Nicotinamide Phosphoribosyltransferase Promotes ...

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Feb 15, 2017 - tested in the monocrotaline and Sugen-hypoxia models of PH. The effects on NAMPT activity on proliferation, migration, apoptosis and calcium ...
10.1161/CIRCULATIONAHA.116.024557

Nicotinamide Phosphoribosyltransferase Promotes Pulmonary Vascular Remodeling and is a Therapeutic Target in Pulmonary Arterial Hypertension Running Title: Chen et al.; NAMPT in Pulmonary Arterial Hypertension

Jiwang Chen, PhD1,2; Justin R. Sysol, BS1,3; Sunit Singla, MD1; Shuangping Zhao, MD, PhD1; Aya Yamamura, PhD1,4; Daniela Valdez-Jasso, PhD5; Taimur Abbasi, MD1,6; Krystyna M. Shioura, PhD1; Sakshi Sahni, MD1; Vamsi Reddy, BS1; Arvind Sridhar, BS1; Hui Gao, MD, PhD1,7; Jaime Torres, MD1; Sara M. Camp, BS8; Haiyang Tang, PhD8; Shui Qing Ye, MD, PhD9; Suzy Comhair, PhD10; Raed Dweik, MD10; Paul Hassoun, MD11; Jason X.-J Yuan, MD, PhD8; Joe G.N. Garcia, MD8 *; Roberto F. Machado, MD1, 3 * Downloaded from http://circ.ahajournals.org/ by guest on April 16, 2017

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Division of Pulmonary, Critical Care Medicine, Sleep and Allergy, Department of Medicine, University of Illinois at Chicago, Chicago, IL; 2 Institute of Precision Medicine, Jining Jiini n ngg Medical Med edic ical University, Jining, P. R. China; 3Department of Pharmacology, University of Illin Illinois nois i at at Chicago, Chic Ch icago, ic Chicago, IL; 4Department of Pharmacy, College of Pharmacy, Kinjo Gakuin University, Univer errsity, Na N Nagoya, goya 5 6 Japan; Department of Bioengineering, University of Illinois at Chicago, Chicago, IL; Departmen Department of Medicine, Mercy Hospital and Medical Center, Chicago, IL; 7Department of Obstetrics and Gynecology Gyne neco ne colo logy lo gyy , U Union n on Hospital, Tongji Medical Co ni College, Huazhong Un University of Science and Technology Tec Te chnologgy (HU (HUST), HUST), Wuhan,, PR China; 8Depa Department parrtment of Med Medicine, diccine, University University of Arizona, 9 Tu ucson, AZ; De Departments eparrtme ment me n s of B nt Biomedical iom io medi dical an and nd He Health th h IInformatics nfor forrma m tics tii s & D Department ep par artm tmen tm en nt of o P Pediatrics, ediiatr ed tric tr ics, ic Tucson, The Th Children Children’s n’s M Mercy errcy Hos Hospital ospital and Un os U University iverssitty off Missouri-Kansas Miiss ssou ourri-Kaansas Ci ou C City ty yS School chool of of Medicine, Medicin ne, Kansas Kans Ka n as Cit City, ty, M MO; O; 10Depa Department art r ment ooff Pa Pathobi Pathobiology, iology gy, Lerner Le Research R eseearrch In Inst Institute, tit itut u e, Pul Pulmonary ulmo ul m nary and mo nd Crit Cr Critical itticall Care Car aree Me Medicine, edi d cine n , Re Resp Respiratory spir sp irat ir ator at o y In or Inst Institute, stit st ittut utee, Cleveland Cle leve vela land la nd Clinic, Cli lini n c, Cleveland, ni Clev Cl evel e an el and, d OH; d, OH; 1111Divi Division D ivi v si sion on n of of Pulmonary and Critical Care Medicine Medicine, Johns Hopkins University School of Medicine, Medicine Baltimore, MD *Co-senior and corresponding authors Address for Correspondence: Roberto F. Machado, MD Division of Pulmonary Critical Care Medicine, Sleep and Allergy Department of Medicine University of Illinois at Chicago 909 South Wolcott Avenue, M/C 719 Chicago, IL 60612 Tel: (312) 996-8039 Fax: (312) 996-4665 Email: [email protected]

Joe G.N. Garcia, MD University of Arizona 1295 N Martin Ave, Room B207 Tucson, AZ 85721 Email: [email protected]

Journal Subject Terms: Pulmonary Biology 1

10.1161/CIRCULATIONAHA.116.024557

Abstract

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Background—Pulmonary arterial hypertension (PAH) is a severe and progressive disease, a hallmark of which is pulmonary vascular remodeling. Nicotinamide phosphoribosyltransferase (NAMPT), is a cytozyme which regulates intracellular NAD levels and cellular redox state, regulates histone deacetylases, promotes cell proliferation and inhibits apoptosis. We hypothesized that NAMPT promotes pulmonary vascular remodeling, and that inhibition of NAMPT could attenuate pulmonary hypertension. Methods—Plasma and mRNA and protein levels of NAMPT were measured in the lungs and isolated pulmonary artery endothelial cells (PAECs) from PAH patients, as well as in lungs of rodent models of pulmonary hypertension (PH). Nampt+/- mice were exposed 10% hypoxia and room air for 4 weeks and the preventive and therapeutic effects of NAMPT inhibition were tested in the monocrotaline and Sugen-hypoxia models of PH. The effects on NAMPT activity on proliferation, migration, apoptosis and calcium signaling were tested in human pulmonary artery smooth muscle cell (hPASMC). Results—Plasma and mRNA and protein levels of NAMPT were increased in the lungs and isolated pulmonary artery endothelial cells (PAECs) from PAH patients, as well as in lungs of rodent ode models ode s oof pu pulmonary o a y hypertension ype e s o ((PH). ). Nampt+/- mice ce were we e pprotected o ec ed from o hypoxiaypo a mediated PH. NAMPT activity promoted human pulmonary artery smooth muscle le ccell elll el (hPASMC) hPASMC) proliferation via a paracrine effect. In addition, recombinant NAMPT T sstimulated timu ti mu ulaate tedd hPASMC proliferation via enhancement of store-operated calcium entry by enhancing expression of Orai2 and STIM2. Finally, inhibition of NAMPT activity attenuated monocrotaline and Sugen hypoxia induced PH in rats. Conc Conclusions—Our clu lusi sion si ons ns—Ouur data provide evidence that NAM NAMPT AMPT plays a rol role ole in ppulmonary ulmonary vascular remodeling emo moddeling and mo nd itss iinhibition nhhib ibit itio it io on could coul co uldd be ul b a poten potential nti tial th therapeutic heraape peut utic ut ic tar target arge gett fo ge fforr PA P PAH. H. Key Key-Words: y-W -Words: pu pulm pulmonary monar ary hypertension hyypertension

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Clinical Perspective

What is new? x

Nicotinamide phosphoribosyltransferase (NAMPT), a pleiotropic protein with extracellular proinflammatory cytokine-like activity and intracellular enzymatic activity as a phosphoribosyltransferase, is upregulated in human and experimental pulmonary hypertension and promotes pulmonary vascular remodeling in vivo and in vitro.

What are the clinical implications? x

Inhibition of NAMPT enzymatic activity is a potential novel therapeutic strategy for

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pulmonary arterial hypertension.

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Introduction Pulmonary arterial hypertension (PAH) is a progressive disease caused by functional and structural changes in the pulmonary vasculature, which lead to increased pulmonary vascular resistance and, eventually right ventricular failure and death. PAH is characterized by vasoconstriction, in-situ thrombosis, vascular inflammation and pulmonary artery smooth muscle cell (PASMC) and pulmonary artery endothelial cell (PAEC) proliferation and apoptosis resistance 1. PAEC dysfunction and injury are believed to play a critical role in the pathogenesis of the disease and can trigger PASMC proliferation and migration through activation growth Downloaded from http://circ.ahajournals.org/ by guest on April 16, 2017

factors or disruption of cell survival mediators 2, 3. Although important progress has been made n recent years, the precise cellular and molecular basis of pulmonary vascular remodeling remo mo ode deli liing iin n in PAH continues to evolve and the discovery of therapeutic targets that attenuate vascular emodelingg is still ongoing. remodeling Nicoti Nicotinamide ina nami mide phosphoribosyltransferase mi pho hosp spho hori ho ribo ri bosy bo syltra sy ransferaase s (NAMPT), (NAMP MPT) MP T), also T) a soo called al cal alledd visf visfatin fat atin in and and n pre-B pree-B ce cell l colo colony-enhancing ony-enhanccin ing factor facttor (PBEF), (PB BEF), was first firrst characterized chaaractterizzed aass a ccytokine yto okine aacting cttin ing on ear early a ly B ar B-lineage -lineag age precursor cells, andd is a pleiotropic protein withh extracellular proinflammatory cytokine-like activity and intracellular enzymatic activity as a phosphoribosyltransferase 4-10. It has been demonstrated as the rate-limiting component of the mammalian NAD biosynthesis pathway from nicotinamide 11. NAMPT activity has been linked to several processes involving cellular adaptation to stress responses. These include resistance to senescence, apoptosis and increase in cell proliferation and regulation of cellular redox state 6-8, 12, 13. At the pathological level NAMPT promotes inflammatory responses by increasing inflammatory cell survival and increasing proinflammatory cytokine production 8, 14-16. NAMPT is also up regulated in many types of malignancies where it promotes cell survival, proliferation and resistance to chemotherapeutic

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agents 7, 17-21. In vascular cells NAMPT promotes endothelial cell survival and angiogenic activity as well as smooth muscle cell survival 10, 13, 22-24. We therefore hypothesized that NAMPT may play an important role in PAH pathobiology by promoting pulmonary vascular remodeling during pulmonary hypertension development, and that inhibition of NAMPT could attenuate experimental pulmonary hypertension.

Methods Human Lung Tissues, Human PAECs and Human PASMCs (hPASMCs) Downloaded from http://circ.ahajournals.org/ by guest on April 16, 2017

Approval for the collection of plasma and the use of human lung tissues and cells was granted by Institutional nstitutional Review Boards of participating centers. Plasma samples were obtained obtain ned d ffrom r m 10 ro 1033 patients with PAH defined by the 2013 Nice classification 25 and 53 controls including patients with hypothyroidism, osteoporosis, nephrolithiasis, diabetes mellitus, osteoarthritis and hypertension. hype perrtension. De-identified pe De-iide d nt n if ifie iedd human ie huma hu mann explanted ma e pl ex plan a ted peripheral p riiph pe p erral llung ungg tiss un tissues sssue uess an and nd PA PAECS AEC ECS S us uused ed d iin n th this is study tuddy were fro from om fo fou four ur co control ontrol ol subjects (2 unsui unsuitable itaablle organ orrga gann donors donnors and do and 2 chronic chr hroonic obstructive hr obbstruc uctive pulmonary disease d sease pati di patients ients without PH) and d patients with idiopathi idiopathic h c PA PAH AH (IPA (IPAH, P H, H 4 patients diagnosed on the basis of National Institutes of Health PAH Registry criteria) obtained at the Cleveland Clinic Foundation. PAECs were isolated as previously described 26. In addition, a primary hPASMC and hPAEC cell lines from Lonza (CC-2581 and CC-2530) were used for cell transfection and proliferation assays. Cells were cultured at 37ºC in their complete medium with 10% fetal bovine serum and studied at passages 5 to 8. Reagents, Pharmacologic Inhibitors and Antibodies The information for reagents, pharmacologic inhibitors and antibodies is provided in the supplementary Materials and Methods.

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Western Blotting Solubilized protein lysates isolated from lung tissues and cells were used to detect NAMPT, STIM2, ORAI2 and E-actin. Cells were lysed in a modified radioimmunoprecipitation assay (mRIPA) lysis buffer with a protease and phosphatase inhibitor cocktail (Sigma Aldrich, St. Louis, MO), and protein quantification and Western blot analysis were performed according to standard procedures. RNA Isolation and Real-time PCR Downloaded from http://circ.ahajournals.org/ by guest on April 16, 2017

Total RNA from lung tissues or cells was isolated using the RNeasy Plus Mini Kit (Cat.#74134, Qiagen). Two micrograms of purified RNA was attenuate transcribed to single-stranded cDNA using the Taqman RNA attenuate transcription kit (Cat.#N8080234, Applied Bios sysste tems ms,, In ms Inc. c c. Biosystems, ABI]). Real-time PCR was performed on an ABI 7900HT machine. Specific Taqman [ABI]). quantitative real-time PCR assays were ordered from ABI (specific assay IDs available upon equ quuest). The re rel lati t ve mRNA ti mRN RNA A ex expr pres res ession on levels we ere nnormalized orma or mali ma lize li z d to the ze the expression exppre ress ssio ss ionn of a io request). relative expression were hou use eeping ggene, usek e e, gl en lucosee-6e6 phossph p at ate dehy ydrog ogen nas asee (G ((G6PDH), 6PDH 6P H), and nd ddetermined ettermineed by housekeeping glucose-6-phosphate dehydrogenase FDOFXODWLQJWKHǻǻ&WYDOXHDVGHWDLOHGLQWKHPDQXIDFWXUHU¶VJXLGHOLQHV Mouse PASMC isolation A mixture of 5 ml of medium 199 (M199) growth medium containing 5 g/l low-melting-point DJDURVHW\SH9,, 6LJPD6W/RXLV02 JOLURQEHDGV GLDPHWHUȝ06LJPD DQG antibiotics (penicillin and streptomycin) was slowly injected over a period of 60 seconds through the RV, thereby perfusing the PA. M199 growth medium (1 ml) containing 5 g/l agarose type VII was injected in airways through the trachea. The lungs were plunged in cold PBS to cause the agarose to gel. Because of the rapidly solidifying nature of the agarose and the size of the iron particles, the likelihood of traversing the capillary space is minimized. All the lobes were

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then isolated and finely minced in a Petri dish. The tissue was further disrupted by passing through a 16-gauge followed by an 18-gauge needle approximately five times. The suspension was then mixed in M199 growth medium containing 80 U/ml type IV collagenase (Sigma) and incubated at 37°C for 90 min. With the use of a magnetic column (Invitrogen), the arteries or arterial tissues containing the iron beads were collected. The supernatant was aspirated and the arteries were washed and suspended in 5 ml M199 containing 20% FBS. Aliquots of the suspension were transferred to T25 culture flasks. Cells from the hypoxic group were incubated at 3% O2, whereas cells from the normoxic control were cultured in air. Smooth muscle cell Downloaded from http://circ.ahajournals.org/ by guest on April 16, 2017

SXULW\ZDVGHWHUPLQHGE\LPPXQRVWDLQLQJZLWKVPRRWKPXVFOHVSHFLILFĮ-actin antibody. Cell Proliferation Assays Cell proliferation was determined using either a 5-bromo-2’-deoxyuridine (BrdU) incorporation assay or cell counting. BrdU assays (QIA58, Calbiochem, San Diego, CA) were performed in a 96-well 96-w well format att aaccording cccor o di d ng tto o ma manu manufacturer’s nufa nu factur fa urer’s ins instructions, n tr ns t uc uctionns, using usi sing n starting start tart r in ingg cell c ll densities ce den ensi siti si ties of ti of 40 4000 00 cells/well. cellls/ s/well. Forr ccell ell cou counting, ounting, g, cells were werre trypsi trypsinized inizeed and and resuspended r suusppen re nded after aftterr eexperimental af xperime m nttal me M d with a TC C100TM automated cell counter (Bi (Bio-Rad). B o-R Rad). F FBS BS or procedures; densities were counted TC10

PDGF was used as positive controls. Overexpressing NAMPT in hPASMCs and hPAECs A human wild-type NAMPT expression sequence was cloned downstream of the ubiquitin promoter in a third generation lentiviral plasmid. Separately, a control plasmid was generated by cloning a GFP expression sequence in the same position. Co-transfection of either control-GFP or NAMPT-expressing constructs with three other packaging plasmids in HEK 293FT cells produced lentivirus capable of expressing either GFP or NAMPT. Viral concentration was determined by qPCR using primers spanning the HIV 5'LTR region. hPASMCs and hPAECs

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were infected with control or NAMPT virus at an MOI of 5 for experiments performed 72 hours later. Plasma and cell culture medium NAMPT measurement Plasma NAMPT levels were measured using Bio-Plex Pro Human Diabetes Visfatin Set (BioRad, cat.#171-B7012M). NAMPT secreted from hPAECs during the culture was measured using a Nampt (Visfatin/PBEF) (human) ELISA Kit (Adipogen, cat.#AG-45A-0006YEK-KI01). [Ca2+]cyt Measurement [Ca2+]cyt was measured in PASMC loaded with fura-2 (4PM) in a Nikon digital fluorescent Downloaded from http://circ.ahajournals.org/ by guest on April 16, 2017

LPDJLQJV\VWHP&HOOVZHUHORDGHGZLWKȝ0IXUD-2 acetoxymethyl ester (fura-2/AM) for 60 min at 25°C and [Ca2+]cyt was measured using a ratiometric method at 32°C. Cyclepiazonic Cycle lepi piaz pi azon az onic on ic aacid cd ci (CPA, CPA, a specific Ca2+-ATPase inhibitor) was used to induce store-operated calcium entry SOCE)). (SOCE). Transfection Tran ansfection ooff Sm an Smal Small ll Interfering Inte In terf rfer rf erin er in ng RN RNA A (siR (siRNA) RNA N ) in h hPASMCs PASM PA SMCs SM Css M Usin ingg Lipofect in ctam ct a ineTM am RNAiMAX RNA NAiMAX NA X Reagent Reagent (I (Invitrogen, Invi vitrog ogen og en, cat.# en caat.# 13 13778-1 13778-150), 150) 0), hPAS 0) hPASMCs AS SMC MCs weree Using Lipofectamine

transfected ransfected with NAMPT siRNA (L-000458-00), STIM2 siRNA (L-013166-01-0005), ORAI2 siRNA (L-015012-00) or scrambled siRNA (D-001810-02), which were purchased from Dharmacon (Thermo Fisher Scientific, Lafayette, CO, USA) according to the manufacturer’s protocol. Animal Models of Pulmonary Hypertension and Hemodynamic Measurements All experiments were approved by the Ethics and Animal Care Committee of the University of Illinois at Chicago. Three rodent models of PH were used in this study. They included a mouse model of hypoxia-induced PH, a rat model of monocrotaline (MCT)-induced PH and a rat model of Sugen-hypoxia induced severe PH. Nampt -/- mice exhibit embryonic lethality thus Nampt+/-

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mice were used in this study. Male mice (8-week old Nampt-/- mice in C57BL/6 background and their WT siblings) were exposed to hypoxia (10% O2) in a ventilated chamber for four weeks. Male Sprague-Dawley rats (190-200 g) were used for the MCT and Sugen-hypoxia induced PH studies. In the MCT-induced PH model, MCT was dissolved in 0.5N HCl to 200 mg/ml, neutralized to pH 7.4 with 0.5N NaOH, and then diluted with sterile water to 60 mg/ml. One dose of MCT (60 mg/kg body weight) was subcutaneously injected to rats. Control rats were injected with the equivalent volume of dissolvent solution according to their weights. Food and water were provided ad libitum and the rats are checked once per day. For the prevention Downloaded from http://circ.ahajournals.org/ by guest on April 16, 2017

experiment, FK866 (2.5 mg/kg) was started in day 2 after the MCT injection and continued for two wo weeks; for the therapeutic experiment, FK866 (2.5mg/kg every 48 hours) was as sstarted tart ta r ed dday rt ay 14 after the MCT injection and continued for two weeks. Our historical data shows that rats in day 14 after MCT injection develop pulmonary hypertension as demonstrated by significantly increased ncrrea eased RVSP SP aand n R nd RVH VH (Su (Supplemental Supp Su pple pp leme le m nt ntal Figure Figu g ree S1). gu ). E Each achh gr ac grou group ouup in iincluded clud cl u ed d 6 tto o 8 rats rats. t . In tthe ts he Suge Sugen-hypoxia genn-hypoxiaa P ge PH H mo model, odel, one one dose off S Sugen ugen (2 (20 20 m mg/kg) g/kg g/ kg)) wa kg wass given give ven subcutaneously suubcut utaneously ut ly aatt the fir first rstt day of hypoxia hypoxia exposure (10% O2). ) After three-week chronic hy hhypoxia poxia exposure, the rats were placed back to room air. FK866 (2.5 mg/kg every 48 hours) was then started two weeks after reoxygenation and continued for three weeks. Each group included 10 to 12 rats. Hemodynamic data after two-week reoxygenation indicates that rats develop significant PH at this time point (Supplemental Figure S2). Right ventricular systolic pressure (RVSP) was determined by right heart catheterization using a Millar pressure transducer catheter. A weight ratio of the right ventricle divided by the sum of left ventricle and septum (RV/(LV+S)) was measured to determine the extent of right ventricular hypertrophy (RVH). The RV contractility index was calculated as (dp/dt)max /instantaneous RV pressuremax, as previously described.27 Pulmonary

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artery remodeling was assessed using Aperio ImageScope software (version 11) after lungs were stained with hematoxylin and eosin (H&E). A minimum of 10 microscopic fields were examined for each slide. Approximately twenty muscular arteries with diameters (D) 50 -ȝPRU' ȝPSHUOXQJVHFWLRQZHUHRXWOLQHGDQGPHDVXUHG9HVVHOUHPRGHOLQJZDVFDOFXODWHGDV H[WHUQDO vessel area – internal vessel area) / external vessel area), as previously described 28. Occlusive pulmonary arteries were counted from H&E stained lung slides from each group. Statistical Analysis Statistical analysis of experimental data was performed using GraphPad Prism 5.1 (GraphPad Downloaded from http://circ.ahajournals.org/ by guest on April 16, 2017

Software, Inc., La Jolla, CA). Results are expressed as mean ± standard error of mean (SEM), except where noted, from at least three experiments. Student t - test and ANOVA Aw eree us er uused ed tto o were compare two and three groups, respectively except for the comparison of NAMPT plasma levels between control and PAH subjects where a Mann-Whitney test was used. A P < 0.05 was con nsid idered sta id ati tissticcal a ly y ssignificant. igni ig n fi fica cant ca nt. nt considered statistically

Results NAMPT is Up-regulated in Patients with PAH and in Experimental Rodent PH Models. Plasma NAMPT levels were significantly elevated in patients with PAH (n=103, median [IQR] 3197 [1493-7280]) when compared to controls (n=53, median [IQR] 1686 [990-2608]; p 0.05

NAMPT

SM-ACTIN

Merged

Normoxia

Hypoxia

Figure S5.

NAMPT expression in a mouse model of hypoxia induced PH.

Immunofluorescence staining of lung tissues from mice after 4-week hypoxia exposure (10% O2) when compared to normoxia demonstrate increased NAMPT expression in the pulmonary vasculature mainly in PAECs and the adventitia, but not in the media, as well as in alveolar and airway epithelial cells. A representative pulmonary vasculature pointed by an arrow was highlighted in the right corner of each image. Paraffinembedded lung tissue sections were deparaffinized with xylene and rehydrated. Antigen retrieval was used before blocking in PBS with 10% normal goat serum, 0.1%BSA, 0.3% TX-100. The antigen retrieval solution is Tris-EDTA buffer (10 mM Tris Base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0). The dilutions for rabbit against NAMPT (Bethyl Laboratories, Inc. catalog# A300-372A) and Cy3-labelled mouse against smooth muscle actin are 1:100 and 1:300, respectively. Secondary antibody for NAMPT was Alexa Fluor® 488 Donkey anti-Rabbit IgG antibody (1:500). An anti-fade mounting media with DAPI (Life Science Inc) was used to fix the coverslip to a slide. The slides were examined using a Nikon Eclipse E800 fluorescence microscope, and the images were processed by MetaMorph software (Molecular Devices, Inc.). Size bar: 50 µm.

NAMPT

SM-ACTIN

Merged

Control

MCT

Hyp+sugen

Figure S6. NAMPT expression in rat models of PH. Immunofluorescence staining of lung tissues from rats after monocrotaline treatment (2 weeks after monocrotaline injection) and rats after hypoxia+sugen treatment (3 weeks hypoxia exposure + 2 weeks reoxygenation) when compared to control healthy rats, demonstrate increased NAMPT expression in the pulmonary vasculature mainly in PAECs and the adventitia, but not in the media, as well as in alveolar macrophages, alveolar and airway epithelial cells. A representative pulmonary vasculature pointed by an arrow was highlighted in the right corner of each image. Paraffin-embedded lung tissue sections were deparaffinized with xylene and rehydrated. Antigen retrieval was used before blocking in PBS with 10% normal goat serum, 0.1%BSA, 0.3% TX-100. The antigen retrieval solution is Tris-EDTA buffer (10 mM Tris Base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0). The dilutions for rabbit against NAMPT (Bethyl Laboratories, Inc. catalog# A300-372A) and Cy3labelled mouse against smooth muscle actin are 1:100 and 1:300, respectively. Secondary antibody for NAMPT was Alexa Fluor® 488 Donkey anti-Rabbit IgG antibody

(1:500). An anti-fade mounting media with DAPI (Life Science Inc) was used to fix the coverslip to a slide. The slides were examined using a Nikon Eclipse E800 fluorescence microscope, and the images were processed by MetaMorph software (Molecular Devices, Inc.). Size bar: 50 µm.

WT Nampt+/A

C

M

* **

4 3 2 1 0

Size (kDa) Nampt

43

β-actin WT

Nampt+/-

1.5

1.0

*** 0.5

D

NAD levels in lungs (X10 -2pmol/µg)

Normoxia

52

Nampt protein levels in lungs (fold change)

B

NAD levels in hearts (X10 -2pmol/µg)

+/+ +/+ +/- +/+ +/-

5

Hypoxia *

8 6 4 2 0

Normoxia

0.0

WT

Hypoxia

Nampt+/-

Figure S7. NAD levels in Nampt+/- mice. Compared to wild-type siblings, Nampt+/mice exhibited significantly lower baseline levels of NAMPT in the lung tissue homogenates. NAD levels in the lung and heart tissues are significantly lower in Nampt+/- mice after 4-week hypoxia (10% O2) exposure. A. Mouse genotyping data via PCR and agarose gel electrophoresis shows that the individuals with double bands (284 bp and 458 bp) are NAMPT heterozygous, with one band wild-type (458 bp). PCR primers: forward: 5’-CAGCAGCAGACCATTTTCAA - 3’;

reverse primer: 5'-

GGGAGTGACACAGCAAATCA-3'. B. Western blotting using lung homogenates shows NAMPT+/- mice exhibited significantly lower baseline levels of NAMPT in the lung tissue homogenates; C. NAD levels are significantly lower in the heart tissues from Nampt+/mice under normoxia or hypoxia conditions; D. NAD levels are significantly lower in the lung tissues from Nampt+/- mice under hypoxia exposure. At baseline, NAD levels have a non-significant trend to be lower in the lung tissues from Nampt+/- mice. *, P < 0.05; **, p < 0.01; ***, p