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Cellular Biology Uncoupling Protein 2 Impacts Endothelial Phenotype via p53-Mediated Control of Mitochondrial Dynamics Yukio Shimasaki, Ning Pan, Louis M. Messina, Chunying Li, Kai Chen, Lijun Liu, Marcus P. Cooper, Joseph A. Vita, John F. Keaney Jr Rationale: Mitochondria, although required for cellular ATP production, are also known to have other important Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 functions that may include modulating cellular responses to environmental stimuli. However, the mechanisms whereby mitochondria impact cellular phenotype are not yet clear. Objective: To determine how mitochondria impact endothelial cell function. Methods and Results: We report here that stimuli for endothelial cell proliferation evoke strong upregulation of mitochondrial uncoupling protein 2 (UCP2). Analysis in silico indicated increased UCP2 expression is common in highly proliferative cell types, including cancer cells. Upregulation of UCP2 was critical for controlling mitochondrial membrane potential (Δψ) and superoxide production. In the absence of UCP2, endothelial growth stimulation provoked mitochondrial network fragmentation and premature senescence via a mechanism involving superoxide-mediated p53 activation. Mitochondrial network fragmentation was both necessary and sufficient for the impact of UCP2 on endothelial cell phenotype. Conclusions: These data identify a novel mechanism whereby mitochondria preserve normal network integrity and impact cell phenotype via dynamic regulation of UCP2. (Circ Res. 2013;113:891-901.) Key Words: angiogenesis ■ endothelium ■ endothelial function ■ ischemia ■ mitochondrial uncoupling proteins T ■ mitochondria ■ superoxides proteins, such as cytochrome c,9 catalase,10 and cytochrome oxidase.11 The mitochondrial release of proteins, such as apoptosis-inducing factor and the second mitochondrial activator of caspase/DIABLO gene product, into the cytosol also regulates genes important for apoptosis.12 Thus, mitochondrial events are readily communicated to the nucleus to affect gene regulation. The cellular responses to environmental stimuli also involve mitochondria. Proteolytic processing of cell-surface receptors, such as epidermal growth factor receptor tyrosine kinase B4, can release intracellular domains that trigger mitochondrial release of proapoptotic proteins.13 Energy deprivation responses, such as AMP kinase activation, induce mitochondrial gene upregulation that is critical for cellular stress adaptation.14 Upregulation of endothelial vascular endothelial growth factor involves perinuclear mitochondrial clustering and the local production of mitochondrial •O2−.15 The latter has also been linked to the control of mitogen-activated protein kinase phosphatases and the coordination of nuclear factor κB–dependent inflammatory responses.16 Finally, mitochondrial electron transport is known to impact the activation state of cell-surface growth factor receptors.17 Thus, he cellular requirement for ATP as a high-energy intermediate to fuel many critical cellular reactions is universal. The production of ATP depends on an extracellular source of carbohydrates or lipids that are initially metabolized in the cytosol to products that are then transported into mitochondria for oxidative phosphorylation to produce a proton gradient (ie, membrane potential, Δψ) that powers ATP generation.1 Known mitochondrial functions extend beyond ATP production to include other cellular processes, such as apoptosis,2 heme synthesis,3 calcium homeostasis,4 inflammation,5 and development.6 Thus, mitochondria have the capacity to impact cellular phenotype via energy-dependent and energy-independent mechanisms. Editorial, see p 846 One means by which mitochondria can impact cellular phenotype is through participation in signaling paradigms, and there is ample evidence from multiple organisms that mitochondria signal to the nucleus. Respiratory deficiency induced by either pharmacologic7 or genetic8 means is known to affect gene expression in the nucleus. Mitochondrial-derived heme coordinates nuclear regulation of genes encoding heme-dependent Original received March 5, 2013; revision received June 27, 2013; accepted July 2, 2013. In May 2013, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15 days. From the Division of Cardiovascular Medicine, Department of Medicine (Y.S., N.P., C.L., K.C., L.L., M.P.C., J.F.K.); Division of Vascular Surgery, Department of Surgery, University of Massachusetts Medical School, Worcester, MA (L.M.M.); and the Department of Medicine, Boston University School of Medicine (J.A.V.). The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA. 113.301319/-/DC1. Correspondence to John F. Keaney Jr, MD, Division of Cardiovascular Medicine, University of Massachusetts Medical School, 55 Lake Ave N, Room S3-855, Worcester, MA, 01655. E-mail [email protected] © 2013 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.113.301319 891 892 Circulation Research September 13, 2013 Nonstandard Abbreviations and Acronyms Δψ BAEC Drp1 Fis1 MLEC Mfn1 Mfn2 Opa1 ROS UCP2 mitochondrial membrane potential bovine aortic endothelial cell dynamin-related protein 1 fission 1 murine lung endothelial cell Mitofusin 1 Mitofusin 2 optic atrophy 1 reactive oxygen species uncoupling protein 2 Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 the mitochondrion is emerging as an important organelle for the coordination of cellular environmental responses. Although mitochondria clearly impact how cells respond to the environment, the mechanisms involved in this process are not well-understood. Many mitochondrial actions are linked to the electron transport chain and the resultant membrane gradient, Δψ. Control of Δψ involves the relative availabilities of substrates for electron transport (nicotinamide adenine dinucleotide and flavin adenine dinucleotide-2), respiration (O2), and ATP synthesis (ADP),1 as well as any proton leak.18 The latter is largely regulated via uncoupling proteins that belong to the mitochondrial carrier superfamily.18 Herein, we report that endothelial cell proliferation and angiogenesis involve upregulation of mitochondrial uncoupling protein 2 (UCP2) to reduce Δψ and limit mitochondrial •O2− that otherwise promotes p53-dependent mitochondrial fragmentation resulting in premature senescence. These findings suggest a new function for UCP2 that has broad implications for processes that involve the vascular endothelium. Methods Endothelial Cell Culture and Transfection Bovine aortic endothelial cells (BAECs) were purchased from Genlantis (San Diego, CA) and were cultured in endothelial basal medium supplemented with EGM-MV Bullet Kit from Lonza (Walkersville, MD). Before each experiment, BAECs were made quiescent by 24-hour incubation in low-serum medium (endothelial basal medium supplemented with 0.1% or 0.4% fetal bovine serum). Human aortic endothelial cells were from Lonza and were cultured as described.14 Murine lung endothelial cells (MLECs) from mice of both sexes were isolated as described,19 and cultured on gelatinor collagen-coated plates and grown in MLEC medium containing 20% fetal bovine serum, 38% Dulbecco's modified eagle medium, 38% Ham’s F-12 with 100-μg/mL endothelial cell growth supplement (ECGS, Biomedical Technologies, Stoughton, MA), 4-mmol/L L-glutamine, 100-μg/mL heparin, and penicillin/streptomycin. Endothelial purity was confirmed by staining with 1,1'-dioctadecyl – 3,3,3',3'-tetramethyl-indocarbocyanine perchlorate acetylated lowdensity lipoprotein (Biomedical Technologies, Stoughton, MA) and anti-CD31 antibody (BD Biosciences, San Jose, CA). Quantitative Real-Time Polymerase Chain Reaction Total RNA was extracted from cells and tissues with the RNeasy Mini Kit (Qiagen) or TRIzol reagent (Invitrogen), and 1 μg of total RNA was reverse transcribed with oligo(dT) primers for cDNA synthesis. Realtime polymerase chain reaction was performed in the iQ5 real-time polymerase chain reaction detection system (Bio-Rad Laboratories) and the products were detected using either SYBR Green dyes (Bio-Rad Laboratories) or TaqMan probes of the TaqMan Gene Expression Assays for specific genes (Applied Biosystems, Foster City, CA). Transfections For gene overexpression, BAECs or MLECs were incubated with adenoviruses at 50 to 100 multiplicity of infection as described.14,20 For gene silencing, BAECs were transfected with 1.3 μg of small interfering RNA oligonucleotides (Thermo Scientific Dharmacon, Lafayette, CO) against human UCP2 (Cat. Nos. D-005114-01, -02, -03, and -04); human superoxide dismutase 2 (SOD2; D-009784-03, -04, -19, and -20); or negative control (D-001210-02, -03, -04, and -05) as described.14 Likewise, MLECs were transfected with mouse mitofusin 1 (Mfn1) (J-065399-09, -10, -11, and -12); mouse mitofusin 2 (Mfn2) (J-046303-05, -06, -07, and -08); mouse p21 (J-058636-05, -06, -07, and -08); mouse p53 (J-04064209, -10, -11, and -12); or the nontargeting pool (D-001810-10-05). Cell Proliferation and Migration Assays DNA synthesis was directly measured via the 3H-thymidine incorporation assay. Cell proliferation was determined with CyQUANT GR fluorescent dye (Invitrogen Molecular Probes, Eugene, OR) to determine the relative cell number with a fluorescence microplate reader (Gemini XPS, Molecular Devices). Cell migration was assayed with the in vitro scratch assay in which a monolayer of quiescent cells was uniformly scratched and the rate of cell migration to close the void was evaluated 20 hours after wounding using ImageJ. Mitochondrial Membrane Potential and Superoxide Measurements Mitochondrial membrane potential (Δψ) was determined using 2 complementary fluorescent methods. Cells were incubated with 1-μmol/L 5, 5´, 6, 6´-tetrachloro-1, 1´, 3, 3´-tetraethylbenzimidazolcarbocyanine iodide (JC-1) for 30 minutes and Δψ estimated as the fluorescence ratio of JC-1 aggregates (red, excitation 550 nm, emission 600 nm) to monomers (green, excitation 485 nm, emission 535 nm) formed as a function of inner mitochondrial membrane potential.21 For measurement of mitochondrial superoxide (•O2−), cells were loaded with 5-μmol/L MitoSOX Red (Invitrogen) for 10 minutes and the fluorescence (excitation 396 nm; emission 510 nm) determined. Live cells were labeled with 0.5-μmol/L MitoSOX for 20 minutes and the fluorescence images at both 405- and 514-nm excitation were captured using an Eclipse TE2000-S fluorescence microscope (Nikon, Melville, NY) with a CCD camera using a SPOT Insight 2MP Firewire Color Mosaic (Diagnostic Instruments, Sterling Heights, MI). Antibodies and Immunoblotting Antibodies against cytochrome c oxidase subunit IV, p21cip/waf, p16ink16a, phospho-p53 (mouse Ser18), phospho-c-Jun (Ser63), phospho p38 mitogen-activated protein kinase (Thr180/Tyr182), and -rabbit or mouse IgG were purchased from Cell Signaling Technology. Antibodies against cytochrome c were from BD Biosciences. We obtained antibodies against UCP1, UCP2, and UCP3 from Fitzgerald Industries International (Acton, MA) and specific UCP2 antibodies (N-19 and A-19) and native p53 antibody from Santa Cruz Biotechnology (Santa Cruz, CA). We obtained SOD2 antibody from Millipore (Temecula, CA) and actin antibody from SigmaAldrich (St. Louis, MO). Protein extracts in DTT-containing SDS sample buffer were separated in 10% or 12% SDS-polyacrylamide gels and transferred to Hybond ECL nitrocellulose membranes (GE Healthcare, Piscataway, NJ). Immunoblotting was then performed and quantified as described.22 In cases where loading controls produced paired bands, both were used for quantification. Oxygen Consumption and Lactate Using a Clark-type oxygen electrode (Hansatech), respiration in whole cells was quantified as previously described.23,24 Briefly, 1×106 cells were resuspended in 1 mL of respiration medium (Dulbecco`s phosphate-buffered saline, 2 mmol/L glucose, 1 mmol/L pyruvate, 2% fatty acid-free bovine serum albumin). To obtain proton leak, oligomycin was added to a final concentration of 2 µmol/L. To measure maximal respiration, carbonyl cyanide 4-(trifluoromethoxy) Shimasaki et al UCP2 and Endothelial Function 893 phenylhydrazone was added to a final concentration of 2.4 µmol/L. Nonmitochondrial respiration, obtained by adding myxothiazol to a final concentration of 4 µmol/L, was subtracted from total respiration measurements. For lactate, accumulation was determined by a lactate fluorometric assay kit (MBL International, Woburn, MA) according to the manufacturer’s instructions and the concentration of secreted lactate was normalized to the cell number in each sample. Capillary Sprouting Assay in Aortas Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 Thoracic aorta was placed in endothelial basal medium-2 as described,25 periaortic tissue was carefully removed, and then the aorta was cleaned and sliced into 1 mm-long rings. Rings were then embedded in liquid collagen gel in 48-well plates (BD Biosciences), incubated at 37°C for 1 hour to polymerize the collagen, and the solid gel covered with MLEC medium diluted 1:2 with Dulbecco's modified eagle medium. Each aortic ring was examined daily and capillary sprouts counted along the sample perimeter under ×100 and ×200 magnification. Vascular sprouts were distinguished from fibroblasts via morphology as described26 and CD31 staining. To infect aortic rings with adenoviral vectors, each ring was embedded in liquid collagen gel containing respective vectors (adenovirus–UCP2, adenovirus–SOD2, etc) at 1.0×108 pfu before polymerized. Transfection was validated in separate experiments with green fluorescent protein adenovirus (AdGFP) transfection and after 4 days by fixation of collagen gel-embedded tissue with 4% formaldehyde in phosphate-buffered saline for 10 minutes at 4°C. Fixed sections underwent immunofluorescence staining with anti-GFP and anti-CD31 antibody and counterstained with 4',6-diamidino-2-phenylindole before fluorescence imaging. Animals and Hindlimb Ischemia Model Heterozygous UCP2-null animals27 on the C57 background were obtained from Dr Bradford Lowell (Harvard Medical School) and bred to homogeneity with age-matched controls. SOD2 heterozygous mice were obtained from Jackson Laboratories and bred to obtain heterozygous and control animals. All experimental protocols were approved by the Institutional Animal Care and Use Committee of University of Massachusetts Medical School. Male mice at 8 to 12 weeks of age were anesthetized with intraperitoneal injection of combination of 100 mg/kg ketamine hydrochloride and 5 mg/kg xylazine (Webster Veterinary, Devens, MA) before surgery. Unilateral hindlimb ischemia in the left leg was introduced in the mice as described.28 In selected experiments, 100 µL (2.0×108 pfu) of adenoviral vectors encoding UCP2, or β-glactosidase (LacZ) was injected into 5 different sites of the ischemic thigh muscles, such as adductor longus, adductor magnus, and adductor brevis muscles. Hindlimb tissue perfusion was assessed with Moor LDI2-IR laser-Doppler imaging system (Moor Instruments, Devon, UK). Blood flow images were obtained under conditions of constant body temperature (36±1.0°C) and average hindlimb blood flow was expressed as the ratio of ischemic to nonischemic foot flow to account for minor variations in imaging conditions. Cell Cycle Analysis Endothelial cells at 50% confluence were synchronized in 0.4% serum overnight. Cells were then cultured in complete medium and at 24 and 48 hours harvested, stained with propidium iodide and subjected to fluorescence-activated cell sorting analysis as described.29 Cellular Senescence Assay MLECs were seeded onto 0.1% gelatin-coated 12-well plates and maintained for 14 days under normal conditions. Senescence was assayed as senescence-associated β-galactosidase activity using the senescence β-galactosidase staining kit (Cell Signaling Technology). Both bright-field and phase-contrast pictures were viewed through ×4 or ×10 objective on a microscope on the Eclipse TE2000-S microscope as above and images processed with SPOT Advanced Version 4.6 imaging software (Diagnostic Instruments Inc). Senescence-associated βgalactosidase–positive cells were quantified with the ImageJ software. Mitochondrial Length Measurements Mitochondrial length was measured as an index of mitochondrial fragmentation. Live endothelial mitochondria were labeled with 100-nmol/L MitoTracker Green FM for 30 minutes and the fluorescence images were captured using a ×100 oil immersion objective to acquire high-resolution images of mitochondria as described.30 To determine mitochondrial length, ImageJ was applied in a blinded fashion to the measurement for each frame of a selected region of interest as described for determining DNA contour lengths.31 Statistical Analysis All data are expressed as mean±SE and the numbers of independent experiments are indicated. Statistical comparisons were conducted between 2 groups by use of Student t test or Mann–Whitney U test as appropriate. Multiple groups were compared with either 1-way Kruskal–Wallis or ANOVA with a post hoc Tukey–Kramer multiple comparisons test as indicated in legends. A P value <0.05 was considered significant. All statistics were done using StatView version 5.0 (SAS Institute, Cary, NC) or GraphPad Prism version 5 (GraphPad Software, La Jolla, CA). Results Dynamic Modulation of UCP2 and Δψ With Endothelial Proliferation Quiescent BAEC monolayers stimulated to proliferate with fetal bovine serum exhibited a reduction in Δψ (Figure 1A), whereas increasing cell confluence was associated with an increase in Δψ (Figure 1B and 1C). Because mitochondrial UCPs are implicated in Δψ regulation,18 we probed endothelial UCP expression and observed mRNA and protein only for UCP2 and UCP3 (Figure 1D). We then observed that UCP2 is upregulated with endothelial proliferation (Figure 1A) and downregulated with increasing confluence, with no dynamic regulation of UCP3 (Figure 1B and 1E). We could recapitulate the proliferation-induced changes in Δψ by molecular manipulation of UCP2 (Figure 1F) and UCP2-null cells had increased Δψ (Figure 1G). Thus, our data indicate that the proliferation-induced changes in Δψ can be explained, at least in part, by dynamic regulation of UCP2. UCP2 and Endothelial Metabolism Because we found that UCP2 is dynamically regulated in the endothelium, we probed its implications for basic mitochondrial functions. We found that UCP2-null cells had a trend for lower basal respiration rate (*P<0.07) than wild-type cells and similar rates of basal proton leak. UCP2-null cells also had similar maximal respiration (2.08±0.30) than wildtype cells (2.61±0.58; P=0.23 by 2-tailed t test; Figure 2A). Proliferating UCP2-null cells also produced lesser lactate than wild-type cells (Figure 2B), consistent with a lower rate of glycolysis.32 Suppression of UCP2 was associated with reduced ATP levels (Figure 2C) and endothelial ATP levels seemed greatest in endothelium with the highest proliferation rate (Figure 2D). Collectively, these data indicate that UCP2null cells have more prominent perturbations in glycolysis than respiration, and that endothelial ATP levels vary as a function of proliferation. UCP2 Regulates Endothelial Phenotype via Changes in Δψ To gain insight into the potential function of UCP isoforms, we examined UCP mRNA in silico as a function of cell type. We found that UCP1 exhibited relatively homogeneous mRNA expression across multiple cell types as did UCP3, with the exception of a 3-fold higher expression in skeletal muscle 894 Circulation Research September 13, 2013 (kDa) 33 42 1 0 2 1 0 0.5 5 10 FBS (%) D (%) 200 * 150 100 50 0 50 60 70 80 90 100 50 60 70 80 90 100 Cell Confluence (%) E Cell Confluence (%) F (kDa) UCP2 UCP3 35 30 0.2 0.1 0 G 100 80 * 60 * * * 40 20 0 50 60 70 80 90 100 Cell Confluence (%) Figure 1. Regulation of endothelial uncoupling protein 2 (UCP2) and Δψ with proliferation. Bovine aortic endothelial cells (BAECs) were (A) stimulated with fetal bovine serum (FBS) or (B) cultured to specific confluence before assessment of Δψ by JC-1 fluorescence and expression of UCP2, UCP3, cytochrome c oxidase IV (COX IV), or actin by immunoblotting. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) of 5-μmol/L was used as a control for the lower limit of Δψ. Data represent n=3; *P<0.05 for trend from 1-way ANOVA. C, Mitochondrial Δψ by tetramethylrhodamine ethyl ester (TMRE) corrected for mitochondrial mass. n=3; *P<0.05 for trend from 1-way ANOVA. D, Expression of UCP2 and UCP3 mRNA and protein in BAECs. E, Densitometric analysis of UCP2 protein by immunoblot as a function of confluence. n=5; *P<0.05 vs 50% by ANOVA with Tukey–Kramer post hoc test. F, BAECs (control [CTL]) were transfected with UCP2 (Ad-UCP2 at the indicated multiplicity of infection [MOI]), β-galactosidase (Ad-LacZ; 100 MOI), or the indicated small interfering RNA (siRNA) followed by assessment for Δψ or protein levels of transfected or endogenous UCP2, COX IV, or actin by immunoblotting. n=3; *P<0.05 vs Ad-LacZ or siRNA control (siCTL). G, Mitochondrial Δψ by TMRE as in C. n=3; *P<0.05 by unpaired t test. Ad indicates adenovirus; GADPH indicates glyceraldehyde 3-phosphate dehydrogenase; siUCP2, small interfering UCP2; and WT, wild-type. (Online Figure I). In contrast, UCP2 mRNA expression varied more than 50-fold as a function of cell type with the highest transcript levels in rapidly dividing cells, including those harboring erythroid (CD71), endothelial progenitor (CD34), and endothelial angiogenic (CD105) markers (Online Figure A 250 Lactate (pmol/cell) Relative O2 uptake 2 1 200 150 100 * 50 0 0 Basal Oligo D (%) 100 * 80 60 40 20 0 WT FCCP SiCTL siUCP2 UCP2-/- (%) (Cellular ATP Level) C I). Based on this latter association, we tested known stimuli for angiogenesis such as vascular endothelial growth factor and AMP kinase33 and found UCP2 upregulation (Figure 3A). Serum- and vascular endothelial growth factor–mediated UCP2 upregulation was associated with c-Jun N-terminal B WT UCP2-/- 3 (Cellular ATP Level) Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 UCP2 / Actin (% of 50) * 3 * JC-1 (Δψ) JC-1 (Δψ) Actin 2 UCP2 UCP3 COX IV Actin 33 34 16 UCP2 42 3 C (kDa) (UCPs mRNA / GAPDH mRNA) B (TMRE / MitoGreen) A 100 80 60 40 20 0 * 50 60 70 80 90 100 Oligo Cell Confluence (%) Figure 2. Metabolic signature of uncoupling protein 2 (UCP2)-null endothelium. A, Oxygen consumption was determined in wild-type (WT) and UCP2-null murine lung endothelial cells (MLECs) in the presence or absence of oligomycin (oligo) or carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) using a Clark electrode as described in Methods section. B, Media from wild-type or UCP2-null MLECs in 0.1% serum was analyzed for lactate content and expressed as a function of cell count. n=3; *P<0.01 vs WT by unpaired t test. C, Bovine aortic endothelial cell (BAEC) ATP levels as a function of UCP2 status as described in Methods section. n=3; *P<0.01 vs siRNA control (siCTL) by unpaired t test. D, BAEC ATP levels as a function of confluence or oligomycin (oligo) treatment. n=4; *P<0.01 for trend by 1-way ANOVA. siUCP2 indicates small interfering UCP2. Shimasaki et al UCP2 and Endothelial Function 895 B (kDa) 33 UCP2 24 SOD2 16 COX IV 15 Cyt c 42 Actin D Cell Migration (cell #) 400 * 300 200 * 100 0 Cells / well (x 104) C * 50 0 CTL VEGF Ad- Ad- Control UCP2 LacZ UCP2 siRNA siRNA E 48 WT UCP2-/- 40 32 24 * 16 0 1 3 5 (days) 20 16 12 * 8 4 0 0 4 5 G 6 7 (days) 8 9 UCP2-/- UCP2-/- + Ad-UCP2 UCP2-/- + Ad-LacZ UCP2-/- 20 16 12 * 8 Perfusion (ischemic/non-ischemic) WT WT UCP2-/- * 100 H (Sprouts / aorta) Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 F (Sprouts / aorta) 150 8 Ad- Ad- Control UCP2 LacZ UCP2 siRNA siRNA * 200 Cell Migration (cell #) Cell Proliferation (%CTL) A 7 5 7 (days) * 30 20 10 WT UCP2-/- WT UCP2-/- 1.2 1.0 0.8 0.6 * 0.4 0.2 0 Pre 0 1 3 7 14 21 (Post-Operative Day) Pre-op POD 0 POD 7 POD 14 N N N 28 POD 28 WT UCP2 -/- I 0 3 40 0 9 4 1 50 9 kinase activation and inhibition attenuated both UCP2 upregulation and vascular endothelial growth factor–mediated endothelial cell proliferation (Online Figure II). We also manipulated endothelial UCP2 levels and found that endothelial cell proliferation (Figure 3B) and migration (Figure 3C) were directly related to UCP2. Consistent with these observations, UCP2-null endothelium exhibited impaired proliferation (Figure 3D) and migration (Figure 3E), without any compensatory upregulation of UCP3 (Online Figure IIIA). Because endothelial proliferation and migration are features of angiogenesis, we examined the impact of UCP2 in a capillary sprout formation assay, an in vitro model of early angiogenesis. Wild-type aortic segments exhibited a higher rate and total extent of capillary sprout formation than did UCP2-null aortic segments (Figure 3F), and reconstitution of UCP2 into UCP2-null endothelium rescued the defect in capillary sprout formation (Figure 3G). Similarly, capillary sprout formation from UCP2-null adipose tissue was impaired relative to wildtype, and UCP2 reintroduction also rescued this defect (Online I I N I Figure 3. Uncoupling protein 2 (UCP2) modulates endothelial cell proliferation and migration. A, Wild-type (WT) murine lung endothelial cells (MLECs) were treated with aminoimidazole carboxamide ribonucleotide (AICAR; 0.5 mmol/L) or vascular endothelial growth factor (VEGF; 25 ng/mL) as indicated for 24 hours followed by immunoblots for the indicated proteins. Actin, cytochrome c (Cyt c), and cytochrome c oxidase subunit IV were loading controls for cytosol and mitochondria. B, Bovine aortic endothelial cell (BAEC) proliferation by [3H]-thymidine incorporation in response to VEGF or UCP2 manipulation as indicated. Data are normalized to the VEGF vehicle control. n=4 to 5; *P<0.05 vs respective controls by Mann–Whitney U test. C, BAECs underwent UCP2 manipulation as indicated and migration assessed 20 hours after scratch. n=3 to 5; *P<0.01 vs respective control by Mann–Whitney U test. D, Proliferation of WT and UCP2−/− MLECs by cell count. n=4; *P<0.05 vs WT by 2-way repeated measures ANOVA. E, Migration of WT and UCP2−/− MLECs after scratch wounding. n=5; *P<0.01 vs WT by Mann–Whitney U test. F, Aortic segments from the indicated genotypes implanted in collagen gel after 7 days (bar=500 μm) with capillary sprout counts as a function of time. n=6; *P<0.001 vs WT by 2-way repeated-measures ANOVA. G, Capillary sprouting in UCP2−/− aortic segments in collagen gel containing no additions (contol [CTL]) or the indicated adenoviral vector. n=6 per group; *P<0.01 vs vehicle or Ad-LacZ by 2-way repeated measures ANOVA. (H) Blood flow recovery in WT and UCP2−/− mice with unilateral femoral artery excision expressed as a fraction ratio of the ischemic (I) vs nonischemic (N) limbs with representative images of laser-Doppler tissue perfusion in hindlimbs. n=4; *P=0.02 vs WT by 2-way repeated measures ANOVA. Ad indicates adenovirus; POD, postoperative day; siRNA, small interfering RNA; and SOD2, superoxide dismutase. Figure IIIB and IIIC). To determine the physiological relevance of our in vitro observations, we examined hindlimb ischemiainduced angiogenesis and observed a lower rate of blood flow recovery in UCP2-null mice compared with wild type controls (Figure 3H) that was largely rescued by adenoviral-mediated restoration of UCP2 expression (Online Figure IIID). UCP2 Modulates Endothelial Proliferation Via ΔψDependent Changes in Mitochondrial •O2− Because Δψ modulates mitochondrial •O2− and UCP2 can impact Δψ,18 we investigated mitochondrial •O2− as a function of cell proliferation. Both BAECs (Figure 4A) and murine endothelial cells (Figure 4B) exhibited an inverse relation between mitochondrial •O2− and cell proliferation rate and this response was accentuated in UCP2-null endothelium (Figure 4B). Reconstitution of UCP2 into UCP2-null endothelium (Figure 4C) normalized mitochondrial •O2− and forced overexpression of UCP2 in wild-type cells attenuated mitochondrial •O2− (Figure 4C). Treatment of either wild-type 896 Circulation Research September 13, 2013 Superoxide (RFU) 50% C WT UCP2-/- 80 * 60 40 20 0 50 90% 60 70 80 90 100 * † 2 † 1 0 Ad-LacZ Ad-UCP2 Ad-SOD2 200 10 5 16 12 * 8 * 100 50 0 Ad- Ad- AdLacZ UCP2 SOD2 H Proliferation (%CTL) 15 Superoxide (RFU) Cells / well (x 104) 20 * 150 CTL WT SOD2+/– 20 6 * 4 * 2 * * 150 100 0 50 0 4 0 1 4 7 (days) * * 100 Ad- Ad- Control SOD2 LacZ SOD2 siRNA siRNA * (Sprouts / aorta) 400 16 14 12 10 8 6 4 2 0 SOD2+/- SOD2+/- J Superoxide (RFU) I 10 K Wild-type SOD2+/25 20 15 10 5 * 0 CTL SOD2 siRNA siRNA 0 4 5 6 (days) 7 Perfusion (ischemic/non-ischemic) WT SOD2+/- Cell Migration (cell #) Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 Superoxide (RFU) * 25 0 0 * 3 G F 30 200 † Cell Confluence (%) E 300 4 D WT UCP2-/- Proliferation (%CTL) B Superoxide (RFU) A 1.2 WT SOD2+/- + Ad-UCP2 SOD2+/- + Ad-LacZ 1.0 0.8 * 0.6 † 0.4 0.2 0 Pre 0 1 3 7 14 21 28 (Post-Operative Day) Figure 4. Uncoupling protein 2 (UCP2) dictates endothelial phenotype via mitochondrial •O2−. A, Bovine aortic endothelial cells (BAECs) stained with mitochondrial-targeted hydroethidine at the indicated level of confluence to assess mitochondrial •O2− (bar=50 μm). B, Mitochondrial •O2− in murine lung endothelial cells (MLECs) from the indicated genotype as a function of confluence expressed as relative fluoresence units (RFU). n=4; *P<0.01 for wild-type (WT) vs UCP2−/− by 2-way factorial ANOVA. C, MLEC mitochondrial •O2− in the indicated genotypes transfected with UCP2 (Ad-UCP2), SOD2 (Ad-SOD2), or LacZ. n=3; *P<0.05 vs UCP2−/− Ad-LacZ; †P<0.05 vs WT Ad-LacZ. D, Proliferation of UCP2-null MLECs transfected as in C with data normalized to control (CTL). n=3; *P<0.05 vs Ad-LacZ by Kruskal–Wallis ANOVA. E, Mitochondrial •O2− in WT vs SOD2+/− endothelium. n=4 to 8; *P<0.05 vs WT by Mann–Whitney U test. F, Proliferation of WT and SOD2+/− MLECs as a function of time. n=4; *P<0.05 for trend by 2-way repeated measures ANOVA. Mitochondrial •O2− (G) and proliferation (H) in SOD2+/− MLECs as a function of transfection with control (LacZ), UCP2, or SOD2 adenovirus. n=3; *P<0.05 vs LacZ by Kruskal–Wallis ANOVA. I, left, Migration of BAECs with manipulated SOD2 levels via the indicated adenovirus or small interfering RNA (siRNA). n=6; *P=0.01 vs respective controls by Mann–Whitney U test. I, right, BAEC Mitochondrial •O2− as a function of treatment with CTL or SOD2 siRNA. n=3; *P<0.01 vs control siRNA by Student t test. J, Capillary sprouting in aortic segments from the indicated genotypes. n=6; *P<0.001 vs WT by 2-way repeated measures ANOVA. K, Blood flow recovery in WT and SOD2+/− mice after unilateral hindlimb ischemia with or without hindlimb transfection with UCP2 or LacZ adenovirus. n=5 per group; †P<0.01 vs WT and *P<0.01 vs Ad-LacZ by 2-way repeated measures ANOVA. Ad indicates adenovirus; and SOD2, superoxide dismutase 2. or UCP2-null endothelium with mitochondrial SOD (SOD2) also reduced the mitochondrial •O2− flux (Figure 4C). Finally, attenuation of mitochondrial •O2− in UCP2-null cells with either UCP2 or SOD2 (Figure 4C) enhanced endothelial cell proliferation (Figure 4D). These data indicate UCP2 modulates endothelial cell proliferation via mitochondrial •O2− . If mitochondrial •O2− explains the UCP2-null phenotype, then independent mitochondrial •O2− manipulation should produce qualitatively similar effects. To this end, we found that SOD2+/− endothelium exhibited increased mitochondrial •O2− (Figure 4E) and impaired proliferation (Figure 4F) that were both corrected by either UCP2 or SOD2 transfection (Figure 4G and 4H). Forced overexpression of SOD2 in BAECs enhanced migration in the scratch assay (Figure 4I), whereas SOD2 suppression inhibited migration and increased mitochondrial •O2− (Figure 4I). Capillary sprouting in aortic segments from SOD2+/− animals was impaired compared with wild-type animals (Figure 4J). In the hindlimb ischemia model, we found less blood flow recovery in SOD2+/− mice than in wild-type mice that was rescued via adenoviral overexpression of UCP2 (Figure 4K) in a manner that suppressed mitochondrial •O2− (Figure 4G) and improved proliferation (Figure 4H) in the endothelium. Thus, excess mitochondrial •O2− produces impaired endothelial cell proliferation, migration, and angiogenesis. Shimasaki et al UCP2 and Endothelial Function 897 B 1.5 1.0 0.5 0 Cell Migration (Cell #) C * 250 200 * 10000 5000 0 AdAdLacZ UCP1 D * 150 100 50 0 15000 Ad- AdLacZ UCP1 AdAdLacZ UCP1 (MitoSOX / MitoGreen) JC-1 (Δψ) 2.0 Cell Proliferation (RFU) A 5 4 3 * 2 1 0 AdLacZ AdUCP1 Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 Figure 5. Independent manipulation of Δψ impacts endothelial phenotype. Bovine aortic endothelial cells (BAECs) were transfected with irrelevant (Ad-LacZ) or uncoupling protein 1 adenovirus (Ad-UCP1) and examined for (A) Δψ, (B) proliferation, (C) migration, and (D) mitochondrial •O2− as described in Methods section. n=4 to 6; *P<0.05 vs Ad-LacZ by unpaired t test. UCP2 Does Not Limit Endothelial Antioxidants or Impact Basal NO• Bioactivity Because endothelial antioixdants and •O2− can impact nitric oxide (NO•) bioactivity,34 we investigated both as a function of UCP2. Acute suppression of UCP2 had no material impact on BAEC enzymatic antioxidant levels (Online Figure IVA). In addition, stimulated NO• bioactivity with acetylcholine was not different between wild-type and UCP2-null vessels (Online Figure IVB) and basal NO• bioactivity was actually greater in UCP2-null vessels than wild-type vessels (Online Figure IVC and IVD). Thus, the impact of mitochondrial •O2− on endothelial phenotype is not dependent on reduced antioxidant levels or basal NO• bioactivity. Limiting Δψ Is Sufficient to Explain the Impact of UCP2 To confirm that Δψ changes can account for the impact of UCP2 effects on endothelial phenotype, we force expressed UCP1, a protein not expressed in endothelium, to decrease endothelial Δψ. We found that forced expression of UCP1 decreased Δψ (Figure 5A), increased proliferation (Figure 5B), increased migration (Figure 5C), and limited mitochondrial •O2− (Figure 5D). Thus, independently decreasing Δψ with UCP1 produced qualitatively similar changes in endothelial cell phenotype as Δψ manipulation with UCP2. These data suggest that the impact of UCP2 on endothelial phenotype is because, in part, of its effect on mitochondrial Δψ. UCP2 Regulates Cell Cycle Progression via Mitochondrial •O2− Because NO• bioactivity was not impaired, we examined other mechanisms of impaired endothelial function. Proliferating UCP2-null endothelium exhibited more cells in the G1-phase cells than wild-type endothelium (Figure 6A), suggesting impaired G1-S cell cycle transition. This part of the cell cycle is controlled, in part, by cyclin-dependent kinases, and we found that UCP2-null cells exhibited higher expression of the cyclindependent kinase inhibitors, such as p16ink4a and p21cip/waf, than did wild-type cells (Figure 6B and 6C). Because cell cycle inhibition can lead to senescence, we examined the senescence indicator, β-galactosidase, and found a 4.5-fold increase in senescence in UCP2-null endothelium compared with wild-type cells (Figure 6D and 6E). Transfection of UCP2-null cells with either UCP2 or SOD2 (Figure 6E) attenuated senescence. We observed a similar senescence increase in SOD2+/− cells (Figure 6E) and transfection of either UCP2 or SOD2 also inhibited the development of senescence (Figure 6E). Because p53 is both sensitive to reactive oxygen species (ROS)29 and can control p16ink4a and p21cip/waf expression, we investigated the role of p53 in the UCP2-null phenotype. We found that p53 suppression by small interfering RNA rescued the proliferation defect of UCP2-null endothelium (Figure 6F). Moreover, we observed that proliferating and hypoxic UCP2-null endothelium exhibited p53 phosphorylation at serine 18 (Figure 6G), a residue involved in redox-sensitive p53 activation.29 Thus, UCP2-null endothelium exhibits a dysfunctional phenotype manifest as premature senescence via excess mitochondrial •O2− in a p53-dependent manner. UCP2 Regulates Endothelial Phenotype via Superoxide-Dependent Control of Mitochondrial Fragmentation Because mitochondria undergo dynamic changes in fusion and fission during the G1-S cell cycle transition,35 we examined endothelial mitochondrial morphology as a function of UCP2. Compared with wild-type endothelium, proliferating UCP2null cells exhibited superoxide-dependent mitochondrial fragmentation that was rescued by transfection with either UCP2 or SOD2 (Figure 7A and 7B). Similarly, SOD2 heterozygous cells exhibited greater mitochondrial fragmentation than wildtype cells that was largely rescued by transfection with SOD2 or UCP2 (Figure 7A and 7B). This fragmentation was linked to p53, because p53 RNA interference attenuated fragmentation (Figure 7C), whereas p21cip/waf forced overexpression recapitulated mitochondrial fragmentation (Figure 7D). To gain insight into the mechanism of mitochondrial fragmentation, we examined gene expression important for normal mitochondrial morphology (Figure 7E) and found that genes required for fusion of mitochondria, such as mitofusin 1 and 2 (Mfn1, Mfn2) and optic atrophy 1 (Opa1), were significantly lower than genes required for mitochondrial fission, such as fission 1 (Fis1) and dynamin-related protein 1 (Drp1)—a pattern associated with mitochondrial fragmentation.36 To determine whether mitochondrial fragmentation was sufficient to cause the UCP2-null phenotype, we suppressed mitofusin 1 and 2 expression and found that induction of mitochondrial fragmentation significantly reduced endothelial cell proliferation (Figure 7F). Taken together, these results indicate that endothelial UCP2 is necessary for maintaining mitochondrial morphology, and that disrupting the normal balance between mitochondrial fusion and fission has implications for endothelial function. 898 Circulation Research September 13, 2013 A UCP2-/- 24 hr # Cells G1 B WT G1 65.0% S 19.7% G2 7.1% G1 40.0% S 37.7% G2 12.5% G2 48 hr # Cells G1 74.5% S 6.4% G2 10.3% PI Content G1 49.1% S 20.4% G2 14.5% * 3 3 2 2 1 1 0 WT UCP2-/- 0 Wild type SA-β-gal stained cells (%) E Cell Proliferation Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 (RFU) 10000 UCP2-/- WT UCP2-/- 5000 0 p21 21 Actin 42 WT UCP2-/- * 40 30 20 ‡ ‡ 10 0 Ad- Ad- Ad- Ad- Ad- Ad- AdGFP GFP UCP2 SOD2 GFP UCP2 SOD2 UCP2-/- SOD2+/- G † ‡ Hypoxia Normoxia WT UCP2-/- p-p53 * No Control Treatment siRNA * † † WT UCP2-/- ** (kDa) 16 * WT 15000 WT UCP2-/p16 PI Content D F C p21 mRNA p16 mRNA (kDa) (Ser18) p53 53 Actin 42 Figure 6. Uncoupling protein 2 (UCP2) and endothelial senescence. A, Cell cycle analysis in wild-type (WT) and UCP2-null murine lung endothelial cells (MLECs) at the indicated time based upon propidium iodide (PI) content. B, Expression of WT and UCP2-null mRNA for p16ink4a and p21cip/waf. n=3; *P<0.05 vs WT by Mann–Whitney U test. C, Expression of WT and UCP2null endothelial p16ink4a and p21cip/waf by immunoblotting. D, WT and UCP2-null senescence-associated β-galactosidase (SA-β-gal) via x-gal staining. Bar=750 μm. E, Endothelial SA-β-gal staining as a function of genotype and transfection with either UCP2 or SOD2. n=3 to 6; *P<0.05 vs WT+Ad-GFP, †P<0.05 vs UCP2−/−+AdGFP, and ‡P<0.05 vs SOD2+/−+Ad-GFP by Kruskal–Wallis ANOVA and post hoc comparison. F, Cell proliferation in WT and UCP2−/− MLECs treated with none, control, or p53 small interfering RNA (siRNA). n=4; *P<0.05 vs WT with control siRNA; **P<0.01 vs WT with no treatment; †P<0.01 vs WT with control siRNA; and ‡P<0.01 vs UCP2−/− with control siRNA by 1-way ANOVA with Tukey–Kramer test. G, MLECs of the indicated genotype under normoxic or hypoxic (1% O2) conditions for 16 hours were lysed and probed for the indicated proteins by immunoblot. Ad indicates adenovirus; and SOD2, superoxide dismutase 2. p53 siRNA Discussion The principal finding of this work is that endogenous UCP2 modulates endothelial mitochondrial network morphology that dictates, in part, endothelial cell function. Through UCP2, endothelial Δψ decreases with cell proliferation and, as endothelial growth slows, reduced UCP2 levels produce an increase in Δψ. We found these changes in Δψ moved in parallel with mitochondrial •O2− production that, if left uncontrolled, resulted in p53-dependent mitochondrial network fragmentation that limited endothelial functions important for angiogenesis, including proliferation, migration, and blood flow recovery from tissue ischemia. The central role of Δψ was supported by observations that Δψ manipulation with UCP1 could phenocopy the effect of UCP2. Moreover, the key role of mitochondrial •O2− was consistent with observations that SOD2+/− endothelium, with excess mitochondrial •O2−, demonstrated mitochondrial network fragmentation and impaired endothelial cell function. Collectively, these data indicate that endogenous UCP2 is an important modulator of endothelial cell function, in part, via its impact on mitochondrial network integrity. The precise biological functions of uncoupling proteins remain a matter of debate. UCP1, the predominant uncoupling protein in brown adipose tissue, mediates Δψ proton leak that is critical for adaptive thermogenesis.37 In contrast, UCP238 and UCP339 can mediate proton leak, but are not necessary for adaptive thermogenesis or normal energy metabolism.18,40 These latter 2 uncoupling proteins modulate Δψ to a lesser extent than UCP1,18 even in the presence of requisite coactivators, such as fatty acids and •O2− -derived alkenals.41,42 Multiple models of UCP2 or UCP3 overexpression suggest a protective role against oxidative stress38,43,44 and endothelial dysfunction from diet-induced obesity.45 Considering that endothelial cell proliferation can be associated with excess •O2− and oxidative stress,46 one could argue that our data identify UCP2 as a stress-responsive protein. However, UCP overexpression is prone to artifact from improper membrane insertion,40 casting doubt on the ultimate role of UCPs in limiting •O2−-mediated toxicity. Our data strongly support a role of UCP2 to limit mitochondrial •O2− and deserve particular attention, because we focused on endogenous UCP2 regulation. Moreover, we used loss-of-function models with rescue by UCP2 complementation that are less prone to artifact. Consistent with the idea that UCP2 limits mitochondrial •O2−, we found that independent manipulation of mitochondrial •O2− (via SOD2) copied the UCP2-null phenotype, increasing confidence that endogenous UCP2 importantly regulates mitochondrial ROS. One might expect that mitochondrial •O2− would limit NO• bioactivity in our system to explain the UCP2-null phenotype.34 Rather, we found intact NO• bioactivity in unstressed UCP2-null endothelium, but the cells exhibited premature senescence and upregulation of p21cip/waf and p16ink4a. These 2 gene products have been linked, in part, to activation of the tumor suppressor, p53.47 Our observations that correction of excess mitochondrial •O2− prevented senescence is consistent with data that ROS can upregulate p53 to induce both cell cycle arrest48 and genes that tend to lower cellular ROS levels.49,50 In this context, it is germane to Shimasaki et al UCP2 and Endothelial Function 899 A WT UCP2-/- UCP2-/- + AdUCP2 SOD2+/- * * 3 * * 2 * 1 0 4 3 1 0 WT 4 3 UCP2-/- SOD2+/- E Relative Expression to GAPDH WT + Ad-p21 WT UCP2-/- 2.0 1.5 1.0 * * ** 0.5 ** 0 Mfn1 Mfn2 Opa1 Fis1 Drp1 Ucp2 F 2 1 * 0 siCTL si-p21 WT Ad- AdLacZ p21 CTL siRNA Mfn2 siRNA Cell Number / Field Mitochondrial Length (μm) Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 WT + AdLacZ siCTL si-p53 siCTL si-p53 siCTL si-p53 SOD2+/- UCP2-/- * * 2 WT UCP2-/- Ad- Ad- Ad- Ad- Ad- AdLacZ UCP2 SOD2 LacZ UCP2 SOD2 D Figure 7. Mitochondrial morphology and endothelial phenotype. A, Representative images of murine lung endothelial cell (MLEC) mitochondrial morphology by Mitotracker green staining as a function of the indicated genotype and transfection with the indicated adenovirus (Ad). B, Composite data indicating average mitochondrial length as an index of fragmentation in MLECs from the indicated genotype and transfected with the indicated adenovirus. n=13 to 16; *P<0.01 vs respective wildtype (WT) or LacZ control by Kruskal– Wallis ANOVA. C, Mitochondrial length as an index of fragmentation in the indicated genotypes with and without suppression of p53. n=12 to 16; *P<0.05 vs respective control (CTL). D, MLEC mitochondrial morphology and average length with forced expression of p21cip/waf. Bar=5 μm; n=9 to 13; *P<0.05 vs LacZ by Kruskal– Wallis ANOVA. E, MLEC expression of mitochondrial genes as a function of the indicated genotype. n=3 to 4; *P<0.05, **P<0.01 vs WT by Mann–Whitney U test. F, WT MLEC proliferation with suppression of mitofusin 2 (Mfn2) or both mitofusin 1 (Mfn1) and Mfn2. n=10 each; *P<0.05 vs CTL siRNA by 2-way repeated measures ANOVA. Drp1 indicates dynamin-related protein 1; Fis1, fission 1; opa1, optic atrophy 1; siCTL, siRNA control; and SOD2 indicates superoxide dismutase 2. C 4 Mitochondrial Length (μm) Mitochondrial Length (μm) B SOD2+/- + AdSOD2 CTL siRNA Mfn1+2 siRNA 500 500 400 * 400 * 300 300 0 2 5 Days in Culture note that RNA interference–mediated suppression of prohibitin, an inner mitochondrial protein, also upregulates mitochondrial •O2−, producing premature senescence and impaired endothelial proliferation.51 Studies in other cell types indicate prohibitins are required for proliferation,52 prompting speculation that suppressing mitochondrial •O2− (perhaps to prevent p53 activation) may be a general requirement of cellular proliferation, perhaps via prevention of premature senescence. In this regard, we need to interpret our data with caution as we have not determined whether senescence alone could explain all of our experimental findings. The notion that UCP2 suppression of mitochondrial •O2− may be important for cell proliferation is supported by our observation that UCP2 mRNA is most prominent in very proliferative cells and tissues, such as bone marrow, lymphomas, leukemias, erythroid precursors, T-cells, and CD105+ endothelial cells (Online Figure I). Moreover, UCP2 gene silencing prevented epidermal cell tumor induction in a p53-dependent manner.53 Thus, data from diverse cell types link UCP2 upregulation (and mitochondrial •O2− suppression) to rapid cell 0 2 5 Days in Culture proliferation, suggesting that UCP2 impacts some fundamental component of the proliferative response. Because UCP2 is a target gene for peroxisome proliferator γ coactivator-1α, the principal determinant of mitochondrial biogenesis,54 UCP2 may be needed to expand mitochondrial mass during cell proliferation, perhaps by limiting mitochondrial ROS-mediated damage. Alternatively, UCP2 is known to promote oxygeninsensitive glycolysis (the Warburg effect)55 and glycolysis is required for the high proliferation rate of many cancer cells.32 Thus, UCP2 may be required for glycolysis-dependent growth, a finding consistent with our observations of reduced lactate production in UCP2-null endothelium (Figure 2). We observed excess mitochondrial fragmentation with UCP2-null mice that was linked to mitochondrial •O2−, because it was recapitulated in the SOD2+/− mice and attenuated with maneuvers that reduce mitochondrial •O2−. These findings suggest a novel relation between mitochondrial redox state and the balance between mitochondrial fusion and fission. This relation is consistent with our observations that mitochondrial 900 Circulation Research September 13, 2013 Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 fragmentation in UCP2-null cells involved a p53- and p21cip/wafdependent mechanism, and literature that p53 can be activated by ROS via p53 phosphorylation on serine 20 (mouse Ser18).29 This latter event is known to involve the ataxia-telangiectasia– mutated kinase. Thus, one potential explanation for our data would be ataxia-telangiectasia–mutated kinase activation by mitochondrial •O2−. This contention is supported by observations that ataxia-telangiectasia–mutated and its p53 target (Ser18/20) have important implications for metabolism.56,57 The findings presented here suggest a role for UCP2 in cell proliferation. With the increased metabolic flux that occurs as a result of growth stimulation, it is not surprising that mitochondrial Δψ and •O2− would increase. The fact that forced expression of UCP1 limited Δψ and recapitulated the effect of UCP2 suggests that UCP2 upregulation is an important mechanism for limiting Δψ and the resultant mitochondrial •O2− flux that, if left unchecked, would activate pathways to limit cell proliferation. With regards to the endothelium, this paradigm has important therapeutic implications. For example, our findings suggest that targeting UCP2 may prove to be an important means of limiting tumor angiogenesis. Conversely, promotion of mitochondrial uncoupling could enhance the angiogenic response to ischemia and this strategy could prove helpful in the setting of occlusive vascular diseases. Thus, our data highlight the importance of mitochondrial uncoupling and its dynamic regulation in the endothelial cell proliferative response. Acknowledgments We kindly thank Dr Bradford Lowell for the UCP2-null mice. Sources of Funding This work was supported by National Institutes of Health grants DK089185 (M.P. 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Novelty and Significance What Is Known? • Mitochondria are known to participate in a number of cellular functions beyond that of energy production. • The main driving force for many mitochondrial functions is the electrochemical gradient (Δψ) produced by the mitochondrial electron transport chain. • The regulation of Δψ involves both the electron transport chain and uncoupling proteins that facilitate protein leak across the inner mitochondrial membrane. What New Information Does This Article Contribute? • Mitochondrial Δψ is dynamically regulated in endothelial cells by uncoupling protein 2 (UCP2) in response to external stimuli such signals to promote proliferation. • The decrease in endothelial cell Δψ by UCP2 upregulation during proliferation limits mitochondrial superoxide generation. • In the absence of UCP2, excess mitochondrial superoxide leads to p53-dependent mitochondrial fragmentation that is necessary and sufficient to impair endothelial cell proliferation and angiogenesis. Mitochondria have long been known to be critical for cellular ATP production. More recently, mitochondrial have been implicated in other cellular processes, but their role in the endothelium is largely unknown. Thus, we sought to probe the role of mitochondria in endothelial proliferative responses. We found that endothelial cell proliferation was associated with a reduced Δψ because of upregulation of UCP2, a protein known to regulate Δψ. In the absence of UCP2, proliferating endothelial cells had higher Δψ and excess mitochondrial superoxide that prevented their proliferation, migration, and participation in angiogenesis. Similarly, endothelium lacking mitochondrial superoxide dismutase had excess mitochondrial superoxide and mimicked the UCP2-null phenotype. This excess mitochondrial superoxide lead to p53-dependent mitochondrial fragmentation and endothelial cell senescence. Correction of excess mitochondrial superoxide corrected both the mitochondrial fragmentation and endothelial dysfunction. These data indicate that endothelial UCP2 is important to maintain both normal mitochondrial dynamics and endothelial function. Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 Uncoupling Protein 2 Impacts Endothelial Phenotype via p53-Mediated Control of Mitochondrial Dynamics Yukio Shimasaki, Ning Pan, Louis M. Messina, Chunying Li, Kai Chen, Lijun Liu, Marcus P. Cooper, Joseph A. Vita and John F. Keaney, Jr Circ Res. 2013;113:891-901; originally published online July 2, 2013; doi: 10.1161/CIRCRESAHA.113.301319 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2013 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/113/7/891 Data Supplement (unedited) at: http://circres.ahajournals.org/content/suppl/2013/07/02/CIRCRESAHA.113.301319.DC1 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/ CIRCRES/2013/301319/R1 Supplemental Material Supplemental Experimental Procedures Bioinformatics – The gene expression profiles of human UCP1, 2, and 3 in various tissues were examined using the BioGPS database from microarrays (Genomics Institute of the Novartis Research Foundation; San Diego, CA). Capillary Sprouting Assay in Adipose Tissues – Freshly harvested epididymal fat pads were placed in Krebs-Ringer solution buffered with HEPES (KRH) as described,1 digested for 30 min at 37°C in KRH, pH 7.4, containing 1 mg/ml collagenase type I (Worthington Biochemical; Lakewood, NJ) and 2.5% BSA Fraction V (Sigma-Aldrich). Digested tissue was then filtered through a 100 µm cell strainer (Fisher Scientific BD Falcon; Pittsburgh, PA) and the captured stromal vascular fraction was cut into small (1 mm3) pieces that were embedded in liquid collagen gel in 48-well plates (BD Biosciences), incubated at 37°C for 1h to polymerize the collagen, and the solid gel covered with MLEC medium diluted 1:2 with DMEM. Each adipose tissue was examined daily and capillary sprouts counted along the sample perimeter under 100 and 200 x magnification. Vascular sprouts were distinguished from fibroblasts via morphology as described1,2 and CD31 staining. Vascular Ring Relaxation – To study NO• bioactivity, mice aged 8-12 weeks were euthanized and thoracic aortae were isolated, cleaned of connective tissue, and cut into 2 mm segments. The vessel segments were mounted on stainless-steel holders in organ baths (Danish Myo Technology, Denmark) containing physiological saline solution (PSS; 119 mM NaCl, 4.69 mM KCl, 1.17 mM MgSO4, 1.18 mM KH2PO4, 2.5 mM CaCl2, 25 mM NaHCO3, 0.03 mM EDTA, 5.5 1 CIRCRES/2013/301319/R1 mM Glucose) and aerated with 95% O2 / 5% CO2 for isometric force recording. Preparations were allowed to equilibrate for 30 min under constant passive force of (~1 mN) and synchronized with KCl and phenylephrine. Basal NO• bioactivity was estimated as the difference in contraction to phenylephrine with or without 300 µM L-NAME to inhibit endothelial nitric oxide synthase. Evoked NO• bioactivity was estimated as the relaxation response to acetylcholine (ACh) in phenylephrine-contracted segments. Data were analyzed with PowerLab software (AD Instruments). 2 CIRCRES/2013/301319/R1 Online Figure I Human UCP1 Human UCP2 Human UCP3 10000 0 Median 10 3xM 15 Median 10xM 3xM 30xM 5000 100xM 0 Median 50 3xM 100 Online Figure I. Uncoupling protein mRNA expression as a function of cell type. Gene expression profiles of human UCP1, 2, and 3 as a function of cell type examined with the BioGPS online database. Expression levels are indicated as a multiple of the median expression level. 3 CIRCRES/2013/301319/R1 A 0 WT 5 15 30 60 180 0 C UCP2-/5 15 30 60 180 min P-c-Jun P-p38 B CTL 0 5’ 15’ 24h 0 SB203580 SP600125 5’ 15’ 24h 0 5’ 15’ 24h P-c-Jun * 150 Proliferation (% DMSO) Actin † † 100 50 P-p38 UCP2 0 DM SO DM SO SP 5 0. SP 5 ) 2. (µM VEGF Online Figure II. c-Jun, N-terminal kinase and UCP2 upregulation. (A) Wild-type (WT) and UCP2-null endothelium was exposed to 10% serum for the indicated number of minutes, lysed, and immunoblots performed for actin or phosphorylated (activated) forms of c-Jun (Ser63) or p38 MAP kinase (Thr180/Tyr182). (B) BAECs in the presence or absence of vehicle alone (CTL), the JNK inhibitor SP600125 (0.5 µM), or the p38 MAP kinase inhibitor SB203580 (10 µM) were exposed to 10% serum for the indicated time. Cells were then lysed and examined for UCP2 or activation of JNK or p38 MAP kinase as in (A). (C) BAECs were exposed to vehicle (DMSO) or VEGF (25 ng/mL) with or without SP600125 as indicated for 24h followed by assessment of proliferation by [3H]-thymidine incorporation. N=4; *P<0.05 vs DMSO; †P<0.05 vs. VEGF/DMSO by one-way ANOVA with Tukey post hoc test. 4 CIRCRES/2013/301319/R1 Supplemental Figure 3 Wild-type UCP2-/- 2.0 Perfusion (ischemic/non-ischemic) 1.2 C UCP2-/- adipose tissue 1.0 0.5 Ad-LacZ † WT + Ad-LacZ UCP2-/- + Ad-UCP2 UCP2-/- + Ad-LacZ 40 Ad-UCP2 1.5 * * 30 20 10 0 0 UCP2 D B (Sprouts / fat) (UCPs mRNA / GAPDH mRNA) A UCP3 0 2 4 (days) 6 UCP2-/- + Ad-UCP2 UCP2-/- + Ad-LacZ 1.0 * 0.8 0.6 0.4 0.2 0 Pre 0 1 3 7 14 21 (Post-Operative Day) 28 Online Figure III. Uncoupling protein manipulation and angiogenesis. (A) UCP2 and 3 mRNA levels in mouse lung endothelial cells. Dagger = not detectable. (B) Representative phase contrast images of capillary sprouts from UCP2-/- adipose tissue explants taken with a 40 x objective on the day 4 culture. Scale bar, 500 µm. (C) Ex vivo capillary sprouting in fat pad explants from the indicated genotype embedded in collagen gel 5d after in vivo transfection with UCP2 or LacZ adenovirus. N=5/group; *P < 0.05 vs. UCP2-/- with AdLacZ by two-way ANOVA.. (D) Blood flow recovery in UCP2-/- mice with unilateral hindlimb ischemia and treatment with control (Ad-LacZ) or UCP2 adenovirus. N=5; *P < 0.05 vs. LacZ by two-way ANOVA. 5 CIRCRES/2013/301319/R1 B 2 (kDa) P L L CT UC T i i C s s 33 UCP2 24 SOD2 100" 22 GPx-1 65 Catalase 42 Actin 16 COX IV %"Ini=al"Tension" A WT# UCP2(/(# 50" 25" 0" '9" '8" '7" '6.5" '6" '5" Acetylcholine"(Log"M)" C D 75" 100" WT" 50" UCP2&/&" *" 25" 0" &9" &8" &7" &6.5" &6" &5" Phenylephrine"(Log"M)" %"Constric5on" %"ConstricCon"" 75" (+) L-NAME 75" 75" 50" 25" 0" WT" 50" -/UCP2&/&" 25" '9" '8" '7" '6.5" '6" '5" Phenylephrine"(Log"M)" 0" &9" &8" &7" &6.5" &6" Online Figure IV. UCP2, antioxidant enzymes, and NO• bioactivity. (A) BAECs were treated with CTL or UCP2 siRNA as indicated for 24h followed by immunoblotting for the indicated antioxidant enzymes. (B) Acetylcholine-induced relaxation of aortic segments of the indicated genotype contracted with phenylephrine. N=6; P = NS. (C) Phenylephrine-induced contraction of aortic segments by genotype. N=6; P < 0.05 vs. WT by two-way repeated measures ANOVA. (D) Phenylephrine-induced contraction of aortic rings by genotype in the presence of 300 µM L-NAME (P = NS). 6 &5" CIRCRES/2013/301319/R1 1. Gealekman O, Burkart A, Chouinard M, Nicoloro SM, Straubhaar J, Corvera S. Enhanced angiogenesis in obesity and in response to PPARgamma activators through adipocyte VEGF and ANGPTL4 production. Am J Physiol Endocrinol Metab. 2008; 295:E1056–64. 2. Nicosia RF, Ottinetti A. Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro. Lab Invest. 1990; 63:115–122. 7