Received: 10 June 2021 | Revised: 12 June 2022 | Accepted: 4 July 2022 DOI: 10.1111/acel.13674 RESEARCH ARTICLE Mitochondrial uncoupling protein-­2 reprograms metabolism to induce oxidative stress and myofibroblast senescence in age-­associated lung fibrosis Sunad Rangarajan1 | Morgan L. Locy2 | Diptiman Chanda2 | Ashish Kurundkar2 Deepali Kurundkar2 | Jennifer L. Larson-­Casey2 | Pilar Londono1 | Rushita A. Bagchi3 | Brian Deskin4 | Hanan Elajaili5 | Eva S. Nozik5 | 2 2 6 Jessy S. Deshane | Jaroslaw W. Zmijewski | Oliver Eickelberg | Victor J. Thannickal7 | 1 Division of Pulmonary Sciences and Critical Care, Department of Medicine, University of Colorado, Aurora, Colorado, USA 2 Division of Pulmonary and Critical Care, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA 3 Division of Cardiology, Department of Medicine, University of Colorado, Aurora, Colorado, USA 4 Division of Pulmonary and Critical Care, Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA 5 Cardiovascular Pulmonary Research Laboratories and Pediatric Critical Care Medicine, Department of Pediatrics, University of Colorado, Aurora, Colorado, USA 6 Division of Pulmonary, Allergy and Critical Care, Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA 7 John W. Deming Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA Correspondence Sunad Rangarajan, Pulmonary Sciences and Critical Care Medicine, University of Colorado Anschutz Medical Campus, RC2, Room 9011, Mail Stop C272, 12700 E 19th Ave, Aurora, CO 80045, USA. Email: sunad.rangarajan@cuanschutz.edu Victor J. Thannickal, John W. Deming Department of Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, #8512 New Orleans, LA 70112, USA. Email: vthannickal@tulane.edu Funding information National Heart, Lung, and Blood Institute, Grant/Award Number: K08 HL135399, P01 HL114470 and R01 HL139617; National Institute on Aging, Grant/ Award Number: R01 AG046210; U.S. Department of Veterans Affairs, Grant/ Award Number: I01BX003056 Abstract Mitochondrial dysfunction has been associated with age-­related diseases, including idiopathic pulmonary fibrosis (IPF). We provide evidence that implicates chronic elevation of the mitochondrial anion carrier protein, uncoupling protein-­2 (UCP2), in increased generation of reactive oxygen species, altered redox state and cellular bioenergetics, impaired fatty acid oxidation, and induction of myofibroblast senescence. This pro-­ oxidant senescence reprogramming occurs in concert with conventional actions of UCP2 as an uncoupler of oxidative phosphorylation with dissipation of the mitochondrial membrane potential. UCP2 is highly expressed in human IPF lung myofibroblasts and in aged fibroblasts. In an aging murine model of lung fibrosis, the in vivo silencing of UCP2 induces fibrosis regression. These studies indicate a pro-­fibrotic function of UCP2 in chronic lung disease and support its therapeutic targeting in age-­related diseases associated with impaired tissue regeneration and organ fibrosis. KEYWORDS cellular senescence, fibroblast, fibrosis, myofibroblast, oxidative stress, UCP2, uncoupling protein-­2 Oliver Eickelberg and Victor J. Thannickal contributed equally. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2022 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd. Aging Cell. 2022;21:e13674. https://doi.org/10.1111/acel.13674  wileyonlinelibrary.com/journal/acel | 1 of 18 2 of 18 | 1 I NTRO D U C TI O N | Fibrosis occurs in multiple pathological conditions and is typically associated with an inadequate or failed regenerative response to RANGARAJAN et al. 2 | R E S U LT S 2.1 | UCP2 is highly expressed in IPF lungs and (myo)fibroblasts tissue injury (Duffield et al., 2013; Horowitz & Thannickal, 2019; Thannickal et al., 2004). Aging is an important contributor of In a transcriptomic analysis of mesenchymal stromal cells (MSCs) failed tissue regeneration. The cellular and molecular mecha- isolated from bronchoalveolar lavage of human subjects with IPF, nisms that account for this loss of regenerative capacity are not we observed increased expression of UCP2 in individuals with pro- well understood. While a number of so-­c alled aging “hallmarks” gressive vs. stable IPF and confirmed this higher expression of UCP2 such as stem-­cell exhaustion, cellular senescence, deregulated in these cells grown in vitro (Figure 1a). For this analysis, progres- nutrient-­s ensing, and mitochondrial dysfunction have been impli- sive disease was defined as a loss in forced vital capacity (FVC) of cated (Lopez-­O tin et al., 2013; Rangarajan et al., 2017), how these greater than 10% and stable disease as FVC loss ≤5% in the pre- may be integrated to explain key cellular, tissue and organ-­level ceding 6 months, as determined by pulmonary function testing. We phenotypes are largely unknown. Idiopathic pulmonary fibrosis validated this finding in whole lung tissue and fibroblasts isolated (IPF) is a chronic, progressive fibrosing disease of the lungs with from explanted lungs of individuals undergoing lung transplantation; increasing incidence and prevalence with age (Raghu et al., 2006). IPF subjects demonstrated significantly higher UCP2 gene expres- Susceptibility to pulmonary fibrosis with aging has been linked sion compared to non-­IPF controls (Figure 1b,c). Furthermore, a pub- to mitochondrial dysfunction (Mora et al., 2017), oxidative licly available dataset of human lung tissues from control subjects stress (Hecker et al., 2014), and metabolic derangements (Bueno and both early and advanced IPF revealed the highest expression of et al., 2020; Romero et al., 2016), but cause–­effect relationships UCP2 in advanced IPF (GSE24206) (Meltzer et al., 2011) (Figure S1a). remain unclear. To determine the cellular localization of UCP2, we performed Mitochondrial uncoupling protein-­ 2 (UCP2) belongs to the immunohistochemistry on tissue sections of IPF subjects and found SLC25 family of mitochondrial anion carrier proteins (Brand & higher expression in regions of active fibrosis in comparison to nor- Esteves, 2005). Although UCP2 has been demonstrated to reg- mal lungs (Figure 1d,e). In fibroblastic foci that are a hallmark of IPF ulate cellular energy homeostasis along with glucose and fatty histopathology, the vast majority of fibroblasts within these foci ex- acid (FA) metabolism (Pecqueur et al., 2008; Rousset et al., 2004; press UCP2 and the myofibroblast marker, α-­smooth muscle actin (α-­ Vozza et al., 2014), its exact physiological role remains unknown. SMA) (Figure 1f–­h). Transforming growth factor-­β1 (TGF-­β1) serves While many studies implicate a role for UCP2 as a stress-­inducible as a central mediator of myofibroblast differentiation and fibrogene- protein that mediates antioxidant effects (Brand & Esteves, 2005; sis in vivo (Thannickal et al., 2003). Exogenous stimulation of human Mailloux & Harper, 2011), its participation in aging and patholog- lung fibroblasts with TGF-­β1 induced a greater than 4-­fold induction ical fibrosis has not been well studied. In this study, we explored of UCP2 mRNA at 24 h following treatment (Figure 1i). Higher ex- the role of UCP2 in cellular bioenergetics, mitochondrial reactive pression of UCP2 mRNA was also observed in human lung fibro- oxygen species (ROS) production, and fatty acid oxidation (FAO) blasts subjected to undergo replicative senescence with a population in lung fibroblasts. We uncovered an unanticipated effect of the doubling length >40 (Figure 1j.). Additionally, treatment of non-­IPF chronic elevation of this stress-­inducible protein in myofibroblast lung fibroblasts with bleomycin for 72 h induced a robust expres- redox deregulation, differentiation, senescence, and apoptosis re- sion of UCP2 (Figure 1k). An increase in UCP2 gene expression was sistance that contribute to persistent, non-­resolving lung fibrosis also observed in senescent fibroblasts, either replication-­induced or associated with aging. following bleomycin treatment, when compared to non-­senescent F I G U R E 1 UCP2 is highly expressed in IPF lungs and (myo)fibroblasts. (a) Mesenchymal stromal cells (MSCs) were isolated and cultured from broncho-­alveolar lavage (BAL) fluid of patients with stable (FVC loss ≤5% in the preceding 6 months) or progressive (FVC loss >10% in the preceding 6 months) IPF. qPCR was performed to assess the expression of UCP2. Graph represents mean ± SEM (n = 7–­8); **p < 0.01. (b) Whole lung tissue obtained from explants of rejected donor lungs (non-­IPF) and IPF lungs were assessed for UCP2 expression by qPCR. Graph represents mean ± SEM (n = 4); *p < 0.05. (c) Fibroblasts were isolated from explants of rejected donor lungs (non-­IPF) and IPF lungs and cultured ex vivo. qPCR was performed to assess the expression of UCP2. Graph represents mean ± SEM (n = 6–­8); *p < 0.05. (d–­h) Immunohistochemical staining for UCP2 was performed on non-­IPF and IPF lung sections. Representative images (20× magnification) are shown in (d,e), respectively. A fibroblastic focus was identified in the IPF lung in (e) (dotted rectangle); (f–­h) high magnification (40×) view of the fibroblastic focus stained with UCP2, α smooth muscle actin (α-­SMA) and secondary IgG antibody control, respectively. Black arrowhead in (f) shows fibroblasts in the fibroblastic focus. (i) Serum-­starved human diploid lung (IMR-­90) fibroblasts were treated with transforming growth factor-­β1 (TGF-­β1) 2 ng/ml for 24 h. UCP2 expression was assessed by real-­time PCR. Graph represents mean ± SEM (n = 3); **p < 0.01. (j) IMR-­90 fibroblasts at low population doubling length (low PDL; PDL < 20), and those undergoing replicative senescence at high PDL (PDL > 40) were assessed for UCP2 expression by real-­time PCR. Graph represents mean ± SEM (n = 3); **p < 0.01. (k) Non-­IPF human lung fibroblasts were treated with bleomycin 25 μg/ml for 72 h. UCP2 expression was assessed by real-­time PCR. Graph represents mean ± SEM (n = 3); **p < 0.01 | RANGARAJAN et al. (a) (b) (c) Non-IPF IPF (d) (e) (g) (f) UCP2 (i) 3 of 18 (h) α-SMA (j) IgG Control (k) fibroblasts in a publicly available dataset (GSE13330) (Pazolli expressing organs (https://www.gtexp​ortal.org/home/gene/UCP2; et al., 2009) (Figure S1b). Interestingly, although UCP2 is ubiquitously Figure S1c). In addition, interrogation of a publicly available single-­ expressed in different cells/tissues, the lung is among the highest cell transcriptomics dataset that characterizes fibroblasts in various 4 of 18 | RANGARAJAN et al. disease conditions (fibroXplorer) revealed that UCP2 is preferentially mRNA (Figure 2h). Thus, UCP2 appears to function as an uncoupler of expressed in a subset of fibroblasts with increased frequency in IPF oxidative phosphorylation in IPF lung (myo)fibroblasts, and its silenc- (Figure S1d–­h). Together, these data indicate that UCP2 is highly ex- ing increases ATP synthetic capacity. pressed in the human fibrotic lung disease, IPF, in senescent (myo) fibroblasts and is inducible by the pro-­fibrotic mediator, TGF-­β1. 2.2 | UCP2 uncouples oxidative phosphorylation and decreases ATP synthesis 2.3 | Constitutive high-­level expression of UCP2 impairs fatty acid oxidation and alters cellular redox state Based on our findings that constitutive UCP2 mediates oxidative Despite its name, there is a lack of clarity and debate regarding how phosphorylation uncoupling and decreases ATP production, we ex- UCP2 functions as an “uncoupler” of mitochondrial electron transport plored potential alterations in cellular metabolism of IPF when com- from ATP synthesis. To explore this in IPF and aging, we first deter- pared to non-­IPF control lung fibroblasts with targeted metabolomics mined if high levels of UCP2 correlate with low levels of ATP in iso- analyses. We confirmed lower levels of ATP in IPF (myo)fibroblasts by lated cells and in tissues. ATP content in lung tissues was found to be this mass spectrometric approach in association with a marked shift markedly reduced in IPF compared with non-­IPF controls (Figure 2a) in concentrations of free fatty acids (FAs); additionally, alteration in and was inversely correlated with UCP2 mRNA levels (Figure 2b). cellular redox state was indicated by low levels of the reduced form Furthermore, (myo)fibroblasts isolated from IPF lungs demonstrated of the thiol-­containing tripeptide, glutathione (GSH) (Figure 3a–­c). significantly lower ATP content than non-­IPF lung fibroblasts when To determine if these metabolic perturbations in IPF lung fibroblasts measured on a per cell basis (Figure 2c). To determine whether UCP2 were related to high basal levels of UCP2, we silenced UCP2 for 24 h contributed to the decreased levels of ATP in lung fibroblasts, we by siRNA before subjecting them to the same targeted metabolomics designed an siRNA sequence that was effective in knocking down analysis. UCP2-­silenced IPF lung fibroblasts showed reversal of the UCP2 in human lung fibroblasts (Figure S2a–­ c); orthologous se- effects on fatty acid utilization/oxidation (FAO) and redox state, as quences efficiently knocked down UCP2 mRNA in mouse lung fibro- indicated by lower levels of several free FAs and higher levels of re- blasts and rat lung epithelial cells (Figure S2d,e); greater efficiency duced glutathione (Figure 3d,e; Figure S3). The suppressive effects of siRNA-­mediated knockdown was observed in IPF fibroblasts (with of UCP2 on FAO were confirmed by findings of lower free FAs in the higher baseline mRNA expression) than in non-­IPF fibroblasts (with supernatant of UCP2-­silenced cells by mass spectrometry (data not lower baseline mRNA expression) (Figure S2a). As a result of an un- shown), as well as the intracellular accumulation of neutral lipids by coupling effect, UCP2 would be expected to lower the mitochondrial LipidTox staining (Figure 3f,g). Taken together, these data indicate membrane potential (δψm), while UCP2 silencing should, at least that deficient free-FA utilization/oxidation and heightened oxidative partially, reverse this effect. Indeed, IPF lung fibroblasts subjected stress in IPF lung fibroblasts are mediated by UCP2. to UCP2 silencing demonstrated higher δψm, as evidenced by more intense formation of JC-­1 aggregates (Figure 2d,e). Next, we assessed whether UCP2 silencing reverses the energy deficit in IPF lung fibroblasts. In IPF fibroblasts that harbor higher levels of UCP2 and lower ATP content, UCP2 silencing led to marked recovery in ATP 2.4 | Chronic elevation of UCP2 induces increased production of reactive oxygen species in IPF lung fibroblasts synthesis; this effect was not observed in non-­IPF fibroblasts with lower baseline levels of UCP2 (Figure 2f,g). However, in both groups To further explore the effects of UCP2 on cellular bioenerget- of fibroblasts, cellular ATP content inversely correlated with UCP2 ics and oxidative stress in IPF fibroblasts, we measured oxygen F I G U R E 2 UCP2 uncouples oxidative phosphorylation and decreases ATP synthesis. (a) Whole lung homogenates from non-­IPF and IPF lungs were assessed for ATP content. Graph represents mean ± SEM (n = 4); **p < 0.01. (b) Graphical representation of the correlation between ATP content and UCP2 mRNA expression in lung homogenates. (c) Non-­IPF and IPF lung fibroblasts grown ex vivo were assessed for ATP content. Graph represents mean ± SEM (n = 3); **p < 0.01. (d) IMR-­90 fibroblasts were treated with non-­t argeting (NT) or UCP2-­ targeting siRNA for 72 h and incubated with JC-­1 dye (2.5 μg/ml) for 30 min, with/without prior treatment with carbonyl cyanide-­4-­ (trifluoromethoxy) phenylhydrazone (FCCP) (5 μM, for 30 min prior to JC-­1) for negative control. Orange/green fluorescence (indicative of JC-­1 aggregates/monomers) was captured by fluorescence microscopy. Scale bars = 50 μm. (e) IPF fibroblasts subjected to siRNA-­mediated knockdown of UCP2 for 72 h were incubated with JC-­1 dye (10 μg/ml) for 10 min, and orange/green fluorescence was analyzed by flow cytometry. The representative graphs depict the percentage of fibroblasts over threshold intensity of orange fluorescence on Y axis, with NT siRNA, 85.44 ± 0.8% vs. UCP2 siRNA, 92.12 ± 0.28%; mean ± SEM; p < 0.01; n = 5 replicates per group; 20,000 events recorded for each replicate. (f–­h) Lung fibroblasts isolated and cultured from explants of 3 non-­IPF and 3 IPF subjects were subjected to siRNA-­mediated knockdown of UCP2 for 72 h. Real-­time PCR was performed to assess UCP2 mRNA expression (f); graph represents mRNA expression relative to non-­IPF cells treated with NT siRNA; mean ± SEM (n = 3); **p < 0.01, *p < 0.05. In parallel, ATP content was assessed in these fibroblasts (g); graph represents mean ± SEM (n = 3); **p < 0.01, *p < 0.05. (h) Graphical representation of correlation between ATP content and UCP2 mRNA expression in these fibroblasts | RANGARAJAN et al. (a) (b) (c) ** ** (d) 5 of 18 NT siRNA UCP2 siRNA (e) NT siRNA JC-1 Aggregates No FCCP UCP2 siRNA + FCCP JC-1 Monomers (f) (g) ** * ** * (h) consumption rate (OCR) and extracellular acidification rate (ECAR) of UCP2 resulted in higher ATP-­linked OCR and lower proton leak using an XFe96 extracellular flux analyzer (Seahorse Biosciences, with an increase (either trend or statistically significant) in total North Billerica, MA). Consistent with our findings on δψm, silencing (basal) OCR (Figure 4a–­c; Figure S4a,b); coincidentally, maximal and 6 of 18 | reserve capacities were increased under the same conditions. To determine if these cellular bioenergetic shifts were linked to FAO, RANGARAJAN et al. 2.5 | UCP2 regulates fibroblast senescence and myofibroblast differentiation we first examined whether inhibiting mitochondrial FA uptake with etomoxir (an inhibitor of CPT1a that transports FAs across the mi- Oxidative stress has been linked to pulmonary fibrosis (Otoupalova tochondrial membrane, the rate-­limiting step of FAO) influenced et al., 2020) and to cellular senescence (Hecker et al., 2014). In the effects of UCP2 silencing. Etomoxir decreased basal OCR of IPF fibroblasts subjected to UCP2 silencing, we observed an in- UCP2-­silenced cells to a greater extent than non-­t argeting siRNA crease in cell proliferation with a concomitant induction of cyclin cells, indicating increased metabolic flux through the FAO pathway D1 and phosphorylated Rb, while the expression of myofibro- in UCP2-­deficient cells (Figure 4d; Figure S4c). A similar pattern of blast differentiation markers, α-­ smooth muscle actin (α-­S MA) increased OCR linked to FAO was observed with palmitate loading and collagen 1a1 (COL1a1) was reduced (Figure 5a–­f ). Enhanced of UCP2-­silenced cells (Figure 4e; Figure S4d). To confirm these cell proliferation was confirmed using the Ki-­67 staining in the findings, we employed a genetic strategy to inhibit FAO by silenc- UCP2-­silenced cells (Figure 5g). Furthermore, the expression of ing CPT1a. We observed that CPT1a silencing abrogated the effects senescence-­associated β-­g alactosidase (SA-­β -­g al) was reduced in of elevated reserve capacity seen with UCP2 silencing (Figure 4f; UCP2-­silenced IPF myofibroblasts (Figure 5h), in association with Figure S4e). These studies support inefficient cellular bioenerget- decreased mRNA expression of the senescence-­associated secre- ics, particularly decreased reserve capacity via FAO, when UCP2 tory phenotype (SASP) proteins, interleukin 6 (IL-­6) and interleukin is chronically elevated. In contrast to effects on OCR, basal levels 1β (IL-­1β) (Figure 5i,j). The expression of SA-­β -­g al was also reduced of glycolytic ECAR were reduced with UCP2 silencing (Figure 4g; in UCP2-­silenced senescent IMR-­9 0 fibroblasts (Figure S5a). The Figure S4f,g), supporting the concept of lower demand on glycolysis effects of UCP2 silencing on cellular reprogramming were pri- when FAO-­dependent ATP generation is restored. marily explained by effects on senescent myofibroblasts which Based on the observation that IPF fibroblasts generate high lev- express higher basal levels of α-­S MA and COL1a1 (Figure S5b); a els of oxidized glutathione, an effect reversed by UCP2 silencing, similar effect on the downregulation of these pro-­fibrotic mark- we conducted studies in which the production of ROS was directly ers was also observed with treatment of IPF myofibroblasts with measured. We assayed mitochondrial superoxide production using a pharmacological inhibitor of UCP2, genipin (Zhang et al., 2006) MitoSOX™ staining and flow cytometric analysis; basal levels of (Figure S5c,d). Senescent myofibroblasts have been well character- superoxide production by mitochondria were found to be reduced ized to acquire apoptosis-­resistant properties (Rehan et al., 2021; by UCP2 silencing without a change in mitochondrial number, mito- Zhou & Lagares, 2021). We observed that silencing of UCP2 low- chondrial DNA content, or the expression of mitochondrial electron ers the apoptosis threshold of these myofibroblasts when stimu- transport chain proteins (Figure 4h,i; Figure S4h,i); the decrease in lated with antimycin A which activates the intrinsic mitochondrial superoxide levels was confirmed with electron paramagnetic reso- pathway of apoptosis (Figure 5k,l). Together, these data support a nance studies as measured by production of its reaction product, ni- critical role for UCP2 in regulating the myofibroblastic, senescent, troxide, at early time points after siRNA treatment (Figure 4j). Since and apoptosis-­resistant phenotype of IPF fibroblasts. superoxide anions are spontaneously or enzymatically reduced to hydrogen peroxide (H2O2), we also measured H2O2 production by isolated mitochondria and found a significant reduction in the rate of mitochondrial H2O2 release with UCP2 knockdown (Figure 4k). 2.6 | Therapeutic targeting of UCP2 promotes resolution of experimental lung fibrosis Thus, the higher basal expression of UCP2 in IPF fibroblasts is associated with elevated levels of ROS, thereby contributing to oxidative Decreased regenerative capacity and impaired fibrosis resolution stress and altered cellular redox state. are phenotypic characteristics of aged mice (Caporarello et al., 2020; F I G U R E 3 Constitutive high-­level expression of UCP2 impairs fatty acid oxidation and alters cellular redox state. (a–­c) Non-­IPF and IPF lung fibroblasts (derived from three explants each) were cultured ex vivo and lysates were subjected to metabolomics analyses. (a) Partial least squares discriminant analysis (PLS-­DA) shows significant separation between the two groups of fibroblasts, (b) variable importance in projection (VIP) scores showing the top 25 metabolites that contribute to the PLS-­DA model, and (c) heatmap of the top 50 metabolites that are significantly different between the two groups; metabolites with lower concentrations are in blue and those with higher concentrations are in red. (d,e) IPF fibroblasts were subjected to siRNA-­mediated knockdown of UCP2 for 24 h. Lysates were subjected to metabolomics analyses. (d) PLS-­DA shows significant separation between the fibroblasts treated with non-­t argeting siRNA (siNT) and UCP2-­t argeting siRNA (siUCP2); (e) VIP scores showing the top 25 metabolites that contribute to the PLS-­DA model. (f) IPF fibroblasts were subjected to siRNA-­mediated knockdown of UCP2 for 72 h, stained with LipidTOX™ Red Neutral Lipid Stain and immunofluorescence imaging performed (representative images shown). Scale bars = 50 μm. (g) Quantification of LipidTOX™ fluorescence [of the fibroblasts in (f)] was performed by randomly selecting 25 individual fibroblasts each in NT siRNA and UCP2 siRNA groups and assessing their corrected total cellular fluorescence, CTCF [CTCF = Integrated density−(Area of selected cell × Mean fluorescence of background readings)], depicted graphically; boxes represent median and extend from 25th to 75th percentiles, and whiskers represent minimum to maximum values in arbitrary units (AU), n = 25, ****p < 0.0001. NT, non-­t argeting | RANGARAJAN et al. (a) Non-IPF (c) IPF (b) (d) #1 #2 (f) #3 #1 #2 #3 LipidTOX NT s iRNA UCP2 siRNA (e) (g) **** 7 of 18 | RANGARAJAN et al. (a) (b) (c) * **** ** (d) (e) **** *** (f) (g) **** **** *** (i) (h) UCP2 siRNA Cell number NT siRNA MitoSOX Red 8 of 18 MitoSOX MFI MitoTracker Green (j) (k) * Mitochondrial H2O2 *** | RANGARAJAN et al. 9 of 18 F I G U R E 4 Chronic elevation of UCP2 induces increased production of reactive oxygen species in IPF lung fibroblasts. (a–­c) IPF fibroblasts were subjected to siRNA-­mediated silencing of UCP2 for a total of 72 h. The fibroblasts were seeded 15,000 cells per well prior to measurement of oxygen consumption rate (OCR) in an XFe96 analyzer (Seahorse Bioscience); computed results for ATP-­linked OCR (a), Proton Leak OCR (b), and their ratio depicting the coupling efficiency (c) depicted graphically. Error bars represent mean ± SEM (n = 8); *p < 0.05, **p < 0.01, ****p < 0.0001. (d) IPF fibroblasts were subjected to siRNA-­mediated silencing of UCP2 for a total of 72 h, seeded at 15,000 cells per well, incubated with a substrate-­restricted medium and treated with etomoxir 4 µM or vehicle. Basal OCR due to intrinsic fatty acid oxidation (FAO) was calculated based on the difference between the OCR just prior to treatment with etomoxir and the OCR 30 min after treatment with etomoxir (depicted graphically). Error bars represent mean ± SEM (n = 10); ****p < 0.0001. (e) IPF fibroblasts were subjected to siRNA-­mediated silencing of UCP2 for a total of 72 h, seeded at 15,000 cells per well, incubated with a substrate-­ restricted medium and treated with bovine serum albumin (BSA) alone or with BSA-­Palmitate conjugate as per the kit manufacturer's instructions. Basal OCR due to extrinsic FAO was calculated by the difference between the basal OCR of Palmitate-­treated cells and the cells treated with BSA alone, and subsequently subtracting the OCR due to excess proton leak in the Palmitate-­treated cells; thus, calculated basal OCR due to extrinsic FAO is depicted graphically. Error bars represent mean ± SEM (n = 4); ***p < 0.001. (f) IPF fibroblasts were subjected to siRNA-­mediated silencing of UCP2, or CPT1a, or both, for a total of 72 h. The fibroblasts were seeded 15,000 cells per well and incubated in a substrate-­restricted medium prior to measurement of OCR; computed results for reserve capacity depicted graphically. Error bars represent mean ± SEM (n = 10); ****p < 0.0001. (g) IPF fibroblasts with similar experimental conditions as in (a–­ c) were assessed for extracellular acidification rate (ECAR) in an XFe96 analyzer; ECAR denoting basal glycolysis depicted graphically. Error bars represent mean ± SEM (n = 8); ***p < 0.001. (h) IPF fibroblasts subjected to siRNA-­mediated knockdown of UCP2 for 72 h were incubated with MitoSOX™ Red (5 µM) and MitoTracker™ Green (100 nM) dyes for 15 min, and red–­green fluorescence was analyzed by flow cytometry. The representative graphs depict the percentage of fibroblasts over threshold intensity of red fluorescence on Y axis, with NT siRNA, 19.03 ± 1.45% vs. UCP2 siRNA, 8.90 ± 0.38%; mean ± SEM; p < 0.0001; n = 6 replicates per group; 20,000 events recorded for each replicate. No significant difference noted in green fluorescence between groups. (i) IPF fibroblasts were subjected to siRNA-­mediated knockdown of UCP2 for 72 h. The fibroblasts were incubated with MitoSOX™ Red (5 µM) alone for 15 min, and red fluorescence was analyzed by flow cytometry, with change in intensity depicted graphically and graph representative of 5 replicates. (j) IPF fibroblasts were subjected to siRNA-­mediated knockdown of UCP2 for 24 h. Electron paramagnetic resonance (EPR) spectroscopy was performed with cyclic hydroxylamine spin probes to assess levels of nitroxide formation, reflecting levels of free radicals, chiefly superoxide in the cells (depicted graphically). Graph represents mean ± SEM (n = 6); *p < 0.05. (k) IPF fibroblasts were subjected to siRNA-­mediated knockdown of UCP2 for 72 h. Mitochondria were isolated and the rate of hydrogen peroxide production assessed using the p-­hydroxyphenylacetic acid (pHPA) assay; graph represents mean ± SEM (n = 3); ***p < 0.001. NT, non-­t argeting; CPT1a, carnitine palmitoyl transferase-­1a Hecker et al., 2014). Bleomycin-­induced lung injury is commonly used and static lung compliance (Figure S6d) in mice treated with UCP2 as a rodent model of pulmonary fibrosis, although the influence of siRNA. The fibrotic areas of the lungs showed evidence of neutro- aging is seldom accounted for in this model. In this model, oropharyn- philic inflammation that was not significantly different between the geal (or intratracheal) instillation of bleomycin typically leads to peak treatment groups (Figure S6e,f). Importantly, in the mice treated with fibrosis at 2–­3 weeks post-­injury followed by gradual resolution over UCP2 siRNA after bleomycin injury, we observed lower levels of the several weeks (Izbicki et al., 2002; Moeller et al., 2008). Previous senescence marker p16 colocalizing with myofibroblasts (expressing studies, including those by our group, have shown that, despite similar high levels of α-­SMA) (Figure 6j–­m). The lungs of the mice treated severity of peak fibrosis, resolution of fibrosis is markedly impaired with UCP2 siRNA also demonstrated evidence of higher numbers of in aged mice (≥18 months) in comparison to young mice (≤2 months) apoptotic cells, many of which colocalized with the myofibroblasts (Hecker et al., 2014; Redente et al., 2011). Using this model, we ob- (Figure 6n–­q). Lung fibroblasts isolated from these mice showed served that lung fibroblasts isolated from mice 3 weeks after bleo- stable reductions in steady-­state levels of the pro-­fibrotic markers, mycin injury showed significant upregulation of UCP2, an effect that α-­SMA and COL1a1 (Figure S6g–­i). In bleomycin-­induced lung injury, was more pronounced in aged mice (Figure 6a). The higher base- there is a general overall correlation between levels of COL1a1 and line levels of UCP2 in fibroblasts of aged mice were complemented UCP2 mRNA in isolated lung fibroblasts (Figure S6j). Overall, these re- by the publicly available dataset, GSE6591 showing higher UCP2 sults indicate that suppressing expression of UCP2 in aged mice with expression in lungs with increasing age of mice (Misra et al., 2007) persistent, non-­resolving fibrosis reprograms the fibrotic phenotype (Figure S6a). To test the efficacy of therapeutically targeting UCP2 in of myofibroblasts to effectively promote fibrosis resolution. established lung fibrosis, we initiated treatment of injured aged mice on day 22 after bleomycin administration (1.5 U/kg) with oropharyngeal UCP2 (or non-­targeting, NT) siRNA (Figure 6b). After 3 weeks of 3 | DISCUSSION siRNA treatment (and 6 weeks after initial bleomycin injury), we confirmed the efficacy of UCP2 silencing in lung tissue (Figure S6b). At UCPs belong to a subfamily of solute transporters embed- this delayed time point when aged mice fail to resolve fibrosis, we ded within the inner membrane of mitochondria (Brand & found marked improvement in resolution capacity, as evidenced by Esteves, 2005). While the physiological function of UCP1 as an histopathology and collagen deposition by Masson's trichrome stain- uncoupler of oxidative phosphorylation in association with the ing (Figure ­6c–­h), biochemical measurements of total lung hydroxy- regulated production of heat in brown adipocytes is well recog- proline content (Figure 6i), total lung collagen mRNA (Figure S6c) nized (Argyropoulos & Harper, 2002; Nicholls & Locke, 1984), 10 of 18 | RANGARAJAN et al. roles of the ancestral UCP2 and UCP3 homologs remain unclear. support this pro-­oxidant effect of UCP2 in lung myofibroblasts. UCP2 appears to function as a mild uncoupler and mitigator First, mitochondrial superoxide production, as determined by of mitochondrial ROS production in immune cells (Arsenijevic superoxide-­d ependent oxidation of the MitoSOX™ fluorophore, et al., 2000; Basu Ball et al., 2011). In this report, we demon- was higher in UCP2-­e xpressing cells. Second, electron paramag- strate that the chronic and constitutive elevation of UCP2 in netic resonance studies confirmed the formation of high levels myofibroblasts, paradoxically, mediates pro-­ oxidant effects of nitroxide, a superoxide reaction product in these cells. Third, that serve to sustain the differentiation and pro-­ s enescent steady-­s tate release rates of H2O2 , the dismutation product of phenotype of lung myofibroblasts. Several lines of evidence superoxide anion, were markedly decreased in UCP2-­s ilenced IPF (a) (b) **** NT siRNA (c) UCP2 (d) Cyclin D1 **** * pRb α-SMA COL1a1 GAPDH (e) (f) (g) UCP2 siRNA NT siRNA ** ** DAPI/Ki-67 DAPI/Ki-67 (i) (h) NT siRNA (j) UCP2 siRNA SA-β gal *** **** DAPI (k) (l) siRNA NT *** UCP2 Cleaved PARP β-Actin Antimycin A + + + + + + | RANGARAJAN et al. 11 of 18 F I G U R E 5 UCP2 regulates fibroblast senescence and myofibroblast differentiation. (a–­l) In these experiments, IPF fibroblasts were subjected to siRNA-­mediated silencing of UCP2 for 72 h. (a) Fibroblast cell counting was performed at 72 h (105 cells/well were seeded in both conditions at the start of each experiment); graph represents mean ± SEM (n = 5); ****p < 0.0001. (b) Western blotting was performed to assess the steady-­state expression of markers of cell-­cycling, cyclin-­D1 and phosphorylated Rb; myofibroblast markers, α-­smooth muscle actin (α-­SMA) and collagen 1a1 (COL1a1); representative blots are shown; densitometric analyses are shown in (c–­f ), respectively; graphs represent mean ± SEM (n = 4); *p < 0.05, **p < 0.01, ****p < 0.0001. (g) Cells were fixed and stained for DAPI and Ki-­67, a nuclear marker for cell proliferation; immunofluorescence imaging was performed; representative images (10×) are shown. (h) Senescence-­associated β-­ galactosidase (SA-­β-­gal) staining with representative light microscopy images (10×) shown. (i,j) Senescence-­associated secretory phenotype (SASP) markers, interleukin 6 (IL-­6) (i) and interleukin 1β (IL-­1β). (j) Gene expression was assessed by real-­time PCR; graphs represent mean ± SEM (n = 4); ***p < 0.001, ****p < 0.0001. (k) Antimycin-­A 100 μM was added to the cells for 6 h prior to harvest. Western blotting was performed to assess the steady-­state levels of the apoptosis marker, cleaved poly (ADP-­ribose) polymerase (PARP); densitometric analysis shown in (l); graph represents mean ± SEM (n = 3); ***p < 0.001. The effects of UCP2 silencing on these fibroblast phenotypes were confirmed to be similar in fibroblasts derived from lung explants of at least 3 different IPF patients myofibroblasts. Finally, the relative levels of oxidized vs. reduced OCR, coupling efficiency, maximal OCR and reserve capacity de- glutathione (GSH), a reliable measure of overall cellular redox spite a marked decrease in ROS production when UCP2 is silenced. state, were noted to be higher in IPF myofibroblasts compared Thus, under baseline conditions, the high expression and activity to control (when measured by an unbiased mass spectrometry-­ of UCP2 in myofibroblasts appear to skew oxygen metabolism based metabolomics approach), and this effect was reversed with towards partially reduced ROS relative to its complete reduction silencing of UCP2. Together, these data provide convincing evi- to water per mole of oxygen consumed/reduced. Taken together, dence that the high basal levels of UCP2 mediate constitutively this supports the concept that “electron leak” from mitochon- higher production of ROS and consequent oxidative stress re- drial ETC might occur, not simply due to increased δψm but due to sponses in IPF lung myofibroblasts. other factors that influence the forward flow of electrons towards In addition to higher levels of ROS production in the context of more efficient and complete reduction of oxygen at cytochrome heightened UCP2 expression/activity, our studies support a net c. Interestingly, the incorporation of free FAs into the inner mito- efflux of FAs and reduced FAO in the mitochondria of senescent chondrial membrane may alter membrane fluidity to alter electron myofibroblasts. This is based on our observations of decreased transport function that leads to higher ROS production (Schönfeld FA consumption in IPF myofibroblasts expressing high levels of & Wojtczak, 2007, 2008). While a role for non-­mitochondrial ROS UCP2, an effect that was reversed with UCP2 silencing, detected production by UCP2 cannot be completely excluded, we did not by both mass spectrometry and lipid staining approaches; fur- observe a statistically significant decrease in non-­mitochondrial thermore, increased basal oxygen consumption in these UCP2-­ OCR with UCP2 knockdown; however, this finding does not ex- silenced cells was abrogated with etomoxir, an inhibitor of CPT1a, clude participation of non-­E TC dependent sources of ROS, such as the rate-­limiting enzyme for FAO. Furthermore, CPT1a silencing those derived from the NADPH oxidase family (Bernard et al., 2014; reversed the beneficial effects of UCP2 knockdown on mitochon- Thannickal & Fanburg, 1995). drial reserve capacity. Consistent with recent studies supporting There is substantive evidence linking increased oxidative a link between fatty acid flippase activity and proton transport stress to myofibroblast differentiation and cellular senescence (Berardi & Chou, 2014), we did observe a dissipation of the proton (Cheresh et al., 2013; Hecker et al., 2009; Jain et al., 2013; gradient by UCP2. However, we do not have direct evidence that Velarde et al., 2012; Wiley et al., 2016). The finding that UCP2 translocation of protons occurs through UCP2. Our data would silencing was sufficient to reverse these pro-­f ibrotic phenotypes support a model by which UCP2 functioning as a FA anion trans- supports the concept that constitutive UCP2-­d ependent ROS porter dissipates the proton gradient by neutralization of pro- generation reversibly controls these differentiation-­ i nducing tons in the intermembranous space, thus facilitating net efflux of and pro-­s enescence programs. An interesting feature of this FAs from the mitochondrial matrix to the cytosol (see Graphical metabolic reprogramming is the inefficient and/or defective Abstract). utilization of FAO for energy production in UCP-­e xpressing IPF How might the same protein function as “antioxidant” in one myofibroblasts. Interestingly, restoring FAO by genetic or phar- context while mediating a “pro-­oxidant” effect in another? The macological methods has been shown to protect mice from tub- conventional antioxidant effect of UCP2 has been ascribed to its ulointerstitial fibrosis of the kidney (Kang et al., 2015), and UCP2 actions of increasing proton leak and dissipating δψm, thereby de- deficiency has been linked to reduced lipid deposition and ECM creasing reverse electron transport (Arsenijevic et al., 2000). Other accumulation in an ischemia–­r eperfusion model of kidney fibro- studies suggest that UCP2 has no effect on proton leak or ROS sis (Ke et al., 2020). Our studies support the combined regula- production (Kukat et al., 2014). In contrast, our studies clearly indi- tion of proton leak and FA transport/utilization as an integrated cate an effect of UCP2 on δψm dissipation with increased produc- function of UCP2, serving as both an “uncoupler” and a solute/ tion of ROS. Importantly, we observed an increase in ATP-­linked FA transporter. 12 of 18 | RANGARAJAN et al. (a) (b) * p=0.0572 * (c) (e) (g) (i) (f) (d) *** ** (h) Bleo + NT siRNA Bleo + UCP2 siRNA (j) (k) (l) (m) ** α-SMA p16 DAPI α-SMA Bleo + NT siRNA p16 DAPI Bleo + UCP2 siRNA (n) (o) (p) (q) ** α-SMA TUNEL ns DAPI α-SMA TUNEL DAPI ** | RANGARAJAN et al. 13 of 18 F I G U R E 6 Therapeutic targeting of UCP2 promotes resolution of experimental lung fibrosis. (a) Young (2 months) and aged (18 months old) C57BL/6 mice were subjected to lung injury by instillation of oropharyngeal bleomycin (1.5 U/kg) (or no injury by instillation of PBS control). Lungs were harvested at 3 weeks after bleomycin injury, and fibroblasts were isolated and assessed for gene expression of UCP2 (depicted graphically). Graph represents mean ± SEM (n = 3 in each group); *p < 0.05. (b) Schematic depicting experimental design. 18-­month-­ old C57BL/6 mice were subjected to lung injury by instillation of oropharyngeal bleomycin (1.5 U/kg) (or no injury by instillation of PBS control). They were treated with UCP2-­t argeting or non-­t argeting (NT) siRNA, administered oropharyngeally every other day for 3 weeks, starting on day 22 after injury. Lungs were harvested at 6 weeks after injury, and the following analyses were performed. (c–­h) Masson's trichrome histochemical staining for collagen was performed. Top panels (c,e,g) show whole lung sections, and bottom panels (d,f,h) show 20× magnification of selected areas. Images are representative of n = 3 in each group. (i) Hydroxyproline content of the lungs was assessed (depicted graphically); graphs represent mean ± SEM (n = 6–­13); **p < 0.01, ***p < 0.001. (j,k) Representative images showing fluorescence patterns of α-­SMA positive fibroblasts (green), senescence marker p16 (red) and nuclei (DAPI-­blue). Scale bars = 50 μm. (l,m) Box plots show fluorescence intensity ratios of p16/α-­SMA and p16/nuclei from regions of enhanced fibrotic remodeling, n = 7 per group, 2 mice for each condition. **p < 0.01. (n–­q) Representative images show fluorescence patterns of α-­SMA (green), apoptosis marker TUNEL (red) and nuclei (DAPI-­blue). Scale bars = 50 μm. Box plots show relative fluorescence intensity ratios, n = 7 per each group, **p < 0.01. NT, non-­t argeting; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling A notable strength of the therapeutic approach described here 4.2 | Subject characteristics is the prospect of targeting the clearance of senescent cells after they have been formed, rather than preventing their formation. ID No. Age (y) Sex Condition Center 3007 55 M Non-­IPF UAB 3008 49 M Non-­IPF UAB 3013 61 F Non-­IPF UAB disease (Horowitz & Thannickal, 2019). Except for the spleen and 15046 65 M Non-­IPF UAB whole blood, UCP2 appears to the most highly expressed in the 16029 69 M Non-­IPF UAB Indeed, by reducing UCP2 expression in IPF (myo)fibroblasts, we were able to not only reduce senescence but also lower the apoptosis susceptibility of these recalcitrant cells, a strategy that may prove more effective to induce fibrosis regression in established lung. The lungs are exposed to elevated levels of oxidative stress 2026 60 M IPF UAB due to higher ambient oxygen concentration and exposure to 2032 66 M IPF UAB environmental toxicants; thus, while adaptive mechanisms may 2041 56 M IPF UAB have evolved to protect against such stress, it also renders this 15044 53 M IPF UAB organ more susceptible to chronic diseases associated with oxidative stress. However, UCP2 is ubiquitously expressed and, while it may serve protective role in the context of acute and transient stressors (e.g., mitohormesis), chronic upregulation of this stress-­ responsive protein may give rise to degenerative tissue responses in multiple organ systems. 4 | 15061 63 F IPF UAB LTC-­27 69 M Non-­IPF U Colorado LTC-­29 87 M Non-­IPF U Colorado LTC-­50 66 M Non-­IPF U Colorado LTC-­22 45 M IPF U Colorado LTC-­3 4 64 M IPF U Colorado LTC-­4 0 68 M IPF U Colorado E X PE R I M E NTA L PRO C E D U R E S 4.1 | Source of cells 4.3 | Cell culture Primary human mesenchymal stromal cells were obtained from Fibroblasts were cultured in DMEM, supplemented with 10% fetal bronchoalveolar lavage fluid of patients with IPF at University of bovine serum (FBS), PenStrep (100 units/ml penicillin, 100 μg/ml Michigan hospital; primary human lung fibroblasts were isolated streptomycin), and were incubated at 37°C in 5% CO2 and 95% air. from failed donor lungs or from healthy parts of lungs curatively All experiments with primary cells were performed on cells below resected for cancer (“non-­IPF”) and from explants of patients with the eighth passage. IPF undergoing lung transplantation at University of Alabama at Birmingham (UAB), Birmingham, AL, and at University of Colorado Anschutz Medical Campus –­ all as approved by the respective 4.4 | RT PCR Institutional Review Boards. The characteristics of subjects from whom the lung fibroblasts were obtained for ex vivo studies are de- Fibroblasts (or lung tissues) were washed with PBS. The total RNA scribed in the next section. Normal human fetal lung diploid fibro- was extracted using RNeasy mini kit (Qiagen) according to the blasts (IMR-­90 cells) were obtained from ATCC. manufacturer's instructions. Total RNA was reverse transcribed to 14 of 18 | RANGARAJAN et al. cDNA using iScript reverse transcription kit (Bio-­Rad). Expression of room temperature. Fluorescence signals were measured on a micro- mRNA of genes of interest was determined by using specific prim- plate reader at Ex/Em = 535/587 nm. To generate a standard cali- ers, as listed in the Key Resources Table. The real-­time PCRs were bration curve, serial dilutions of ATP were used. ATP concentrations performed in a 7300 real-­time PCR system (Applied Biosystems) were calculated from the standard curve data and normalized to the using a SYBR Green-­based real-­time PCR assay with SYBR Green corresponding tissue weight or number of cells. PCR master mix (Applied Biosystems). Reactions were carried out for 40 cycles. Data are expressed for each target gene normalized to endogenous GAPDH or β-­Actin as 2−δδCt, and relative mRNA expres- 4.8 | RNA interference sion is represented graphically as fold change compared to control condition. siRNA (UCP2 targeting and non-­t argeting) with sense sequences as described in the Key Resources Table was obtained from Dharmacon 4.5 | Immunohistochemistry (Supporting Information). Fibroblasts were transfected with 100 nM siRNA using Lipofectamine 2000 in OptiMEM medium according to the manufacturer's protocol overnight. This was followed by recov- Paraffin-­embedded lung tissues were cut into 5 μm sections and ery using DMEM with 10% FBS the next day for all experiments last- mounted on glass slides for staining. The sections were subjected ing more than 24 h. to heat-­induced antigen retrieval as described previously (Chanda et al., 2016) followed by processing for immunohistochemical localization of UCP2 or smooth muscle actin in IPF lung sections. Briefly, 4.9 | Flow cytometry xylene was used to deparaffinize the tissue sections which were then hydrated through ethanol series and water. Antigen retrieval Fibroblasts were incubated at 37°C with the indicated fluores- was performed in a 95°C water bath using citrate buffer (at pH 6.0) cent dye(s), as described in the figure legends. The cells were then followed by quenching of endogenous peroxidases using 3% hydro- washed twice with PBS. Data were collected with an LSR-­II flow gen peroxide. The tissue sections were blocked using 5% normal cytometer (Becton Dickinson) and analyzed with FlowJo software goat serum for 1 h and then incubated in primary antibodies over- (version 10.6.1; TreeStar). night at 4°C. IgG isotype controls (with no primary antibody) were utilized as negative control. The Dako Envision Dual Link System was used for secondary antibody. Colorimetric detection was achieved using the DAB/H2O2 kit from the Vector Laboratories. Nuclei were 4.10 | Metabolomics analysis counterstained with hematoxylin (Vector Labs). For mouse lung sec- Metabolites from frozen cell pellets were extracted at 2 × 106 cells/ml tions, after the antigen retrieval step, Masson's trichrome staining in ice-­cold 5:3:2 MeOH:acetonitrile:water (v/v/v). Extractions were for collagen was performed. carried out using vigorous vortexing for 30 min at 4°C. Supernatants were clarified by centrifugation (10 min, 18,000 g, 4°C) and 10 μl 4.6 | Light and immunofluorescence microscopy analyzed using a Thermo Vanquish UHPLC coupled to a Thermo Q Exactive mass spectrometer. Global metabolomics analyses were performed using a 5 min C18 gradient in positive and negative ion Light and immunofluorescence microscopy was performed using a modes (separate runs) with electrospray ionization as described Keyence BZ-­X700 microscope. The images obtained were processed (Gehrke et al., 2019; Nemkov et al., 2019). For all analyses, the MS using Adobe Photoshop CS6. scanned in MS1 mode across the m/z range of 65–­950. Peaks were annotated in conjunction with the KEGG database, integrated, and 4.7 | ATP assay quality control performed using Maven (Princeton University), as described (Nemkov et al., 2015). Analysis of the output was performed using Metaboanalyst version 5. Pre-­weighed lung tissue (~30 mg) or 0.15 × 106 lung fibroblasts were processed for determination of ATP content using a commercially available kit (Abcam; ab833355) as per the manufacturer's instruc- 4.11 | Mitochondrial stress test tions. Briefly, frozen lung tissue or fibroblast cell pellets were resuspended in ice-­cold ATP assay buffer and lysed using Dounce The assay was performed according to Agilent Seahorse Extracellular homogenizer. The resulting lysates were clarified via centrifugation Analyzer manufacturer's instructions. Briefly, fibroblasts were at 13,000 g at 4°C. The supernatant fractions were collected and seeded on a Seahorse XFe96 assay plate at a density of 1.5 × 105 subjected to the deproteinization procedure via TCA precipitation cells per well 24 h prior to the assay. The sensor cartridge was hy- (Abcam; ab204708). The deproteinized samples were then incu- drated with XF calibrant overnight, and the fibroblasts were washed bated with required reaction components for 30 min in the dark at with Agilent Base media supplemented with 10 mM glucose, 1 mM | RANGARAJAN et al. 15 of 18 sodium pyruvate, and 2 mM L-­glutamine just prior to the assay. The spectrofluorometer at Ex/Em = 320/400 nm at 10 min intervals up following drugs were injected sequentially: oligomycin 2.5 μg/ml, to 240 min to calculate rate of production of H2O2. FCCP 5 μM, rotenone 2 μM + antimycin A 4 μM and 2-­deoxy glucose 50 mM. Oxygen consumption rate and extracellular acidification rate were measured simultaneously. 4.14 | Western immunoblotting 4.12 | Electron paramagnetic resonance (EPR) study (Sigma-­Aldrich) followed by addition of protease and phosphatase Cells were lysed in the radioimmune precipitation assay (RIPA) buffer inhibitors. The total protein concentration of the lysates was quantitated using a Micro BCA Protein Assay kit (Pierce). Further RIPA Mitochondrial superoxide production was measured by EPR using the was added to equalize the concentrations of the lysates, and 10× re- mitochondrial spin probe 1-­hydroxy-­4-­[2-­(triphenylphosphonio)-­acet ducing agent along with 4× loading buffer was added to the lysates, amido]-­2,2,6,6-­tetramethylpiperidine, 1-­hydroxy-­2,2 ,6,6-­tetramethy followed by incubation at 95°C for 5 min. The samples were then sub- l-­4-­[2-­(triphenylphosphonio)acetamido]piperidinium dichloride (mito-­ jected to SDS-­PAGE, and Western immunoblotting was performed TEMPO-­H). Fibroblasts were subjected to siRNA-­mediated knock- as described previously (Desai et al., 2014). Immunoblots were im- down of UCP2 24 h prior to the EPR measurements. Mito-­TEMPO-­H aged using Amersham Biosciences 600 Imager (GE Healthcare) at probe was prepared in deoxygenated 50 mM phosphate buffer. The UAB and Biorad ChemiDoc XRS+ imager at University of Colorado. cells were washed and treated with mito-­TEMPO-­H 0.25 mM in Krebs-­ Quantification (densitometry) was performed using Fiji software. HEPES buffer (KHB) containing 100 μM of a metal chelator DTPA to avoid direct oxidation with metal ion or hydroxyl radical generation by Fenton reaction. The cells were incubated for 50 min at 37°C and then 4.15 | Source of mice placed on ice and gently scraped. 50 μl of cell suspension was loaded in an EPR capillary tube, and EPR measurements were performed at Only male mice (C57BL/6) were used for all experiments. Young mice room temperature using a Bruker EMXnano X-­band spectrometer. (8–­10 weeks age) were purchased from The Jackson Laboratory, EPR acquisition parameters were microwave frequency = 9.6 GHz; Bar Harbor, ME. Aged mice (~17 months) were procured from the center field = 3432 G; modulation amplitude = 2.0 G; sweep National Institute on Aging by Dr. Thannickal. The mice were housed width = 80 G; microwave power = 19.9 mW; total number of scans = 5; in vivariums at UAB and CU Anschutz on a 12-­h light–­dark cycle sweep time = 12.11 s; and time constant = 20.48 ms. The mito-­ with access to food and water ad libitum. All experiments were con- TEMPO·(nitroxide) radical concentration was obtained by simulat- ducted after approval by the UAB and CU Anschutz IACUC. ing the spectra using the SpinFit module incorporated in the Xenon software of the bench-­top EMXnano EPR spectrometer followed by the SpinCount module (Bruker), as described previously (Elajaili et al., 2019). Total protein content from the analyzed samples were 4.16 | Murine model of bleomycin-­induced lung fibrosis quantified via a Micro BCA Protein Assay kit (Pierce), and nitroxide concentrations were normalized to total protein. Mice were anesthetized with isoflurane followed by oropharyngeal instillation/aspiration of bleomycin (1.5 U/kg) in 60 μl PBS. Lungs 4.13 | Mitochondrial H2O2 production rate were harvested at 3 weeks after bleomycin instillation for assessing fibrotic burden and UCP2 gene expression in tissue/isolated fibroblasts. In experiments involving therapeutic siRNA administration, Mitochondrial H2O2 production was determined fluorometrically mice received UCP2-­t argeting or non-­t argeting (NT) siRNA (50 μg/ as described previously (Murthy et al., 2010). Briefly, mitochondria dose in 60 μl PBS) administered oropharyngeally under isoflurane were isolated by lysing the cells in a mitochondria buffer containing anesthesia every other day for 3 weeks, starting on day 22 after in- 10 mM Tris, pH 7.8, 0.2 mm EDTA, 320 mM sucrose, and protease jury. Lungs were harvested at 6 weeks after injury for assessing end inhibitors. Lysates were homogenized using a Kontes Pellet Pestle points including hydroxyproline, collagen and UCP2 expression, and Motor and centrifuged at 2000 g for 8 min at 4°C. The supernatant for isolating fibroblasts. was removed and kept at 4°C, and the pellet was lysed, homogenized, and centrifuged again. The two supernatants were pooled and centrifuged at 12,000 g for 15 min at 4°C. The pellet was then re- 4.17 | Hydroxyproline assay suspended in mitochondria buffer without sucrose. The isolated mitochondria were incubated in phenol-­red free Hanks' Balanced Salt Mouse lung tissues were dried in 2 ml microcentrifuge tubes at 70°C solution supplemented with 6.5 mM glucose, 1 mM HEPES, 6 mM so- in a block heater for 48 h and then hydrolyzed in 6 N HCl at 95°C dium bicarbonate, 1.6 mM p-­hydroxylphenyl acetic acid (pHPA), and for further 48 h. The tubes were centrifuged at 13,000 g for 10 min, 0.95 μg/ml HRP. Fluorescence of pHPA-­dimer was measured using a and the debris-­free supernatant was transferred to fresh tubes for 16 of 18 | RANGARAJAN et al. storage prior to hydroxyproline estimation. Fluorometric hydroxy- Brian Deskin proline assay was performed using a commercially available kit Hanan Elajaili (QuickZyme Biosciences) with hydroxyproline as a standard, as per Eva S. Nozik the manufacturer's instructions. https://orcid.org/0000-0001-7850-5146 https://orcid.org/0000-0002-4772-6218 https://orcid.org/0000-0002-7229-5528 Jessy S. Deshane https://orcid.org/0000-0002-6761-5662 Jaroslaw W. Zmijewski Oliver Eickelberg https://orcid.org/0000-0001-7314-6164 https://orcid.org/0000-0001-7170-0360 4.18 | Quantification and statistical analysis Victor J. Thannickal Graphing and statistical analysis were performed with Graphpad REFERENCES Prism ver 9.1. Unpaired t test was used for comparing 2 variables, Argyropoulos, G., & Harper, M. E. (2002). Uncoupling proteins and thermoregulation. Journal of Applied Physiology (Bethesda, MD: 1985), 92(5), 2187–­2198. https://doi.org/10.1152/jappl​physi​ ol.00994.2001 Arsenijevic, D., Onuma, H., Pecqueur, C., Raimbault, S., Manning, B. S., Miroux, B., Couplan, E., Alves-­Guerra, M. C., Goubern, M., Surwit, R., Bouillaud, F., Richard, D., Collins, S., & Ricquier, D. (2000). Disruption of the uncoupling protein-­2 gene in mice reveals a role in immunity and reactive oxygen species production. Nature Genetics, 26(4), 435–­439. https://doi.org/10.1038/82565 Basu Ball, W., Kar, S., Mukherjee, M., Chande, A. G., Mukhopadhyaya, R., & Das, P. K. (2011). Uncoupling protein 2 negatively regulates mitochondrial reactive oxygen species generation and induces phosphatase-­mediated anti-­inflammatory response in experimental visceral leishmaniasis. Journal of Immunology, 187(3), 1322–­ 1332. https://doi.org/10.4049/jimmu​nol.1004237 Berardi, M. J., & Chou, J. J. (2014). Fatty acid flippase activity of UCP2 is essential for its proton transport in mitochondria. Cell Metabolism, 20(3), 541–­552. https://doi.org/10.1016/j.cmet.2014.07.004 Bernard, K., Hecker, L., Luckhardt, T. R., Cheng, G., & Thannickal, V. J. (2014). NADPH oxidases in lung health and disease. Antioxidants & Redox Signaling, 20(17), 2838–­2853. https://doi.org/10.1089/ ars.2013.5608 Brand, M. D., & Esteves, T. C. (2005). Physiological functions of the mitochondrial uncoupling proteins UCP2 and UCP3. Cell Metabolism, 2(2), 85–­93. https://doi.org/10.1016/j.cmet.2005.06.002 Bueno, M., Calyeca, J., Rojas, M., & Mora, A. L. (2020). Mitochondria dysfunction and metabolic reprogramming as drivers of idiopathic pulmonary fibrosis. Redox Biology, 33, 101509. https://doi. org/10.1016/j.redox.2020.101509 Caporarello, N., Meridew, J. A., Aravamudhan, A., Jones, D. L., Austin, S. A., Pham, T. X., Haak, A. J., Moo Choi, K., Tan, Q., Haresi, A., Huang, S. K., Katusic, Z. S., Tschumperlin, D. J., & Ligresti, G. (2020). Vascular dysfunction in aged mice contributes to persistent lung fibrosis. Aging Cell, 19(8), e13196. https://doi.org/10.1111/ acel.13196 Chanda, D., Kurundkar, A., Rangarajan, S., Locy, M., Bernard, K., Sharma, N. S., Logsdon, N. J., Liu, H., Crossman, D. K., Horowitz, J. C., De Langhe, S., & Thannickal, V. J. (2016). Developmental reprogramming in mesenchymal stromal cells of human subjects with idiopathic pulmonary fibrosis. Scientific Reports, 6, 37445. https://doi. org/10.1038/srep3​7445 Cheresh, P., Kim, S. J., Tulasiram, S., & Kamp, D. W. (2013). Oxidative stress and pulmonary fibrosis. Biochimica et Biophysica Acta, 1832(7), 1028–­1040. https://doi.org/10.1016/j. bbadis.2012.11.021 Desai, L. P., Zhou, Y., Estrada, A. V., Ding, Q., Cheng, G., Collawn, J. F., & Thannickal, V. J. (2014). Negative regulation of NADPH oxidase 4 by hydrogen peroxide-­ inducible clone 5 (Hic-­ 5) protein. The Journal of Biological Chemistry, 289(26), 18270–­18278. https://doi. org/10.1074/jbc.M114.562249 Duffield, J. S., Lupher, M., Thannickal, V. J., & Wynn, T. A. (2013). Host responses in tissue repair and fibrosis. Annual Review of Pathology, 8, 241–­276. https://doi.org/10.1146/annur​ev-­patho​l- ­02071​2-­163930 and anova for three or more variables with multiple comparisons. All data are expressed as mean ± SEM, unless otherwise indicated. p < 0.05 were considered statistically significant. AU T H O R C O N T R I B U T I O N S Conceptualization: SR and VJT; Conduct of experiments and data generation: SR, MLL, DC, AK, DK, JLL, PL, RAB, HE, ESN and JSD; Analysis of data and interpretation: SR, JWZ and VJT; Manuscript preparation and editing: SR and VJT; Resources: SR, OE and VJT. AC K N OW L E D G M E N T S We thank the University of Colorado School of Medicine Metabolomics Core for their contributions to this manuscript. We thank Eugene Becker for assistance in experiments. This manuscript was supported by National Institutes of Health (NIH) grants, K08 HL135399 (to SR); P01 HL114470 and R01 AG046210 (to VJT) and R01 HL139617 (to JWZ and VJT); and a Department of Veterans' Affairs (VA) Merit Award I01BX003056 (to VJT). The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the VA. C O N FL I C T O F I N T E R E S T VJT has consulted in the broad area of pulmonary fibrosis for the following companies: Mistrial Therapeutics, Inc., Boehringer Ingelheim Pharmaceuticals, Inc., United Therapeutics, Blade Therapeutics, Versant Venture, Translate Bio and Sunshine Bio. SR and VJT have initiated the process of filing a patent on therapeutic targeting of UCP2 in fibrotic diseases. DATA AVA I L A B I L I T Y S TAT E M E N T The data that support the findings of this study are available from the corresponding author upon reasonable request. ORCID Sunad Rangarajan https://orcid.org/0000-0001-5313-4828 https://orcid.org/0000-0002-3812-5867 Morgan L. Locy Diptiman Chanda https://orcid.org/0000-0002-4835-4460 Ashish Kurundkar https://orcid.org/0000-0001-9078-1039 Deepali Kurundkar https://orcid.org/0000-0001-5223-1431 Jennifer L. Larson-­Casey Pilar Londono https://orcid.org/0000-0001-7238-7986 https://orcid.org/0000-0002-6026-5698 Rushita A. Bagchi https://orcid.org/0000-0002-9075-0766 https://orcid.org/0000-0003-4266-8677 RANGARAJAN et al. Elajaili, H. B., Hernandez-­L agunas, L., Ranguelova, K., Dikalov, S., & Nozik-­Grayck, E. (2019). Use of electron paramagnetic resonance in biological samples at ambient temperature and 77 K. JoVE (Journal of Visualized Experiments), (143), e58461. https://doi. org/10.3791/58461 Gehrke, S., Rice, S., Stefanoni, D., Wilkerson, R. B., Nemkov, T., Reisz, J. A., Hansen, K. C., Lucas, A., Cabrales, P., Drew, K., & D'Alessandro, A. (2019). Red blood cell metabolic responses to torpor and arousal in the hibernator arctic ground squirrel. Journal of Proteome Research, 18(4), 1827–­1841. https://doi.org/10.1021/acs.jprot​ eome.9b00018 Hecker, L., Logsdon, N. J., Kurundkar, D., Kurundkar, A., Bernard, K., Hock, T., Meldrum, E., Sanders, Y. Y., & Thannickal, V. J. (2014). Reversal of persistent fibrosis in aging by targeting Nox4-­ Nrf2 redox imbalance. Science Translational Medicine, 6(231), 231ra247. https://doi.org/10.1126/scitr​anslm​ed.3008182 Hecker, L., Vittal, R., Jones, T., Jagirdar, R., Luckhardt, T. R., Horowitz, J. C., Pennathur, S., Martinez, F. J., & Thannickal, V. J. (2009). NADPH oxidase-­ 4 mediates myofibroblast activation and fibrogenic responses to lung injury. Nature Medicine, 15(9), 1077–­1081. https:// doi.org/10.1038/nm.2005 Horowitz, J. C., & Thannickal, V. J. (2019). Mechanisms for the resolution of organ fibrosis. Physiology (Bethesda), 34(1), 43–­55. https://doi. org/10.1152/physi​ol.00033.2018 Izbicki, G., Segel, M. J., Christensen, T. G., Conner, M. W., & Breuer, R. (2002). Time course of bleomycin-­induced lung fibrosis. International Journal of Experimental Pathology, 83(3), 111–­119. http://www.ncbi. nlm.nih.gov/pubme​d/12383190, http://www.ncbi.nlm.nih.gov/ pmc/artic​les/PMC25​17673/​pdf/iep00​83-­0111.pdf Jain, M., Rivera, S., Monclus, E. A., Synenki, L., Zirk, A., Eisenbart, J., Feghali-­Bostwick, C., Mutlu, G. M., Budinger, G. R., & Chandel, N. S. (2013). Mitochondrial reactive oxygen species regulate transforming growth factor-­beta signaling. The Journal of Biological Chemistry, 288(2), 770–­777. https://doi.org/10.1074/jbc.M112.431973 Kang, H. M., Ahn, S. H., Choi, P., Ko, Y. A., Han, S. H., Chinga, F., Park, A. S., Tao, J., Sharma, K., Pullman, J., Bottinger, E. P., Goldberg, I. J., & Susztak, K. (2015). Defective fatty acid oxidation in renal tubular epithelial cells has a key role in kidney fibrosis development. Nature Medicine, 21(1), 37–­46. https://doi.org/10.1038/nm.3762 Ke, Q., Yuan, Q., Qin, N., Shi, C., Luo, J., Fang, Y., Xu, L., Sun, Q., Zen, K., Jiang, L., Zhou, Y., & Yang, J. (2020). UCP2-­induced hypoxia promotes lipid accumulation and tubulointerstitial fibrosis during ischemic kidney injury. Cell Death & Disease, 11(1), 26. https://doi. org/10.1038/s4141​9-­019-­2219-­4 Kukat, A., Dogan, S. A., Edgar, D., Mourier, A., Jacoby, C., Maiti, P., Mauer, J., Becker, C., Senft, K., Wibom, R., Kudin, A. P., Hultenby, K., Flögel, U., Rosenkranz, S., Ricquier, D., Kunz, W. S., & Trifunovic, A. (2014). Loss of UCP2 attenuates mitochondrial dysfunction without altering ROS production and uncoupling activity. PLoS Genetics, 10(6), e1004385. https://doi.org/10.1371/journ​al.pgen.1004385 Lopez-­Otin, C., Blasco, M. A., Partridge, L., Serrano, M., & Kroemer, G. (2013). The hallmarks of aging. Cell, 153(6), 1194–­1217. https://doi. org/10.1016/j.cell.2013.05.039 Mailloux, R. J., & Harper, M. E. (2011). Uncoupling proteins and the control of mitochondrial reactive oxygen species production. Free Radical Biology & Medicine, 51(6), 1106–­1115. https://doi. org/10.1016/j.freer​adbio​med.2011.06.022 Meltzer, E. B., Barry, W. T., D'Amico, T. A., Davis, R. D., Lin, S. S., Onaitis, M. W., Morrison, L. D., Sporn, T. A., Steele, M. P., & Noble, P. W. (2011). Bayesian probit regression model for the diagnosis of pulmonary fibrosis: Proof-­of-­principle. BMC Medical Genomics, 4, 70. https://doi.org/10.1186/1755-­8794-­4-­70 Misra, V., Lee, H., Singh, A., Huang, K., Thimmulappa, R. K., Mitzner, W., Biswal, S., & Tankersley, C. G. (2007). Global expression profiles from C57BL/6J and DBA/2J mouse lungs to determine | 17 of 18 aging-­related genes. Physiological Genomics, 31(3), 429–­4 40. https://doi.org/10.1152/physi​olgen​omics.00060.2007 Moeller, A., Ask, K., Warburton, D., Gauldie, J., & Kolb, M. (2008). The bleomycin animal model: A useful tool to investigate treatment options for idiopathic pulmonary fibrosis? The International Journal of Biochemistry & Cell Biology, 40(3), 362–­382. https://doi. org/10.1016/j.biocel.2007.08.011 Mora, A. L., Bueno, M., & Rojas, M. (2017). Mitochondria in the spotlight of aging and idiopathic pulmonary fibrosis. The Journal of Clinical Investigation, 127(2), 405–­414. https://doi.org/10.1172/jci87440 Murthy, S., Ryan, A., He, C., Mallampalli, R. K., & Carter, A. B. (2010). Rac1-­mediated mitochondrial H2O2 generation regulates MMP-­9 gene expression in macrophages via inhibition of SP-­1 and AP-­1. The Journal of Biological Chemistry, 285(32), 25062–­25073. https:// doi.org/10.1074/jbc.M109.099655 Nemkov, T., D'Alessandro, A., & Hansen, K. C. (2015). Three-­minute method for amino acid analysis by UHPLC and high-­ resolution quadrupole orbitrap mass spectrometry. Amino Acids, 47(11), 2345–­ 2357. https://doi.org/10.1007/s0072​6-­015-­2019-­9 Nemkov, T., Reisz, J. A., Gehrke, S., Hansen, K. C., & D'Alessandro, A. (2019). High-­throughput metabolomics: Isocratic and gradient mass spectrometry-­based methods. Methods in Molecular Biology, 1978, 13–­26. https://doi.org/10.1007/978-­1-­4939-­9236-­2_2 Nicholls, D. G., & Locke, R. M. (1984). Thermogenic mechanisms in brown fat. Physiological Reviews, 64(1), 1–­6 4. https://doi.org/10.1152/ physr​ev.1984.64.1.1 Otoupalova, E., Smith, S., Cheng, G., & Thannickal, V. J. (2020). Oxidative Stress in Pulmonary Fibrosis. Comprehensive Physiology, 10(2), 509–­ 547. https://doi.org/10.1002/cphy.c190017 Pazolli, E., Luo, X., Brehm, S., Carbery, K., Chung, J. J., Prior, J. L., Doherty, J., Demehri, S., Salavaggione, L., Piwnica-­Worms, D., & Stewart, S. A. (2009). Senescent stromal-­derived osteopontin promotes preneoplastic cell growth. Cancer Research, 69(3), 1230–­1239. https:// doi.org/10.1158/0008-­5472.Can-­0 8-­2970 Pecqueur, C., Bui, T., Gelly, C., Hauchard, J., Barbot, C., Bouillaud, F., Ricquier, D., Miroux, B., & Thompson, C. B. (2008). Uncoupling protein-­2 controls proliferation by promoting fatty acid oxidation and limiting glycolysis-­derived pyruvate utilization. The FASEB Journal, 22(1), 9–­18. https://doi.org/10.1096/fj.07-­8945com Raghu, G., Weycker, D., Edelsberg, J., Bradford, W. Z., & Oster, G. (2006). Incidence and prevalence of idiopathic pulmonary fibrosis. American Journal of Respiratory and Critical Care Medicine, 174(7), 810–­816. https://doi.org/10.1164/rccm.20060​2-­163OC Rangarajan, S., Bernard, K., & Thannickal, V. J. (2017). Mitochondrial dysfunction in pulmonary fibrosis. Annals of the American Thoracic Society, 14(Supplement_5), S383–­S388. https://doi.org/10.1513/ Annal​sATS.20170​5-­370AW Redente, E. F., Jacobsen, K. M., Solomon, J. J., Lara, A. R., Faubel, S., Keith, R. C., Henson, P. M., Downey, G. P., & Riches, D. W. (2011). Age and sex dimorphisms contribute to the severity of bleomycin-­ induced lung injury and fibrosis. American Journal of Physiology. Lung Cellular and Molecular Physiology, 301(4), L510–­L 518. https://doi. org/10.1152/ajplu​ng.00122.2011 Rehan, M., Kurundkar, D., Kurundkar, A. R., Logsdon, N. J., Smith, S. R., Chanda, D., Bernard, K., Sanders, Y. Y., Deshane, J. S., Dsouza, K. G., Rangarajan, S., Zmijewski, J. W., & Thannickal, V. J. (2021). Restoration of SIRT3 gene expression by airway delivery resolves age-­associated persistent lung fibrosis in mice. Nature Aging, 1(2), 205–­217. https://doi.org/10.1038/s4358​7-­021-­0 0027​-­5 Romero, Y., Bueno, M., Ramirez, R., Álvarez, D., Sembrat, J. C., Goncharova, E. A., Rojas, M., Selman, M., Mora, A. L., & Pardo, A. (2016). mTORC1 activation decreases autophagy in aging and idiopathic pulmonary fibrosis and contributes to apoptosis resistance in IPF fibroblasts. Aging Cell, 15(6), 1103–­1112. https://doi. org/10.1111/acel.12514 18 of 18 | Rousset, S., Alves-­Guerra, M. C., Mozo, J., Miroux, B., Cassard-­Doulcier, A. M., Bouillaud, F., & Ricquier, D. (2004). The biology of mitochondrial uncoupling proteins. Diabetes, 53(Suppl 1), S130–­S135. http:// www.ncbi.nlm.nih.gov/pubme​d/14749278, http://diabe​tes.diabe​ tesjo​urnals.org/conte​nt/53/suppl_1/S130.full.pdf Schönfeld, P., & Wojtczak, L. (2007). Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport. Biochimica et Biophysica Acta, 1767(8), 1032–­1040. https://doi.org/10.1016/j. bbabio.2007.04.005 Schönfeld, P., & Wojtczak, L. (2008). Fatty acids as modulators of the cellular production of reactive oxygen species. Free Radical Biology & Medicine, 45(3), 231–­241. https://doi.org/10.1016/j.freer​adbio​ med.2008.04.029 Thannickal, V. J., & Fanburg, B. L. (1995). Activation of an H2O2-­generating NADH oxidase in human lung fibroblasts by transforming growth factor beta 1. The Journal of Biological Chemistry, 270(51), 30334–­ 30338. http://www.ncbi.nlm.nih.gov/pubme​d/8530457, http:// www.jbc.org/conte​nt/270/51/30334.full.pdf Thannickal, V. J., Lee, D. Y., White, E. S., Cui, Z., Larios, J. M., Chacon, R., Horowitz, J. C., Day, R. M., & Thomas, P. E. (2003). Myofibroblast differentiation by transforming growth factor-­beta1 is dependent on cell adhesion and integrin signaling via focal adhesion kinase. The Journal of Biological Chemistry, 278(14), 12384–­12389. https:// doi.org/10.1074/jbc.M2085​4 4200 Thannickal, V. J., Toews, G. B., White, E. S., Lynch, J. P., 3rd, & Martinez, F. J. (2004). Mechanisms of pulmonary fibrosis. Annual Review of Medicine, 55, 395–­417. https://doi.org/10.1146/annur​ ev.med.55.091902.103810 Velarde, M. C., Flynn, J. M., Day, N. U., Melov, S., & Campisi, J. (2012). Mitochondrial oxidative stress caused by Sod2 deficiency promotes cellular senescence and aging phenotypes in the skin. Aging (Albany NY), 4(1), 3–­12. https://doi.org/10.18632/​aging.100423 Vozza, A., Parisi, G., De Leonardis, F., Lasorsa, F. M., Castegna, A., Amorese, D., Marmo, R., Calcagnile, V. M., Palmieri, L., Ricquier, D., Paradies, E., Scarcia, P., Palmieri, F., Bouillaud, F., & Fiermonte, G. (2014). UCP2 transports C4 metabolites out of mitochondria, RANGARAJAN et al. regulating glucose and glutamine oxidation. Proceedings of the National Academy of Sciences of the United States of America, 111(3), 960–­965. https://doi.org/10.1073/pnas.13174​0 0111 Wiley, C. D., Velarde, M. C., Lecot, P., Liu, S., Sarnoski, E. A., Freund, A., Shirakawa, K., Lim, H. W., Davis, S. S., Ramanathan, A., Gerencser, A. A., Verdin, E., & Campisi, J. (2016). Mitochondrial dysfunction induces senescence with a distinct secretory phenotype. Cell Metabolism, 23(2), 303–­314. https://doi.org/10.1016/j. cmet.2015.11.011 Zhang, C. Y., Parton, L. E., Ye, C. P., Krauss, S., Shen, R., Lin, C. T., Porco, J. A., Jr., & Lowell, B. B. (2006). Genipin inhibits UCP2-­mediated proton leak and acutely reverses obesity-­and high glucose-­induced beta cell dysfunction in isolated pancreatic islets. Cell Metabolism, 3(6), 417–­427. https://doi.org/10.1016/j.cmet.2006.04.010 Zhou, Y., & Lagares, D. (2021). Anti-­aging therapy for pulmonary fibrosis. Nature Aging, 1(2), 155–­156. https://doi.org/10.1038/s4358​7-­021-­ 00035​-­5 S U P P O R T I N G I N FO R M AT I O N Additional supporting information can be found online in the Supporting Information section at the end of this article. How to cite this article: Rangarajan, S., Locy, M. L., Chanda, D., Kurundkar, A., Kurundkar, D., Larson-Casey, J. L., Londono, P., Bagchi, R. A., Deskin, B., Elajaili, H., Nozik, E. S., Deshane, J. S., Zmijewski, J. W., Eickelberg, O., & Thannickal, V. J. (2022). Mitochondrial uncoupling protein-2 reprograms metabolism to induce oxidative stress and myofibroblast senescence in age-associated lung fibrosis. Aging Cell, 21, e13674. https://doi.org/10.1111/acel.13674