPT-pGL3-luc with siSTAT5A/B (small interfering RNA for signal transducer and activator of transcription 5A/B) or scrambled siRNA (siControl) was transfected into HPAECs. Cells were exposed to PDGF or vehicle. PDGF-BB 10 ng/ml robustly increased NAMPT promoter activity at 4 hours (*P < 0.01 vs. no PDGF), which was significantly attenuated by silencing of transcription factor STAT5 (^#P < 0.01 vs. PDGF/siCtrl). (F) NAMPT-pGL3-luc with pCMV6-SOX18 (plasmid cytomegalovirus 6–SRY-box transcription factor 18) or siSOX18 was transfected into HPAECs. Cells were exposed to VEGF 100 ng/ml for 4 hours. VEGF significantly increased NAMPT promoter activity with significant attenuation by SOX18 overexpression and enhancement by SOX18 silencing (*P < 0.01 vs. control). (G) NAMPT-pGL3-luc with siSOX17 or scrambled siRNA was transfected into HPAECs. Silencing SOX17 significantly abolished the enhanced NAMPT promoter activity induced by VEGF (100 ng/ml for 4 h). The increase in NAMPT promoter activity was markedly attenuated by siSOX17 compared with siControl (^#P < 0.01). Ctrl = control; RLU = relative light units; siCtrl = scrambled siRNA; siSOX = siRNA for SOX.

Effect of Transcription Factors STAT5, SOX17, and SOX18 on NAMPT Promoter Activity

Several transcription factors and growth factor receptors are recognized to directly or indirectly contribute to vascular remodeling and PAH development (25, 28). For example, STAT5A/B is involved in growth factor receptor–induced gene expression (29). Analysis of STAT5A/B involvement in PDGF-BB–stimulated NAMPT promoter activity in ECs (siRNAs; 48 h) revealed robust PDGF-BB–mediated increases in NAMPT promoter activity that were significantly attenuated by reduced STAT5A/B expression (P < 0.01 vs. PDGF/siCtrl) (Figure 2E).

SOX family transcription factors have previously been reported to be involved in acute lung injury (SOX18) (30), and our recently published PAH genome-wide association study identified SOX17 as a risk allele in PAH (31). Because in silico analysis revealed three highly conserved SOX family binding sites within the NAMPT promoter sequence, we examined both SOX18 and SOX17 involvement in NAMPT promoter activity. These studies revealed significant stimulus- and pathobiology-specific NAMPT promoter activation. The silencing of SOX18 enhanced VEGF-induced EC NAMPT promoter activities (P < 0.01), and SOX18 overexpression reduced NAMPT promoter activities (Figure 2F). In contrast, the effect of the SOX17 transcription factor was found to exert effects on VEGF-induced NAMPT promoter activity that were exactly the opposite of those of SOX18. Silencing of SOX17 expression resulted in attenuation of VEGF-induced increases in NAMPT promoter activity (Figure 2G), indicating a precise and finely regulated mechanism of SOX family transcription factors on NAMPT expression.

Effect of HIF-2α on NAMPT Promoter Activity and Identification of the NAMPT Hypoxia-Responsive Element

We next evaluated the influence of hypoxic exposure (2% and 5% hypoxia for 0–48 h) on NAMPT promoter luciferase reporter activity in transfected human lung ECs and determined significant hypoxia-mediated increases in NAMPT promoter activity. Exposure to 2% hypoxia resulted in a 3.3-fold increase (P < 0.01 vs. 0 h) (Figure 3A), which peaked at 8 hours. Exposure to 5% hypoxia resulted in time-dependent increases in NAMPT promoter activity with a 5.1-fold increase at 48 hours (P < 0.01 vs. 0 h) (Figure 3B). We recently reported that HIF-2α contributes to severe vascular remodeling and occlusive lesions in PAH (25) via increases in EndMT, so we next examined HIF-1α and HIF-2α involvement in NAMPT regulation. The robust increases in NAMPT promoter activities elicited by hypoxia were entirely attributable to HIF-2α (P < 0.01) (Figure 3B) because silencing of HIF-2α abolished the increase in NAMPT promoter activity. These results were confirmed by overexpression of either HIF-1α or HIF-2α in normoxia-exposed lung ECs; that is, HIF-2α overexpression produced a far greater increase in NAMPT promoter activity than HIF-1α (9-fold vs. 2.5-fold) (P < 0.05) (Figure 3C). The specific inhibitor of PHD2 (prolyl hydroxylase domain-containing protein 2), FG-4592, was next used to inhibit PHD2 activity and HIF hydroxylation known to reduce HIF degradation (32). FG-4592 challenge increased EC NAMPT promoter activity compared with in control subjects (∼3-fold) (Figure 3D). Silencing of HIF-2α, but not HIF-1α, expression resulted in significantly decreased (∼90% decrease) NAMPT promoter activity elicited by FG-4592 (Figure 3D). These findings regarding HIF-mediated NAMPT promoter regulation were confirmed by studies of NAMPT protein expression in cellular lysates from FG-4592–challenged ECs in the presence of siRNAs for HIF-2α and HIF-1α (Figures 4A and 4B). Furthermore, measurements of eNAMPT secreted into the cell supernatant media from these same wells after FG-4592 exposure indicated a primary and robust role for HIF-2α, but not HIF-1α, in NAMPT expression and eNAMPT secretion (Figure 4C). These data suggest that HIF-2α is an important transcription factor regulating NAMPT promoter activation, especially in hypoxia-induced NAMPT promoter activation, and eNAMPT expression and secretion.

Figure 3.

Hypoxia increases NAMPT promoter via HIF-2α (hypoxia-inducible factor-2α). NAMPT-pGL3-luc was transfected into HPAECs with FuGENE HD transfection reagent and cotransfected with plasmid for Renilla luciferase reporter for herpes simplex virus thymidine kinase promoter (pRL-TK). Cells were exposed to either 2% or 5% hypoxia for 4 to 48 hours, and luciferase activity was measured using a dual-luciferase assay kit, normalized to control (fold change) (error bars mean ± SEM). (A) Two percent hypoxia significantly increased NAMPT promoter activity in human lung endothelial cells (ECs) from 4 hours to 24 hours, with the peak at 8 hours, compared with control cells (*P < 0.05 vs. control). (B) Cells transfected with NAMPT-pGL3-luc with pRL-TK and cotransfected by siCtrl or siHIF-2α into HPAECs were exposed to 5% hypoxia or normoxia for 24 to 48 hours. Five percent hypoxia significantly increased NAMPT promoter activity in human lung ECs from 24 hours to 48 hours (^#P < 0.01 vs. siCtrl only). siHIF-2α significantly decreased NAMPT promoter activity under normoxic conditions (*P < 0.01 vs. siCtrl only). The effects of 5% hypoxia for 24 and 48 hours on NAMPT promoter activity were significantly abolished by siHIF-2α (*P < 0.01 vs. siCtrl/24 h, siCtrl/48 h, respectively). (C) HIF-1α and HIF-2α overexpression significantly increases NAMPT promoter activity in lung ECs. The plasmids for human HIF-1α (NM_001530) or HIF-2α (NM_001430) in pCMV6-XL6 or pCMV6-XL6 empty vector (control) were transfected into HPAECs, and luciferase activity was measured using a dual-luciferase assay kit and normalized to control (fold change). HIF-1α and HIF-2α overexpression significantly increased NAMPT promoter activity by 2.5- and 9-fold, respectively, compared with controls. Compared with cells transfected by HIF-1α, cells transfected by HIF-2α showed significantly higher NAMPT promoter activity by 3.6-fold (*P < 0.01 vs. HIF-1α and ^#P < 0.05 vs. control). (D) PHD2 (prolyl hydroxylase domain-containing protein 2) inhibition increases NAMPT promoter activities in an HIF-2α–dependent manner. NAMPT-pGL3-luc with pRL-TK was transfected into HPAECs. ON-TARGETplus siRNAs against HIF-1α, HIF-2α, or siCONTROL#2 (Dharmacon) 100 nM were also transfected into HPAECs. After 48 hours, cells were incubated with PHD2 inhibitor FG-4592 (100 μM) or vehicle in HPAECs or vehicle for 8 hours. Then luciferase activities were measured using a dual-luciferase assay kit and normalized by luciferase activities with siCONTROL#2 (fold change). FG-4592 significantly increased NAMPT promoter activity by threefold (*P < 0.01 vs. Ctrl with vehicle). Silencing HIF-2α by siHIF-2α transfection abolished effects of FG-4592 on NAMPT promoter activity compared with luciferase activities with scrambled siRNA and siHIF-1α (^#P < 0.01 vs. Ctrl/FG-4592 and siHIF-1α/FG-4592), respectively. (E) Nest deletion mutations of truncated NAMPT promoter every 300 bp were generated from full-length human NAMPT promoter by subcloning. The full-length and truncated NAMPT-pGL3-luc were transfected into HPAECs with pRL-TK. After 48 hours, cells were exposed to PHD2 inhibitor FG-4592 (100 μM) or vehicle for 8 hours. Then luciferase activity was measured and normalized by full-length NAMPT promoter activity with vehicle (control) and expressed as fold change. Activity of NAMPT promoter −3,028, −2,728, −2,428, −2,128, −1,828, −1,528, −1,228, −928, and −628 bp (upstream of ATG) fragments were significantly increased after incubation with PHD2 inhibitor FG-4592 (100 μM) in HPAECs (*P < 0.05 vs. the same size of promoter with vehicle). HIF-RE = hypoxia-inducible factor-response element; Veh = vehicle.

Figure 4.

HIFs increase NAMPT protein expression and eNAMPT secretion via HIF-2α. ON-TARGETplus siRNAs against HIF-1α, HIF-2α, or siCONTROL#2 (Dharmacon) 100 nM were transfected into HPAECs. After 48 hours, cells were incubated with the PHD2 inhibitor FG-4592 (100 μM) or vehicle in HPAECs for 24 hours. NAMPT proteins were detected by rabbit anti-NAMPT antibody (1:10,000; Bethyl Laboratories) and goat antirabbit horseradish peroxidase (Invitrogen). eNAMPT concentrations in supernatant media were measured by sandwich ELISA (18). (A) Knockdown HIF-1α slightly decreases PHD2 inhibitor FG-4592–induced NAMPT protein expression compared with scrambled siRNA. (B) Knockdown of HIF-2α, in contrast, significantly decreases FG-4592–induced NAMPT protein expression in ECs. (C) Secreted eNAMPT protein concentrations were significantly increased in ECs overexpressing HIF-1α and HIF-2α, with eNAMPT protein concentrations significantly greater in HIF-2α–overexpressing ECs (*P < 0.05 vs. control and ^#P < 0.05 vs. HIF-1α). Silencing of HIF-2α, but not HIF-1α, significantly decreased eNAMPT proteins induced by FG-4592 (error bars mean ± SEM) (*P < 0.05 vs. FG-4592/siCtrl). pHIF-1α = plasmid carrying HIF-1α gene; pHIF-2α = plasmid carrying HIF-2α gene, siCONTROL#2 = nontargeting siRNA #2 (Dharmacon).

We next determined the core promoter sequence and regulatory elements for HIFs within the NAMPT promoter using Genomatix (www.genomatix.de) and TESS software (www.cbil.upenn.edu/tess). We identified five regions representing potential HIF-binding promoter genomic regions and next assessed FG-4592–induced NAMPT luciferase reporter promoter activation using a nested deleted promoter with varying DNA length (Figure 3E) and determined that both full-length and truncated NAMPT promoter reporter activities (−3,028 bp to −628 bp) were increased, reflecting FG-4592’s well-known capacity to increase HIF availability to drive transcription. In contrast, the truncated NAMPT promoter (−328 bp) failed to respond to PHD2 inhibition (Figure 3E), indicating that the HIF-binding region is located between −628 bp and −328 bp upstream of the NAMPT ATG site. In silico analysis indicated highly conserved NAMPT promoter HIF-binding sites (conserved in mice and human) in these regions: −654 to −640 bp, −600 to −586 bp, −426 to −410 bp, −370 to −356 bp, and −355 to −340 bp upstream of the ATG site, and further promoter interrogation confirmed that PHD2-mediated increases in NAMPT promoter activity involve HIF-2α binding between −600 bp and −340 bp upstream of ATG.

eNAMPT Significantly Increases EndMT

EndMT is a pathologic feature of PAH and is characterized by the loss of cell–cell adhesion and cell phenotype conversion from a cobblestone pattern to a spindle-shaped morphology. Molecular changes associated with EndMT include decreases in PECAM1 (platelet endothelial cell adhesion molecule 1) and increased expression of the EndMT transcription factor, SNAI1 (snail family transcriptional repressor 1). Comparison of EC protein expression levels of EndMT biomarkers (immunofluorescence) after exposure to rheNAMPT (1 μg/ml, 24 h) showed significantly increased SNAI1 expression (EndMT driver) (Figure 5A) and significantly decreased PECAM1 expression (EC marker) in eNAMPT-treated ECs (Figure 5B) with SNAI1 and PECAM1 lung immunofluorescence quantified by fluorescence density (Figures 5D and 5E). eNAMPT also significantly increased discontinuous adherens junction staining in lung ECs (Figures 5C and 5F), again consistent with eNAMPT-enhanced EndMT. We recently reported that TGF-β1 induces EndMT in human lung ECs to concentrations observed in lung ECs isolated from patients with IPAH (25). Lung ECs, coincubated with TGF-β1 10 ng/ml and either eNAMPT or vehicle (7 d), showed that eNAMPT significantly increased SNAI1 mRNA concentrations with or without TGF-β1 (qRT-PCR) (Figure 5G), consistent with eNAMPT’s contribution to EndMT in human lung ECs. These results were further supported by Western blotting of SNAI1 and PECAM1 in EC lysates after exposure to eNAMPT; that is, eNAMPT elicited a significant increase in SNAI1 protein expression and a significant reduction in PECAM1 expression quantified by densitometry (Figure 5H).

Figure 5.

eNAMPT significantly increases endothelial-to-mesenchymal transition. Human lung ECs were treated with 1 μg/ml recombinant human NAMPT (rhNAMPT) (PeproTech Inc.) or vehicle for 24 hours. Nuclei were counterstained with Hoechst (blue). (A and D) eNAMPT exposure significantly increased SNAI1 (snail family transcriptional repressor 1) in ECs. Representative images (A) and summarized data by quantification with fluorescence density (D) show the mean fluorescence intensity of SNAI1 (green) in rhNAMPT- or vehicle-treated ECs (n = 3 per group; 3 fields per view; 10 regions of interest per field of 27 views) (*P < 0.01 vs. vehicle) (error bars mean ± SEM). (B and E) eNAMPT exposure significantly decreased PECAM1 (platelet endothelial cell adhesion molecule 1) in lung ECs. Representative images (B) and the summarized data by quantification with fluorescence density (E) show the mean fluorescence intensity of PECAM1 (green) in rhNAMPT- or vehicle-treated ECs (n = 3 per group) (*P < 0.01 vs. vehicle). (C and F) eNAMPT exposure significantly increased discontinuous adherens junctions in lung ECs (C), and the summarized data by quantification with fluorescence density (F) show the percentage of discontinuous adherens junctions (green) in rhNAMPT- or vehicle-treated ECs (n = 3 per group) (*P < 0.01 vs. vehicle). The white arrows indicate the cell–cell tight adherens junctions with high PECAM1 expression, and yellow arrows indicate the gap between cells with low PECAM1 expression. (G) Human lung ECs were coincubated with TGF-β1 10 ng/ml or vehicle and either rhNAMPT 1 μg/ml or control for 7 days. Real-time RT-PCR analysis of SNAI1 (normalized to GAPDH) in ECs (n = 3 per group) shows that TGF-β1 and rhNAMPT both significantly increased SNAI1 mRNA transcription (**P < 0.01 vs. vehicle and ***P < 0.05 vs. control), with the highest concentration observed when ECs were exposed to both TGF-β1 and rhNAMPT. (H) SNAI1 and PECAM1 protein concentrations detected after EC exposure to rhNAMPT (1 μg/ml) for 24 hours by immunoblotting (insets). The SNAI1 protein concentrations were significantly increased, and the concentrations of PECAM1 were decreased, by eNAMPT exposure (*P < 0.05 vs. vehicle control). Scale bars: 50 μM.

An eNAMPT-Neutralizing pAb Significantly Attenuates MCT-induced PAH in Rats

We next investigated eNAMPT as a therapeutic PAH target using a well-validated eNAMPT-neutralizing pAb (8, 12, 13) in a rat preclinical model of pulmonary hypertension produced by MCT exposure (60 mg/kg, one dose, 4 wk). Delivery of the eNAMPT-neutralizing pAb (three times per wk, i.p.) did not alter basal pulmonary hemodynamics (Figures 6A and 6B), but it produced significant (∼30%) reductions in MCT-induced increases in PAP, RVSP, and RV hypertrophy, reflected by alterations in the Fulton index (ratio of the right ventricular weight to left ventricular plus septal weight) (Figures 6A and 6B). There were no significant effects of control IgG compared with vehicle on right ventricular pressure (data not shown). In addition, the dramatic MCT-mediated pulmonary vascular remodeling reflected by pulmonary arterial wall thickness in small (50 μm) and intermediate (100 μm) arteries was abolished by the eNAMPT-neutralizing pAb (Figure 6C), indicating that eNAMPT neutralization significantly attenuates in vivo vascular injury and remodeling in a preclinical model of PAH. Finally, given the intimate relationship between inflammation and pulmonary hypertension outcomes (3, 5, 33), total NF-κB and phosphorylated (activated) NF-κB (p-NF-κB) expression was assessed in MCT-exposed human lung ECs. MCT significantly increased p-NF-κB, which was abolished by the eNAMPT-neutralizing pAb (Figures 6D and 6E), indicating direct attenuation of MCT-induced TLR4 signaling and PAH-associated inflammation.

Figure 6.

eNAMPT neutralization significantly reduces hemodynamic alterations and lung vascular remodeling in preclinical monocrotaline (MCT) PAH model. Rats were grouped into four experimental groups, which received vehicle (n = 8), goat anti-NAMPT antibodies (Ab; n = 6), MCT (n = 8), or MCT with an eNAMPT-neutralizing polyclonal antibody (pAb) (n = 6), respectively. Two experimental rat groups received a single dose of MCT (60 mg/kg body weight) injected subcutaneously. A goat anti-eNAMPT pAb (1 mg/kg/d) (LAMPIRE Biological Laboratories) or vehicle was injected i.p. on Day 14 after the MCT injection and continued daily for 2 weeks. (A) After 4 weeks, right ventricular pressure (RVP) and right ventricular systolic pressure (RVSP) were measured by right-sided heart catheterization with a Millar pressure transducer catheter in four groups of rats (control, eNAMPT Ab, MCT, and MCT with eNAMPT Ab). (B) Quantification of RVSP and the Fulton index (ratio of the right ventricular weight to left ventricular plus septal weight [RV/(LV + S)]) in groups with vehicle, NAMPT Ab, MCT injection, or MCT injection with NAMPT Ab. Administration of eNAMPT-neutralizing Ab to rats did not change the normal, but significantly reversed the increased, RVSP and RV/(LV + S) induced by MCT (^#P < 0.01 vs. control and *P < 0.01 vs. MCT, respectively) (error bars mean ± SEM). Each group included six to eight rats. (C) The lung vascular medial thickness in small (50 μm) and intermediate (100 μm) arteries was measured by quantitative immunohistochemistry (qIHC) in four groups of rats (control, NAMPT Ab, MCT, and MCT with NAMPT Ab). NAMPT Ab reduces lung vascular medial thickness in small (50-μm) arteries (*P < 0.05 MCT vs. control and ^#P < 0.05 MCT with NAMPT Ab vs. MCT). NAMPT Ab reduces lung vascular medial thickness in intermediate (100-μm) arteries as revealed by qIHC assessment (*P < 0.05 MCT vs. control and ^#P < 0.05 MCT with NAMPT Ab vs. MCT). (D) eNAMPT-neutralizing pAb significantly reduced MCT-induced NF-κB activation in human lung ECs. The phospho-NF-κB RelA/p65 (pSer536) and NF-κB p65 were detected by rabbit anti-phospho-NF-κB-p65 and NF-κB-p65 antibodies, respectively. HPAECs were treated with MCT, MCT with goat anti-NAMPT Ab (LAMPIRE Biological Laboratories), or vehicle for 24 hours, and protein expression of p-NF-κB and total NF-κB was detected by immunoblotting. (E) Densitometric analysis and quantification of p-NF-κB and total NF-κB protein expression. Compared with controls, MCT significantly increased p-NF-κB, and eNAMPT Ab significantly decreased or abolished MCT-induced increases in p-NF-κB (n = 6; *P < 0.05 vs. control and ^#P < 0.05 vs. MCT) (error bars mean ± SEM).

Discussion

Our prior genomics-intensive approaches in human peripheral blood mononuclear cells and in preclinical models of inflammatory lung injury (9, 10, 34, 35) identified NAMPT as a novel candidate gene, biomarker (11, 12, 14), and therapeutic target in ventilator-induced lung disease and ARDS (7, 8), with NAMPT promoter variants conferring risk and severity of disease (11, 13, 36). More recently, we identified NAMPT as a candidate gene in PAH, a fatal disease without effective curative therapies. Our genomic studies in peripheral blood mononuclear cells from subjects with PAH revealed NAMPT as a top dysregulated gene, and eNAMPT plasma concentrations are increased in subjects with PAH and are linked to PAH severity, particularly RV diastolic dysfunction (Figure 1). Expression of NAMPT, a cytozyme with dual roles as a proinflammatory cytokine and as a NAMPT regulating nicotinamide adenine dinucleotide metabolism (16), is markedly increased in remodeled vessels from subjects with PAH and in ECs isolated from preclinical PAH models (Figure 1) (14). While a complete understanding of NAMPT involvement in PAH pathophysiology and vascular remodeling remains elusive, we focused on the critical role of eNAMPT in PAH pathobiology and embarked on a series of investigations to detail mechanisms of transcriptional regulation (transcription factors, PAH-responsive promoter elements), the role of eNAMPT in EndMT and lung vascular remodeling, and the potential for eNAMPT as a PAH therapeutic target.

The findings of significantly elevated plasma eNAMPT in human subjects with IPAH and increased NAMPT protein expression in obliterated human IPAH pulmonary arteries and plexiform lesions strongly implicate tissue-specific dysregulation of NAMPT expression in human PAH. Several signaling pathways affecting pulmonary vasoconstriction and remodeling, such as PDGF (37, 38), VEGF (33), and the profibrotic tissue-remodeling TGF-β1 (39), have been implicated in PAH pathogenesis and, as we now show, elicit robustly enhanced EC NAMPT promoter activity and subsequently significantly increased NAMPT protein expression after 24 hours, consistent with eNAMPT as a therapeutic target in PAH pathobiology.

We also evaluated specific transcription factors potentially influencing PAH stimuli-mediated NAMPT gene and protein expression and corroborated our prior work in mechanically stressed (18% cyclic stretch) endothelium showing that STAT5 (23), a transcription factor altered by multiple cytokines and growth factors, is involved in NAMPT regulation in response to PAH stimuli such as PDGF. PDGF expression is increased in IPAH small pulmonary arteries (37), and PDGF induces STAT5 activation via the PDGF β-receptor (24). PDGF receptor antagonism reverses development of vascular remodeling and cor pulmonale in experimental severe pulmonary hypertension (38). Our study highlights that PDGF-mediated increases in NAMPT promoter activity are STAT5 dependent.

In addition to STAT5, we evaluated two SOX family transcription factors—SOX18 and SOX17—in specific responses to mechanotransduction- and PAH-specific stimuli. SOX18 regulates vascular development (40) and EC-specific claudin-5 gene expression (41) and preserves lung barrier integrity (42). We found that SOX18 overexpression significantly attenuated 18% cyclic stretch–induced mechanical stress and NAMPT promoter activity. These results are diametrically opposite to our results with SOX17, a transcription factor required for normal pulmonary vascular morphogenesis and a positive feedback regulator of VEGF signaling (43). SOX17 genetic variants are associated with PAH susceptibility to congenital heart disease (44), and we recently reported SOX17 as a novel PAH gene via a PAH genome-wide association study (31). In our present study, we demonstrate that involvement of SOX18 and SOX17 in NAMPT promoter activity is significantly stimulus and pathobiology specific, with SOX17 being critically involved in VEGF-induced NAMPT promoter activity. These data indicate differential roles of SOX17 and SOX18 in NAMPT regulation and provide potentially novel molecular insights into PAH pathobiology.

Robust EC NAMPT transcriptional activation was observed in response to hypoxic exposures, a well-known stimulus for pulmonary artery remodeling (45), and was determined to be highly dependent on HIF-2α. This was validated by PHD2 inhibition with FG-4592, which results in reduced HIF-2α hydroxylation and, therefore, inhibition of HIF ubiquitination and degradation. FG-4592 was further used as a tool to define the NAMPT hypoxia-responsive element residing between −628 bp and −328 bp upstream of ATG. Because we recently reported that the EC PHD2–HIF-2α axis is an important contributor to severe pulmonary hypertension via EC epithelial-to-mesenchymal transition–mediated vascular remodeling (25), these findings implicate NAMPT in EC obliterative vascular remodeling and severe PAH in mice and humans via HIF-2α (46).

Another finding of the present study was the involvement of eNAMPT in EndMT, a process whereby EC transition to a mesenchymal or smooth muscle cell phenotype and that plays a pivotal role in PAH initiation, initiation of remodeling, and formation of obliterative PAH lesions (47). Although the concentration of eNAMPT used (1 μg/ml) for these in vitro epithelial-to-mesenchymal transition studies is somewhat higher than physiological concentrations noted, we found eNAMPT to contribute to SNAI1 and PECAM1 immunoreactivity in cells as an indicator of EndMT development and to alterations in SNAI1 and PECAM1 expression. Because eNAMPT directly ligates TLR4 and lung NF-κB transcriptional activities (17) that induce EndMT (48), and because NAMPT also induced EC barrier dysfunction and loss of EC monolayer integrity (49), our findings highlight a potential mechanism by which eNAMPT influences PAH susceptibility and severity.

In summary, our studies are highly consistent with an essential role for eNAMPT, a uniquely functioning cytozyme, as a highly novel PAH therapeutic target in amelioration of the inflammatory cascade activation, vascular remodeling, and RV dysfunction, essential features of PAH pathobiology. The role of iNAMPT in inflammatory injury has been substantiated by using inhibitors of iNAMPT enzymatic activity (19, 50). In the present work, we have focused on eNAMPT as a unique damage-associated molecular pattern protein that, via binding of the pattern recognition receptor TLR4, is a primary mechanism for human lung EC resistance to apoptosis (18) and potentially in pulmonary vascular remodeling (33). TLR4 mice are resistant to chronic hypoxia–induced PAH (51), and NF-κB activation is a feature of end-stage PAH in humans (52). Furthermore, elevations in inflammatory cytokines predict survival in IPAH and familial PAH (53). Our preclinical PAH studies using an eNAMPT-neutralizing pAb that reduced NF-κB phosphorylation and PAH severity in vivo provide compelling support for eNAMPT as a contributor to PAH pathobiology. However, despite the compelling support for eNAMPT as a novel PAH candidate, several limitations exist. First, although comparable to other PAH biomarker studies, the sample size of our study cohort is relatively small, both in the number of subjects with PAH and in the number of patients without PAH undergoing cardiac catheterization with inflammatory lung disease. Currently, efforts are underway to greatly expand this sample size to validate our initial findings. A second limitation of our study is that, owing to its role as a novel damage-associated molecular pattern protein, eNAMPT plasma concentrations are not (and would not be expected to be) uniquely increased in PAH, because eNAMPT plasma concentrations are known to be elevated in other inflammatory disorders such as ARDS (11, 12, 17). A second potential limitation of our work is that the interrogation of NAMPT expression in PAH may also be epigenetically regulated by DNA methylation (54) and by miRNAs (24). These approaches were not explored in this study but are studies that are clearly warranted. Furthermore, both growth factors and HIFs, each of which contribute to PAH development, enhanced NAMPT promoter activity and protein expression. The long-term effects of these factors on NAMPT’s contribution to PAH in vivo will be confirmed in future investigations. Finally, the in vivo evaluation of STAT5, SOX17, and HIF-2α in PAH models has not yet been addressed, a necessity for realizing the full translational impact of this work. These studies are currently underway. Despite these shortcomings, taken together, our studies shed important light on the molecular mechanisms of PAH pathogenesis and indicate that the cytozyme eNAMPT is a major PAH target and strongly warrants consideration as a highly novel therapeutic target for amelioration of vascular remodeling and severity in PAH.