Hypoxia-inducible factor 1� stabilization by carbonmonoxide results in cytoprotective preconditioningBeek Y. Chin*, Ge Jiang†, Barbara Wegiel*, Hong J. Wang*, Theresa MacDonald*, Xu Chen Zhang†, David Gallo*,Eva Cszimadia*, Fritz H. Bach*, Patty J. Lee†, and Leo E. Otterbein*‡

*Beth Israel Deaconess Medical Center, Department of Surgery, Transplant Center, Harvard Medical School, Boston, MA 02215;and †Department of Medicine, Yale University School of Medicine, New Haven, CT 06520

Edited by Louis J. Ignarro, University of California School of Medicine, Los Angeles, CA, and approved February 1, 2007(received for review October 29, 2006)

The most salient feature of carbon monoxide (CO)-mediated cyto-protection is the suppression of inflammation and cell death. One ofthe important cellular targets of CO is the macrophage (m�). Manystudies have shown that exposure of m� to CO results in thegeneration of an antiinflammatory phenotype; however, these re-ports have ignored the effect of CO alone on the cell before stimu-lation. Most investigations have focused on the actions of CO inmodulating the response to noxious stimuli. We demonstrate herethat exposure of m� to CO results in a significant and transient burstof reactive oxygen species (ROS) arising from the mitochondria(mitochondria-deficient m� do not respond to CO to produce ROS).The ROS promote rapid activation and stabilization of the transcrip-tion factor hypoxia-inducible factor 1� (HIF-1�), which regulatesexpression of genes involved in inflammation, metabolism, and cellsurvival. The increase in HIF-1� expression induced by CO results inregulated expression of TGF-�, a potent antiinflammatory cytokine.CO-induced HIF-1� and TGF-� expression are necessary to preventanoxia/reoxygenation-induced apoptosis in m�. Furthermore, block-ade of HIF-1� using RNA interference and HIF-1�-cre-lox m� resultedin a loss of TGF-� expression and CO-induced protection. A similarmechanism of CO-induced protection was operational in vivo toprotect against lung ischemia-reperfusion injury. Taken together, weconclude that CO conditions the m� via a HIF-1� and TGF-�-dependent mechanism and we elucidate the earliest events in m�signaling that lead to and preserve cellular homeostasis at the site ofinjury.

ischemia reperfusion � macrophage � heme oxygenase-1

There is increasing awareness of the salutary effects of CO atlow concentrations (15–250 ppm) in preclinical animal mod-

els of disease, including shock (1), postoperative ileus (2), organtransplantation (3), airway hyperresponsiveness (4), necrotizingenterocolitis (5), and ischemia/reperfusion (I/R) injury (IRI)(6). Initially thought of as a highly toxic molecule, CO ispresently considered a novel therapeutic. Elucidation of themolecular mechanism(s) that explain the actions of CO is still inits infancy. We and others have recently shown that CO increasesthe generation of reactive oxygen species (ROS), includingsuperoxide and hydrogen peroxide, via its interaction withmitochondrial oxidases (7) that initiate immediate signal trans-duction and activates oxidant stress response factors (8, 9).

One of the key cellular targets of endogenous ROS is the stressresponse transcription factor, hypoxia-inducible factor 1� (HIF-1�), which regulates numerous genes involved in angiogenesis,metabolism, and cell survival (10, 11). HIF-1� is tightly regulatedby two hemoproteins, prolyl 4-hydroxylase (PHD) (12, 13) andasparaginyl-hydroxylase factor inhibiting-HIF-1�, both of whichfunction as cellular O2 sensors (14, 15). In the presence of hypoxiaor elevated levels of ROS, PHD becomes hydroxylated, resulting inthe activation and stabilization of HIF-1� (16, 17), which furtherregulates expression of stress response genes (18–20). This plasticityallows HIF-1� to behave as either protective or detrimental duringinflammation, which continues to evoke discussion with strong

evidence implicating HIF-1� as an important mediator in theimmune response (19, 20). This concept is supported by the fact thatmacrophages (m�) lacking HIF-1� show impaired bacterial killingand persistent inflammation (19). Additionally, expression of aconstitutively active HIF-1� hybrid can protect cardiomyocytesagainst IRI (21).

One of the key genes regulated by ROS and HIF-1� (22, 23) isthe pleiotropic cytokine, TGF-�. TGF-� belongs to a family ofproteins that regulate cellular proliferation, differentiation, andmatrix remodeling (24). Exogenous administration of TGF-� at-tenuates myocardial necrosis (25), enhances arterial oxygenationafter lung transplantation (26), and promotes the restoration ofepithelial monolayers in regenerating tubules in postischemic kid-neys (27). Conversely, inhibition of TGF-� leads to growth dereg-ulation in disease states such as cancer, cirrhosis, and fibrosis (28,29). Some of the cytoprotective actions of TGF-� necessitate‘‘deactivation’’ of m�, i.e., suppressing excessive generation ofm�-derived ROS and subsequent cytokine release at the site ofinjury, so as to minimize further damage (31).

We hypothesized that CO exerts its effects in the cell rapidly, andtherefore we explored events that occurred early into CO exposurethat would explain subsequent protective effects. We demonstratethat CO rapidly stabilizes HIF-1�, leading to up-regulation ofTGF-�, which are necessary events to rescue m� from cell deathand sustain tissue homeostasis in a model of IRI in mice.

ResultsCO Induces HIF-1� Activation in m�. Exposure of m� [RAW 264.7and THP-1 (data not shown) to CO (250 ppm) and a CO-releasingmolecule (CORM) (80 �M)] resulted in rapid up-regulation ofHIF-1�, initially observed at 30 min with peak expression at 1–2 hby Western blot analysis (Fig. 1A). Mobility-shift assays showedDNA binding of HIF-1� after 15 min of exposure that wasmaintained for 30 min before returning to control levels (Fig. 1B).M� transiently transfected with pHIF-1�-luc and pTranslucent andexposed to CO gas or a CORM for 1 h induced HIF-1� promoteractivity 3- to 4-fold compared with vector alone showing thefunctional consequences of CO (P � 0.01; Fig. 1C). Additionally theeffects of CO were NO-independent, as blockade with N-nitro-L-arginine methyl ester had no effect. The relative role of ROS in theinduction of HIF-1� are made clear by the ability of peroxide toinduce HIF-1� and that the effects of CO on HIF-1� stabilization

Author contributions: B.Y.C., P.J.L., and L.E.O. designed research; B.Y.C., G.J., B.W., H.J.W.,T.M., X.C.Z., D.G., and E.C. performed research; H.J.W. contributed new reagents/analytictools; B.Y.C., F.H.B., and L.E.O. analyzed data; and B.Y.C. and L.E.O. wrote the paper.

Conflict of interest statement: L.E.O. is a paid consultant of Linde Healthcare.

This article is a PNAS direct submission.

Freely available online through the PNAS open access option.

Abbreviations: m�, macrophage; BMDM, bone marrow-derived m�; HO, heme oxygenase;HIF-1�, hypoxia-inducible factor 1�; ROS, reactive oxygen species; I/R, ischemia/reperfusion;IRI, I/R injury; CORM, CO-releasing molecule; A/R, anoxia/reoxygenation.

‡To whom correspondence should be addressed. E-mail: lotterbe@bidmc.harvard.edu.

© 2007 by The National Academy of Sciences of the USA

www.pnas.org�cgi�doi�10.1073�pnas.0609611104 PNAS � March 20, 2007 � vol. 104 � no. 12 � 5109–5114

MED

ICA

LSC

IEN

CES

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

17, 2

020

are abrogated in the presence of catalase and superoxide dismutase(Fig. 1C).

CO-induced HIF-1� expression also occurred in vivo. Wide-field3D-reconstructed fluorescent images showed immuno-positivestaining for HIF-1� in the nuclei (Fig. 1D) of isolated alveolar m�(F4/80�; data not shown) of C57BL/6 mice exposed to CO or airfor 2 h. A similar pattern was seen in vitro in RAW 264.7 m�exposed to CO (Fig. 2C).

CO Induces ROS Generation in m�. CO efficiently increases oxidativestress (31–33). M� preloaded with DCF-DA and exposed to CO

showed a transitory and significant burst of ROS after 5 min of COthat continued for 30–60 min (Fig. 2A Upper). The signal thenrapidly declined, suggesting that the ROS generation by CO istightly regulated. Mitochondria-deficient m� (�o) exposed to COproduced insignificant levels of ROS (Fig. 2A Lower) comparedwith WT cells, implicating the mitochondria as the source of ROS.Residual or nonmitochondrial ROS production may likely beattributed to other oxidant sources such as NAD(P)H oxidase.

Mitochondria-Derived ROS Mediate CO Induced HIF-1� Activity. PO2and ROS control HIF-1� stability (34). We show that it ismitochondria-derived ROS that mediate CO-induced HIF-1�expression because CO was unable to induce HIF-1� expressionat any time point in �o m� (Western blot analysis (Fig. 2B) ornuclear translocation (Fig. 2C).

CO Induces the Expression of TGF-�. In addition to activating HIF-1�,CO modulated the expression of TGF-�, a m�-derived cytokineregulated by HIF-1� that is essential for lung healing and remod-eling after injury. M� exposed to CO showed increased expressionof TGF-� with mRNA expression peaking after 1 h and returningto control levels over 24 h (Fig. 3A). Correlative protein expressionin the media showed significant increases by 24 h in CO- orCORM-treated cells, which was NO-independent (Fig. 3B; P �0.001). Immunofluorescent detection of TGF-� is seen in 3Dreconstructed sections of freshly isolated CO-treated mouse lungm� versus air-treated m� (Fig. 3C). Similar effects were observedin vitro in RAW cells exposed to CO (data not shown).

Induction of TGF-� by CO Requires HIF-1� and Is Independent of IL-10.There are HIF-1� consensus binding sites on the TGF-� promoter(35). To determine whether HIF-1� mediates CO-induced TGF-�expression, we generated a stable line of m� expressing HIF-1�-miRNA (LMP-HIF-1�-miRNA). These cells when exposed to COwere unable to express HIF-1� protein and up-regulate TGF-�mRNA expression (Fig. 4) when compared with LMP vector-treated control cells. These data show that HIF-1� is critical forCO-induced TGF-� expression in m�. The necessary role of

Fig. 1. Induction of HIF-1� expression and activity after administration of CO.(A) Kinetics of HIF-1� protein via Western blot analyses, in m� exposed to CO ora CORM. �-Tubulin and �-actin were used as loading controls. (B) Detection ofHIF-1� DNA binding by EMSA. (C) HIF-1� luciferase reporter activity in indicatedgroups. *, P � 0.01 vs. control; *#, P � 0.05 vs. CO. (D) Immunofluorescentdetection of cytoplasmic and nuclear HIF-1� (green punctate dots, denoted byarrows) in situ in 3D-reconstructed isolated lung m� (air control, Upper; panel-COexposed, Lower). Radiographs and luciferase data are representative of three tofive independent experiments. Images are representative of 10 fields of viewfrom air- and CO-exposed animals (n � 3 each). (Scale bar: 1 �m.)

Fig. 2. Role of mitochondria in CO-induced HIF-1� expression. (A) Mitochon-dria-deficient (�0) and WT m�s were characterized by their ability to generateROS by 2�,7�-dichlorodihydrofluorescein diacetate (DCF) fluorescence. (Upper)WT. (Lower) �0. Cells were preloaded with DCF � CO, and fluorescence wasdetermined by FACS (filled, 0 min; unfilled dashed line, 5 min; unfilled bold line,60 min). (B) Western blot showing kinetics of HIF-1� protein in �0 cells exposed toCO; �-tubulin is shown as a loading control. (C) Immunofluorescent staining forHIF-1� (red, denoted by arrows) in cultured WT m�s (left-air and right-CO; Upper)and �0 cells (left-air and right-CO; Lower). Images shown are representative of atleast 10 fields of view. Results shown represent one of three independent exper-iments. (Scale bar: 1 �m.)

Fig. 3. Induction of TGF-� expression after exogenous administration of CO.(A) Kinetics of TGF-� mRNA expression in mxz by PCR analyses. Results werenormalized to the �-actin (*, P � 0.001). (B) Secretion of TGF-� in response toCO gas (� N-nitro-L-arginine methyl ester) or CORM in m� (*, P � 0.002).Shown is the mean � SD of six wells from three independent experiments. (C)Immunofluorescent localization of TGF-� in mouse lung � CO. Wide-field, 3Dreconstruction of lung sections stained for TGF-� shows primarily localizationin alveolar m�s. Images are representative of 10 sections from three or fourmice per group. (Left) Air. (Right) CO. Blue are nuclei and red is intracellularTGF-� expression denoted by arrows. (Scale bar: 1 �m.)

5110 � www.pnas.org�cgi�doi�10.1073�pnas.0609611104 Chin et al.

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

17, 2

020

HIF-1� and TGF-� in CO-induced protection was also observed invivo (see Fig. 6). Exposure to CO alone does not induce IL-10expression (data not shown) and thus TGF-� expression is mostlikely regulated by HIF-1�.

CO Increases HIF-1� Activity and Promotes Survival of m� Exposed toAnoxia-Reoxygenation (A/R). HIF-1� activation markedly enhancescell survival in many models of hypoxia and cancer (19, 36–38).M�s exposed to A/R (8 h of anoxia followed by 16 h of reoxygen-ation) demonstrated a 5-fold increase in propidium iodide stainingcompared with untreated (Fig. 5A; P � 0.01) In contrast, m�sexposed to the same A/R regimen treated with CO only duringreoxygenation were protected from cell death (Fig. 5A; P � 0.01).Similar effects were observed when measuring caspase-3 activity asa marker of apoptosis. A/R induced a 6-fold increase in activity[5.8 � 0.3% in control to 33.6 � 10% (P � 0.009)], which wasabrogated in CO-treated cells to 4.8 � 0.3% (P � 0.01). CO-induced TGF-� is at least in part responsible for the cytoprotection.Conditioned media from RAW cells exposed to CO for 24 h andplaced onto cells exposed to A/R were completely protected fromapoptosis (Fig. 5A), similar to the effects observed with COtreatment during reoxygenation. CO induces secretion of TGF-�,which then mediates protection against A/R; neutralization ofTGF-� with an antibody partially but significantly abrogated theeffects observed with CO (Fig. 5A). Finally, addition of recombi-nant TGF-� to RAW cells in the absence of CO preventedAR-induced apoptosis, mimicking the CO effects (Fig. 5A). Theseresults show that TGF-� is a critical mediator in the protectiveeffects of CO in preventing A/R-induced cell death.

To further test the role of HIF-1� in the protection of CO against

A/R, we performed identical experiments as above using bonemarrow-derived m� (BMDM) obtained from WT and HIF-1�-loxp mice. After purification and differentiation, BMDM wereinfected with an Adeno-Cre recombinase (CVL) virus to knockdown HIF-1�. Efficiency of infection was �85%. Controls includedWT cells infected with Y5. All cells were then exposed to A/R �CO. Unlike animals with WT-infected cells, CO was unable toprotect animals with HIF-1�-deficient m� against A/R-induced celldeath (Fig. 5B). Alveolar m�s isolated from HIF-1�-loxp mice andtreated with the same regimen as described above yielded similarresults (data not shown). These data further support a role forHIF-1� in mediating CO-induced cytoprotection against A/R-induced cell death. Based on reports of CO modulating IL-10, andIL-10 regulating TGF-� expression, we evaluated a potential rolefor IL-10 in the A/R model. We observed an increase in TGF-�expression in response to A/R, which was augmented in thepresence of CO. These effects were independent of IL-10, asaddition of an IL-10 neutralizing antibody had no effect on CO-induced TGF-� expression (Fig. 5C).

CO Inhibits IRI in the Lung: The Role of HIF-1� and TGF-�. HIF-1� andTGF-� play vital roles in attenuating IRI (39–41). Based on thesereports, we tested whether the molecules identified in our in vitrofindings in m� were involved in vivo in a model of lung IRI whereCO has been shown to be an effective treatment (42, 43). Unlike theprevious reports that did not evaluate the effects of CO during thepretreatment period (42, 43), we show that CO exposure tononinjured mice resulted in increased expression of both HIF-1�and TGF-�, primarily seen in alveolar m�. We exposed mice to COalone before IRI to ascertain whether HIF-1� and TGF-� wereinvolved in preconditioning the lung to induce resistance to sub-sequent injury. CO exposure to uninjured mice resulted in in-creased expression of both HIF-1� and TGF-�, which was primarilylocalized to alveolar m� (Fig. 6A). We demonstrate that COattenuated I/R-induced cell death as measured by TUNEL staining(�50% over untreated animals) compared with I/R alone (Fig. 6B–G). Additionally, pretreatment with HIF-1�-siRNA intratrache-ally negated CO-induced protection, implicating CO-inducedHIF-1� in mediating lung cytoprotection. There were also signif-icantly more TGF-�-positive m�s [colocalization of m� marker(F4/80�; data not shown) and TGF-�] in lungs exposed to I/R inthe presence of CO (Fig. 6H) compared with I/R alone, supportinga protective role of TGF-� similar to the in vitro studies. FewerTGF-�-positive m�s were observed in CO-treated, IR-injuredanimals where HIF-1� was silenced with siRNA (Fig. 6H), con-firming the in vivo role of HIF-1� in regulating TGF-�. Silencing ofTGF-� in the lung with specific siRNA (TGF-� null mice areembryonic lethals) resulted in a similar abrogation of CO-inducedprotection (Fig. 6I), validating in vivo the requirement for TGF-�induction by CO.

DiscussionProtection by exogenous CO in lung IRI (43, 44) involves modu-lation of p38 and ERK1/2 MAPK as well as downstream activation

Fig. 4. Induction of TGF-� expression by CO is HIF-1�-dependent. A stableHIF-1�-miRNA m� cell line was generated to study the relationship be-tween HIF-1� activation and TGF-� expression. (A) CO is unable to induceHIF-1� in HIF-1�-miRNA-infected m� (open bars) compared with LMP vectorcontrol (filled bars) as shown by Western blot (Inset) (2 h) and PCR (0–4 h)analyses. (B) The ability of a HIF-1�-miRNA m� cell line (filled bars) to expressTGF-� in response to CO was compared with control and LMP vector controlcells (empty bars) by Western blot. �-Actin was used as a loading control.Results shown mean � SD from three independent experiments of n � 3 perexperiment (*, P � 0.001 vs. LMP-miRNA control).

Fig. 5. Role of HIF-1� in CO-induced inhi-bition of apoptosis. (A) M�s were exposed toa regimen of A/R to induce cell death (asdescribed in Methods) in the presence of CO,conditioned media from cells exposed to COfor 24 h (CM), recombinant TGF-�, and acombination of CO plus neutralizing TGF-�(�TGF-�). Apoptosis was assessed by FACSanalyses of propidium iodide. Results repre-sent mean � SD of four independent exper-iments. (B) A/R-induced apoptosis in m�sdeficient inHIF-1�,Ad-CVL(BMDMfromHIF-1�-Loxp mice � Ad-Cre), and vector control Ad-Y5 � CO. Results represent mean � SD from four independent experiments. (C) CO augments A/R-induced TGF-�expression, which is independent of IL-10. RAW 264.7 cells were pretreated with a neutralizing antibody to IL-10 (�IL-10) and then exposed to A/R � CO as describedabove. Results are mean � SD of four to six wells from three independent experiments *, P � 0.02 vs. Untx; *#, P � 0.001 vs. Air�A/R.

Chin et al. PNAS � March 20, 2007 � vol. 104 � no. 12 � 5111

MED

ICA

LSC

IEN

CES

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

17, 2

020

of EGR-1, caspases, and PAI-1 (34, 43, 44). In addition, exogenousCO (15 ppm) prevented endothelial cell apoptosis via p38/STAT3-mediated inhibition of Fas and caspase-3 (42). CO (1,000 ppm)preconditioning for 16–24 h before lung transplantation suppressedERK1/2-mediated proinflammatory and prothrombotic factor se-cretion (43). In all of these cases, the effects of CO were in thepresence of a stressor. In this study, we elucidate the sequence ofevents that occur earlier during preconditioning with CO anddefine the specific downstream signaling and gene regulation thatresult in the observed inhibition of subsequent IRI.

We document one of the earliest events observed in cells andtissues exposed to a low concentration of CO, i.e., transitoryproduction of ROS within 5–10 min of exposure. More importantly,that the CO-derived ROS that have also been shown to increaseperoxisome proliferator-activated receptor � (PPAR�) (31), rap-idly stabilize HIF-1� within minutes, leading to regulated TGF-�expression that subsequently protects m� and lung tissue againstA/R injury and IRI. M�s are a major target of CO in this modeland show the highest expression of HIF-1� and TGF-�. CO rapidlyconditions the m� through HIF-1� and secretion of TGF-� into a

‘‘prosurvival’’ mode, preventing tissue damage ordinarily elicited byIRI. PPAR� is increased after 2–3 h of CO, in stark contrast toCO-induced HIF-1� activation, which is observed as early as 15–30min postexposure. Ongoing studies are evaluating potential PPAR�regulation by HIF-1�. If no link is found, these two molecules mayfunction in different signaling cascades, each mediating its owneffects.

We and others (1, 2, 5, 31, 42, 43) have shown that administeringlow concentrations of CO (15–500 ppm) has profound cytoprotec-tive effects in vivo in rodents and swine and in vitro, modulatingvasodilatation, inflammation, apoptosis, and immune tolerance.These concentrations are well tolerated with no adverse effects onthe animals noted. These effects are mediated by activation ofMAPKs (42, 43), peroxisome proliferator-activated receptor � (31),and STAT (41), as well as the heme-containing molecules solubleguanylate cyclase (44) and the mitochondrial oxidases (7, 31, 45).The latter conclusion is most strongly supported by our demon-stration that CO is ineffective when using a mitochondria-deficientm� line (�0). Recent reports suggest that increases in ROS arisingfrom mitochondria (9, 16) and/or from NAD(P)H oxidase (45) also

Fig. 6. CO-dependent HIF-1� and TGF-� expression are nec-essary to prevent lung IRI. (A) C57BL/6 mice were exposed toCO alone (250 ppm) for 2 and 24 h (n � 4 per group). Lungswere harvested and stained for HIF-1� (2-h exposure) or TGF-�(24-h exposure). Images are representative of six to eightsections per lung. IgG isotype control is shown. (B–G) Silencingof HIF-1� abrogates CO-induced protection against lung IRI.HIF-1�-siRNA or saline were administered to mice intratrache-ally as described in Methods. CO was administered 1 h beforeIRI. Lung injury was assessed 48 h after reperfusion by TUNELstaining. TUNEL-positive cells can be seen in C–F. (B) Air/HIF-1�-siRNA. (C) Air/I/R. (D) CO/I/R. (E) Air/I/R/HIF-1�-siRNA. (F)CO/I/R/HIF-1�-siRNA. Note that CO-treated animals (D) show amarked reduction in TUNEL-positive cells compared withthose in C, E, and F. Images shown are representative of 8–10sections from n � 3–5 animals per group. (G) Histogram rep-resentation of B–F. Results are mean � SD of four to six miceper group (*, P � 0.01 vs. CO). (H) TGF-� expression fromIRI-injured lungs. Air�I/R-injured mice (Upper Left), CO�I/R-injured mice (Lower Left), air�HIF-1�-siRNA�I/R-injured mice(Upper Right) and CO�HIF-1�-siRNA�I/R-injured mice (LowerRight). Note that the majority of positive staining is localizedto m� (arrows) and a greater number of TGF-�-positive m� ispresent in lungs exposed to CO�I/R (Lower Left) when com-pared with that of CO�HIF-1�-siRNA�I/R-injured mice (LowerRight). Images are representative of 8–10 sections from threeto five animals per group. (I) TGF-� expression by CO is re-quired for protection. CO was unable to protect against IRI inmice treated with TGF-�-siRNA as described in H. Results aremean � SD of four to six mice per group (*, P � 0.002 vs. CO).(Scale bar: 40 �m.)

5112 � www.pnas.org�cgi�doi�10.1073�pnas.0609611104 Chin et al.

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

17, 2

020

stabilize HIF-1� (46) to regulate downstream gene expression.Generation of endogenous ROS, especially hydrogen peroxide, hasbeen postulated as a means of ‘‘cellular communication’’ (47),which is supported in Fig. 1. Activation of HIF-1� by ROS appearsto act upstream of prolyl hydroxylases (48) in the mitochondria atthe Rieske Fe-S clusters on complex III (49). Interfering with theseclusters may be one possible target for CO because of the affinityof CO for iron. Another possibility to explain the effects of CO onHIF-1� is that low concentrations of NO promote HIF-1� stability(50), perhaps by binding to the prolyl hydroxylases and interferingwith O2 sensing. However, NO in this model, does not play a keyrole in mediating CO-induced HIF-1� activation.

The potential relevance of the effects of CO as studied here tothat generated endogenously by heme oxygenase (HO)-1 wasrecently supported by D’Amico et al. (45) where comparisons weremade between exogenous CO at concentrations similar to onesused here with those generated endogenously by HO. They foundremarkable similarities in the effects on cellular respiration. Similarto our in vitro findings, exogenous CO disrupted mitochondrialrespiration that led to increased ROS, an effect that was magnifiedwith decreases in cellular PO2. In our study CO was administeredunder normoxic conditions, which may then account for thetransitory nature of the ROS burst. The transient effects on ROSgeneration might also be explained by an increase in the activity ofantioxidant enzymes such as MnSOD and glutathione, which areactivated in response to CO (unpublished observations).

IRI is associated with cell death from oxygen deprivation anduncontrolled release of ROS and inflammatory cytokines duringreperfusion. Elevated HIF-1� and TGF-� expression imparts anti-apoptotic, ant-fibrotic, and antiinflammatory properties in heartand kidney models of IRI (51–56). We and others have shown thatTGF-� can function to promote activation of prosurvival genes suchas HO-1, acting to block inflammation (29, 51, 55). The ability toup-regulate HIF-1� and TGF-� before the onset of IRI wouldameliorate the undesirable consequences of IRI. In this study, wehave elucidated a sequence of events that occurs in response to COresulting in a rapid phenotypic switch toward a protective pheno-type that prevents lung IRI. These studies support the concept thatHIF-1� and TGF-� function as protective molecules in preparingthe cells for imminent injury.

Elucidation of the signaling pathways involved in the protectiveeffects of CO is important for both our basic understanding of themechanism of action of CO and the creation of new therapeuticapproaches to IRI. Activation of HIF-1� and TGF-� to imparttherapeutic effects in preventing injury sustained during IRI wouldbe important clinically during surgical procedures, organ transplan-tation, balloon angioplasty, or hemorrhagic shock. The fact thatwithin 15 min of exposure CO markedly induces HIF-1� activation,preceding all reported signaling events to date, positions HIF-1� asa central regulator in conditioning of the m�. We observed that COdid not reduce the influx of m� to the site of injury, but ratherreprogrammed their state of activation toward one of protectionversus aggression. Harnessing the immune system is in part how COand HO-1 act to maintain homeostasis. Taken together, these dataprovide critical information related to the biology of CO and themechanisms of its action. Clinical testing has begun evaluating theefficacy of CO in organ transplantation to prevent the sequelae ofIRI that lead to chronic rejection.

Materials and MethodsCell Culture. Murine RAW 264.7 m�, human THP-1 m�, andHEK293 cells from ATCC (Rockville, MD) were maintained inDMEM supplemented with 10% FBS and penicillin/streptomycin(Invitrogen, Carlsbad, CA). Primary bone marrow-derived m�were harvested as described (31) from myelogenic specific HIF-1�Double Floxed (loxP) B.129/C57BL/6 mice (generous gift fromRandall Johnson, University of California at San Diego, La Jolla,CA). RAW and THP-1 mitochondria-deficient m� (�o cells) were

generated and maintained as described (17). Confirmation ofmitochondria deficiency was assessed by the absence of cytochromeoxidase-2 transcript by PCR (R & D Systems, Minneapolis, MN).

Adenoviral Infections of BMDM. Peripheral blood mononuclear cellswere differentiated into m� and infected with recombinant ade-novirus for Cre recombinase or control virus (Y5) at a concentra-tion of 50 multiplicity of infection per cell. Infections were per-formed as described (31). Experiments were performed on day 5.Cre activity in knocking down HIF-1� loxP BMDM was assessed byPCR (Qiagen, Valencia, CA).

Reagents. Antibodies for HIF-1�, TGF-�, and �-actin were pur-chased from Novus Biochemicals (Littleton, CO), R&D Systems,and Cell Signaling Technology (Beverly, MA), respectively, andvisualized with HRP-conjugated anti-mouse antibodies (Cell Sig-naling Technology). DAPI was from Molecular Probes, Eugene,OR. CORM was purchased from Sigma (St. Louis, MO). Recom-binant human and neutralizing TGF-� antibodies were from R &D Systems. HIF-1�-siRNA and TGF-�-siRNA were purchasedfrom Dharmacon (Lafayette, CO). RT-PCR and RNAeasy kitswere from Ambion (Austin, TX) and Qiagen, respectively. HIF-1�oligonucleotides with consensus sequence, 5�-TCT GTA CGTGAC CAC ACT CAC CTC–3�, was from Santa Cruz Biotechnol-ogy (Santa Cruz, CA).

Transfections and Transient Reporter Assays. THP-1 m�s weretransfected with 1 �g/ml pHIF-1�-luc and pTranslucent responsiveplasmids (Panomics, Freemont, CA) using FuGene 6 (RocheDiagnostics, Alameda, CA) according to the manufacturer’s direc-tion. After 24–36 h, cells were washed with serum-free media andexposed to CO or CORM for 0–2 h, and luciferase activity(Promega, Madison, WI) was assessed as described (31). Lumines-cence was normalized to total protein levels and presented as foldinduction against control pTranslucent reporter activity.

HIF-1� and TGF-� Silencing. THP-1 m�s were transfected withHIF-1� or TGF-� siRNA-SMARTpool reagent (Dharmacon) withFuGene (Roche Diagnostics). Four siRNA sequences were tested,and one was chosen for further experimentation based on its abilityto block CO and hypoxia-induced HIF-1� and or TGF-� expressionby Western blot.

Stable Transfection with mir-HIF-1�-shRNA. shRNAmir (microRNA-adapted shRNA) against mouse HIF-1� was generated in pSM2vector (Open Biosystems, Huntsville, AL). shRNAmirHIF-1� wassubcloned into MSCV-LTRmiR30-PIG (LMP) vector (Open Bio-systems) with XhoI and EcoRI (Invitrogen) restriction enzymes.Confirmation was verified by restriction site analysis and sequenc-ing. For production of retrovirus, HEK293 cells were transfectedwith shRNAmirHIF-1�LMP, VSVG, and Gag-Pol plasmids atratio 2:1:1 using Lipofectamine 2000 (Invitrogen). After 12 h,medium containing the virus was centrifuged at 2,000 � g for 10 minat 4°C, and the supernatant was filtered by using 0.45-�m filters(Millipore, Phoenix, AZ). After 14-h incubation with viruses, RAWcells were selected with 5 �g/ml of puromycin (Sigma) for 2 weeksand the expression of GFP and knockdown was tested by Westernblot.

CO Exposure. Cells were exposed to CO as described (30).

FACS Analyses of 2�,7�-Dichlorodihydrofluorescein Diacetate and Pro-pidium Iodide Staining as Markers of ROS Production and Cell Death.RAW 264.7 and THP-1 m�s were incubated with 0.05 mMDCFH-DA (Invitrogen) for 30 min at 37°C before the harvest timepoint. The cells were then washed, exposed to air or CO for 5–60min, and resuspended in FACS buffer (PBS � 1% FBS), and theintensity of fluorescence was measured as described (33). Cell cycle

Chin et al. PNAS � March 20, 2007 � vol. 104 � no. 12 � 5113

MED

ICA

LSC

IEN

CES

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

17, 2

020

and DNA content as markers of cell death were detected withpropidium iodide and the data were analyzed with CellQuestsoftware (BD, San Diego, CA).

Western Blot Analysis. Cellular protein extracts, concentrations, andthe methodology used for Western analyses were obtained asdescribed (31). The membranes were incubated with HIF-1� andTGF-� antibodies followed by incubation with HRP-conjugatedanti-mouse and anti-rabbit secondary antibodies, respectively. Sig-nal development was carried out with an enhanced chemilumines-cence detection kit (Pierce, Rockford, IL).

Immunofluorescence Staining. Cells were grown on Met-Tek slides(Met-Tek, Ashland, MA), exposed as described, and fixed inice-cold methanol/acetic acid (75%: 25%). Cells were then perme-abilized with 0.1% Nonidet P-40 (Sigma) and incubated withnonimmune IgG. Expression of HIF-1�, and TGF-� were detectedwith antibodies as described and visualized via conjugation toAlexa-488 and Alexa-592 antibodies (Molecular Probes). Nucleiwere stained and visualized with DAPI. Micrographs were ob-tained with an Axiovert 200M Apotome wide-field microscope(Zeiss, Thornwood, NY) and Axiovision software. Lungs from micetreated as described were immersed in either zinc or mercuryfixative (BD Pharmingen), sectioned in 5-�m slices, and stained forHIF-1� and TGF-�. Primary antibodies were visualized by usingHRP-conjugated anti-mouse antibody, and images were capturedby using the Zeiss light microscope.

EMSA. Cultured m�s were exposed to CO for 0–2 h after which thecells were lysed, nuclear protein was extracted, and electrophoresiswas performed as described (31).

SemiQuantitative RT-PCR. Primers for HIF-1� and TGF-� werepurchased from Santa Cruz Biotechnology and R & D Systems,respectively. Total RNA was extracted and reverse-transcribed per

the manufacturer’s instructions (Ambion) on an iCycler (Bio-Rad,Carlsbad, CA) with the following conditions: start at 94°C for 4 minfollowed by 30–35 cycles of amplification (94°C for 45 s, 55°C for45 s, and 72°C for 45 s), with a final extension of 72°C for 10 min.Amplified PCR products were fractionated with a 1% agarose gel.

Real-Time PCR. Total RNA (1 �g) was reverse-transcribed intocDNA by Moloney murine leukemia virus enzyme (Promega,Mannheim, Germany) with random hexamers (1 �g/�g total RNA.All PCRs were performed with the SYBR Green kit (BioRad,Hercules, CA). Primers were purchased from Biosource Interna-tional, Camarillo, CA. A Mx3000P QPCR System (Stratagene, LaJolla, CA) was used with the following cycling conditions: initialdenaturation at 95°C for 10 min, followed by 40 cycles at 94°C for30 s, 58°C for 15 s, and 72°C for 30 s and a 10-min terminalincubation at 72°C. Expression of target genes was normalized to�-actin or tubulin expression levels.

In Vivo siRNA Administration and Murine Lung IRI. Normal C57BL/6male mice at 6–8 weeks weighing 20–25 g (Jackson Laboratory, BarHarbor, ME) were anesthetized with methoxyfluorane (Sigma).Animal protocols were approved by the Beth Israel DeaconessMedical Center and Yale University institutional animal care anduse committees. After anesthetization 3 nmol/20 g in a volume of50 �l of HIF-1� or TGF-� siRNA (Dharmacon) was administeredintranasally. This protocol was repeated twice at 48 and 24 h beforeI/R. Lung IRI was induced as described (42). Lungs were extractedfor TUNEL analyses 24–48 h post-IRI. Quantitation of TUNEL-positive cells was performed by counting the number of positivecells in 8–10 random fields of view per section.

We thank Martin Bilban for assisting with the silencing technology and theJulie Henry Fund at the Transplant Center of the Beth Israel DeaconessMedical Center for their support. This work was supported by NationalInstitutes of Health Grants HL-071797 and HL-076167 (to L.E.O.).

1. Mazzola S, Forni M, Albertini M, Bacci ML, Zannoni A, Gentilini F, Lavitrano M, BachFH, Otterbein LE, Clement MG (2005) FASEB J 14:2045–2047.

2. Moore BA, Overhaus M, Whitcomb J, Ifedigbo E, Choi AM, Otterbein LE, Bauer AJ (2005)Crit Care Med 6:1317–1326.

3. Neto JS, Nakao A, Kimizuka K, Romanosky AJ, Stolz DB, Uchiyama T, Nalesnik MA,Otterbein LE, Murase N (2004) Am J Physiol 287:F979–F989.

4. Ameredes BT, Otterbein LE, Kohut LK, Gligonic AL, Calhoun WJ, Choi AM (2003) Am JPhysiol 285:L1270–L1276.

5. Zuckerbraun BS, Otterbein LE, Boyle P, Jaffe R, Upperman J, Zamora R, Ford HR (2005)Am J Physiol 289:G607–G613.

6. Nakao A, Neto JS, Kanno S, Stolz DB, Kimizuka K, Liu F, Bach FH, Billiar TR, Choi AM,Otterbein LE, Murase N (2005) Am J Transplant 5:282–291.

7. Taille, C. El-Benna J, S, Boczkowski MJ, Motterlini R (2005) J Biol Chem 280:25350–25360.8. Emerling BM, Platanias LC, Black E, Nebreda AR, Davis RJ, Chandel NS (2005) Mol Cell

Biol 25:4853–4862.9. Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, Schumacker PT (1998)

Proc Natl Acad Sci USA 95:11715–11720.10. Bacon AL, Harris AL (2004) Ann Med 36:530–539.11. Stoeltzing O, McCarty MF, Wey JS, Fan F, Liu W, Belcheva A, Bucana CD, Semenza GL,

Ellis LM (2004) J Natl Cancer Inst 96:946–956.12. Bruick RK, McKnight SL (2001) Science 294:1337–1340.13. Yu F, White SB, Zhao Q, Lee FS (2001) Proc Natl Acad Sci USA 98:9630–9635.14. Hewitson KS, McNeill LA, Riordan MV, Tian YM, Bullock AN, Welford RWD, Elkins JM,

Oldham NJ, Battacharya S, Gleadle J (2002) J Biol Chem 277:26351–26355.15. Lando D, Peet DJ, Gorman JJ, Whelan DA, Whitelaw ML, Bruick RK (2002) Genes Dev

16:1466–1471.16. Schroedl C, McClintock DS, Budinger GR, Chandel NS (2002) Am J Physiol 283:L922–L931.17. Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, Schumacker PT (1998)

Proc Natl Acad Sci USA 95:11715–11720.18. Albina JE, Mastrofrancesco B, Vessella JA, Louis CA, Henry WL, Jr, Reichner JS (2001)

Am J Physiol 281:C1971–C1977.19. Cramer T, Yamanishi Y, Clausen BE, Forster I, Pawlinski R, Mackman N, Haase VH,

Jaenisch R, Corr M, Nizet V, et al. (2003) Cell 112:645–657.20. Blouin CC, Page EL, Soucy GM, Richard DE (2004) Blood 103:1124–1130.21. Date T, Mochizuki S, Belanger AJ, Yamakawa M, Luo Z, Vincent KA, Cheng SH, Gregory

RJ, Jiang C (2005) Am J Physiol 288:C314–C320.22. Zarember KA, Malech HL (2005) J Clin Invest 115:1702–1704.23. Hong YH, Peng HB, La Fata V, Liao JK (1997) J Immunol 159:2418–2423.24. Massague J, Blain SW, Lo RS (2000) Cell 103:295–309.25. Chen H, Li D, Saldeen T, Mehta JL (2003) Am J Physiol 284:H1612–H1617.26. Daddi N, Kanaan SA, Suda T, Tagawa, T, D’Ovidio F, Grapperhaus K, Kozower BD, Ritter

JH, Mohanakumar T, Patterson GA (2003) J Heart Lung Transplant 22:1323–1334.27. Docherty NG, Perez-Barriocanal F, Balboa NE, Lopez-Novoa JM (2002) Ren Fail 24:391–406.

28. Fogar P, Pasquali C, Basso D, Floreani A, Piva MG, De Paoli M, Melis A, Sperti C,Pedrazzoli S, Plebani M (1998) J Med 29:277–287.

29. Biswas S, Criswell TL, Wang SE, Arteaga CL (2006) Clin Cancer Res 12:4142–4146.30. Tsunawaki S, Sporn M, Ding A, Nathan C (1988) Nature 334:260–262.31. Bilban M, Bach FH, Otterbein SL, Ifedigbo E, de Costa d’Avila, J., Esterbauer H, Chin BY,

Usheva A, Robson SC, Wagner O, Otterbein LE (2006) Immunity 24:601–610.32. Chin BY, Trush MA, Choi AM, Risby TH (2003) Am J Physiol 284:L473–L480.33. Piantadosi CA, Carraway MS, Suliman HB (2006) Free Radical Biol Med 40:1332–1339.34. Jiang BH, Zheng JZ, Leung SW, Roe R, Semenza GL (1997) J Biol Chem 272:19253–19260.35. Sanchez-Elsner T, Ramirez JR, Sanz-Rodriguez F, Varela E, Bernabeu C, Botella LM

(2004) J Mol Biol 336:9–24.36. Li J, Shi M, Cao Y, Yuan W, Pang T, Li B, Sun Z, Chen L, Zhao RC (2006) Biochem Biophys

Res Commun 342:1341–1351.37. Suzuki A, Kusakai G, Shimojo Y, Ogura T, Kobayashi M, Esumi H (2005) J Biol Chem

280:31557–31563.38. Cai Z, Manalo DJ, Wei G, Rodriguez ER, Fox-Talbot K, Lu H, Zweier JL, Semenza GL

(2003) Circulation 108:79–85.39. Natarajan R, Salloum FN, Fisher BJ, Kukreja RC, Fowler AA (2006) Circ Res 98:133–140.40. Gross CE, Bednar MM, Howard DB, Sporn MB (1993) Stroke 24:558–562.41. Lefer AM, Ma XL, Weyrich AS, Scalia R (1993) Proc Natl Acad Sci USA 90:1018–10122.42. Zhang X, Shan P, Alam J, Fu XY, Lee PJ (2005) J Biol Chem 280:8714–8721.43. Mishra S, Fujita T, Lama VN, Nam D, Liao H, Okada M, Minamoto K, Yoshikawa Y,

Harada H, Pinsky DJ (2006) Proc Natl Acad Sci USA 103:5191–5196.44. Verma A, Hirsch DJ, Glatt CE, Ronnett GV, Snyder SH (1993) Science 259:381–384.45. D’Amico G, Lam F, Hagen T, Moncada S (2006) J Cell Sci 119:2291–2298.46. Ateghang B, Wartenberg M, Gassman M, Sauer H (2006) J Cell Sci 119:1043–1052.47. Droge W (2002). Physiol Rev 82:47–9548. Brunelle JK, Bell EL, Quesada NM, Vercauteren K, Tiranti V, Zeviani M, Scarpulla RC,

Chandel NS (2005) Cell Metab 1:409–414.49. Guzy RD, Hoyos B, Robin E, Chen H, Liu L, Mansfield KD, Simon MC, Hammerling U,

Schumacker PT (2005) Cell Metab 1:401–408.50. Hagen T, Taylor CT, Lam F, Moncada S (2003) Science 302:1975–1978.51. Chin BY, Petrache I, Choi AM, Choi ME (1999) J Biol Chem 274:11362–11368.52. Bergeron M, Gidday JM, Yu AY, Smenza GL, Ferriero DM, Sharp FR (2000) Ann Neurol

48: 285–296.53. Zhang B, Tanaka J, Yang L, Sakanaka M, Hata R, Maeda N, Mitsuda N (2004) Neuroscience

126:433–440.54. Yang BC, Zander DS, Mehta JL (1999) J Pharmacol Exp Ther 291:733–738.55. Kuruvilla AP, Shah R, Hochwald GM, Liggit HD, Palladino MA, Thorbecke GJ (1991) Proc

Natl Acad Sci USA 88:2918–2921.56. Horowitz JC, Lee DY, Waghray M, Keshamouni VG, Thomas PE, Zhang H, Cui Z,

Thannickal VJ (2004) J Biol Chem 279:1359–1367.

5114 � www.pnas.org�cgi�doi�10.1073�pnas.0609611104 Chin et al.

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

17, 2

020