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Research Report
Peroxisome proliferator activated receptor (PPAR)-γco-activator 1-α and hypoxia induced factor-1α mediateneuro- and vascular protection by hypoxic preconditioningin vitro
Jia Zhaoa, Ling Lib,⁎, Zhong Peib, Chaoying Lib, Huan Weic, Bo Zhangd, Ying Penga,Ying Wangb, Yuqian Taob, Ruxun Huangb
aDepartment of Neurology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, No. 107 Yanjiangxi Road, Guangzhou, 510120, PR ChinabDepartment of Neurology, First Affiliated Hospital, Sun Yat-Sen University, No. 58 Zhongshan Road 2, Guangzhou, 510080, PR ChinacDepartment of Neurology, Kunming Yan-an Hospital, Kunming, No. 64 Renmindong Road, Kunming, 650100, PR ChinadDepartment of Neurology, The Second Affiliated Hospital, Guangzhou Medical University, No. 250 Changgangdong Road, Guangzhou,510260, PR China
A R T I C L E I N F O
⁎ Corresponding author. Fax: +86 20 87606221E-mail addresses: [email protected]
[email protected] (C. Li), weihuan2010@[email protected] (Y. Wang), yuqiantao@yah
Abbreviations: PGC-1α, Peroxisome Prolifervascular endothelial growth factor; ROS, reac
0006-8993/$ – see front matter © 2012 Elseviedoi:10.1016/j.brainres.2012.01.059
A B S T R A C T
Article history:Accepted 24 January 2012Available online 1 February 2012
Preconditioning-induced cellular adaptation is a new therapeutic strategy for ischemicstroke. This research aims to examine the role of peroxisome proliferator activatedreceptor (PPAR)-γ co-activator 1-α (PGC-1α) and hypoxia induced factor-1α (HIF-1α) in hyp-oxic preconditioning-induced protection. In this study, rat artery endothelial cells and neu-ronal PC12 cells were preconditioned with hypoxia before oxygen–glucose deprivation(OGD) insult. Cell viability, protein expression and oxidative stress were then evaluated.PGC-1α and HIF-1α were knocked down by RNA interference. We found that hypoxic pre-conditioning significantly reduced cell damage, enhanced the expression of PGC-1α,HIF-1α and VEGF and attenuated oxidative stress in endothelial and PC12 cells in OGDmodel. The protective effects of hypoxic preconditioning were hardly detected in HIF-1αor PGC-1α deficit cells. The loss of protection was accompanied with a significant loss ofVEGF expression in HIF-1α or PGC-1α deficit PC12 cells and PGC-1α deficit endothelialcells as well as a considerable decrease of anti-oxidative effects in PGC-1α knocked-downendothelial cells. The present study demonstrated that both PGC-1α and HIF-1α playedcrucial roles in hypoxic preconditioning in endothelial and neuronal cells.
© 2012 Elsevier B.V. All rights reserved.
Keywords:Hypoxic preconditioningIschemic strokeNeuronal cellEndothelial cellReactive oxygen speciesVascular endothelial growth factor
.(J. Zhao), [email protected] (L. Li), [email protected] (Z. Pei),oo.cn (H. Wei), [email protected] (B. Zhang), [email protected] (Y. Peng),oo.com.cn (Y. Tao), [email protected] (R. Huang).ator Activated Receptor (PPAR)-γ Co-activator 1-α; HIF-1α, Hypoxia induced factor-1α; VEGF,tive oxygen species; PC12, rat pheochromocytoma; OGD, Oxygen–glucose deprivation
r B.V. All rights reserved.
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1. Introduction
Stroke is a major cause of disability and mortality worldwide(Dirnagl et al., 2009). Thrombolysis is the only approved ther-apy available for acute ischemic stroke. However, the shorttherapeutic window limits its utility (Deb et al., 2010). There-fore, attention has turned to the endogenous strategies bywhich the brain can protect itself against ischemic damage.It has shown that a brief exposure to sublethal stresses suchas mild hypoxia can result in transient but strong resistanceto subsequent lethal ischemic insults (Gidday, 2006). Thetolerance of the brain activated by hypoxia usually refers to“hypoxic preconditioning” (Sharp et al., 2004). Hypoxic pre-conditioning provides the experimental paradigm that ad-dresses the heterogeneity and complexity inherent in strokepathophysiology (Baker, 2004; Pignataro et al., 2009).
Hypoxic preconditioning is presumed to activate a funda-mental genomic program presenting endogenous cytoprotec-tion and survival (Stenzel-Poore et al., 2007). The involvementof complex cellular signaling cascades has been well docu-mented in experimental models of hypoxic preconditioning(Zhang et al., 2004). However, the precise mechanism hasnot been fully understood. Although it has been acceptedthat transcription was mainly regulated by hypoxia inducedfactor-1α (HIF-1α) during hypoxia (Huang and Bunn, 2003),the involvement of non-HIF-1 dependent processes in thehypoxic stress response has also been demonstrated(Baranova et al., 2007; Bernaudin et al., 2002).
Recent studies have reported that peroxisome proliferatoractivated receptor (PPAR)-γ co-activator 1-α (PGC-1α) is an im-portant regulator of preconditioning (Gutsaeva et al.,2008). PGC-1α is a transcriptional co-activator enriched inmitochondria-rich tissues with high oxidative capacity andserves critical roles in the regulation of cellular energy metab-olism through specific interaction with certain transcriptionfactors (Finck and Kelly, 2006). Several studies have demon-strated that PGC-1α can activate gene regulatory programs tomeet the energy demands of a changing environment (Aranyet al., 2008; Uldry et al., 2006). Furthermore, PGC-1α has beenshown to act as a potent modulator of oxidative metabolismduring ischemia (Arany et al., 2008).
Currently, most investigators in the field of ischemicstroke primarily focus on neurons to study the neuroprotec-tion. In contrast, very few studies investigate themechanismsof vascular protection. A number of clinical trials on neuro-protectants have failed, implying that treatments focusingon neurons alonemay not be sufficient. Indeed, when the sur-vival of neurons is threatened during cerebral ischemia, so isendothelial cells (Hertz, 2008). More importantly, the cerebralendothelium dysfunction may worsen overall ischemicbrain damage, leading to aggravation of clinical outcomes(Carbonell and Rama, 2007).
In this study, we investigated the role of HIF-1α andPGC-1α in hypoxic preconditioning in cultured endothelialcells and neuronal PC12 cells. RNA interference was usedto further explore whether HIF-1α and PGC-1α can influencethe cellular vascular endothelial growth factor (VEGF)expression and reactive oxygen species (ROS) scavengingsystem.
2. Results
2.1. Hypoxic preconditioning attenuated OGD-induced celldeath and protein oxidization
As shown in Fig. 1, OGD significantly decreased cell viability inboth PC12 and endothelial cells in a time-dependent manner(Fig. 1A). In PC12 cells, the cell death rates were 62.31% and77.61% in OGD group and preconditioning plus OGD group,respectively. Preconditioning by exposing PC12 cells tohypoxia for 30 min significantly attenuated OGD 2 h plus24 h reperfusion-induced cell death. In endothelial cells, thedeath rates were 61.21% and 77.48% in OGD group and precon-ditioning plus OGD group, respectively. Preconditioning byexposing endothelial cells to hypoxia could significantlyreduce OGD 10 h plus 24 h reperfusion-induced endothelialcell damage.
Oxyblot showed that OGD significantly induced proteinoxidization in both neuronal and endothelial cells whereashypoxic preconditioning effectively attenuated OGD-induceexcessive protein oxidization (Figs. 2A–C). Similarly, OGDcaused excessive ROS generation from mitochondria as evi-denced by MitoSOXwhereas hypoxic preconditioning stronglyinhibited OGD-induced ROS production (Fig. 2D).
2.2. Hypoxic preconditioning increased protein levels ofHIF-1α, PGC-1α and VEGF in PC12 and endothelial cells
In endothelial cells, the level of HIF-1α increased to 160.70%and 177.12% of control at 0 and 48 h following 30 min hypoxicpreconditioning (all P<0.01. n=3, One-way ANOVA). Similarly,the level of PGC-1α increased to 126.82% and 190.82% of con-trol at 0 and 48 h following 30 min hypoxic preconditioning(P0h<0.05 and P48h<0.01, n=3, Kruskal–Wallis Test). The ex-pression of VEGF time-dependently increased to 209.78%,213.81% and 272.60% and 263.51% of control, at 0. 24, 48 and72 h following hypoxic preconditioning (All P<0.01, n=3, Krus-kal–Wallis Test). Hypoxic preconditioning induced similar ex-pression patterns of HIF-1α, and PGC-1α in PC12 cells: HIF-1αincreased to 195.79% at 0 h and 243.46% at 48 h following hyp-oxic preconditioning (P<0.01 in both cases, n=3, Kruskal–Wal-lis Test); PGC-1α increased to 153.54% at 48 h and 120.53% at72 h after hypoxia (P<0.01 in both cases, n=3, Kruskal–WallisTest); VEGF increased to 114.66% at 0 h and 193.91% at 48 hafter hypoxia (P0h<0.01 in both cases, n=3, Kruskal–WallisTest).
Hypoxic preconditioning-induced expressions of HIF-1α,PGC-1α and VEGF were associated with preconditioning-mediated protection of cell viability in both endothelial andPC12 cells exposed to OGD (Figs. 3 and 4).
2.3. Knockdown of PGC-1α, HIF-1α diminished theprotective effect of hypoxic preconditioning in both endothelialand PC12 cells
Either HIF-1α or PGC-1α knocked-down diminished the pro-tective effect of hypoxic preconditioning in both endothelialand PC12 cells (Figs. 5A and B). Compared to wild type cells,the expression of VEGF significantly decreased in HIF-1α
Fig. 1 – Hypoxic preconditioning decreased OGD-induced cell death (A). OGD insult time-dependently decreased cell viability inneuronal cells (PC12) and endothelial cells (EC). Hypoxic preconditioning was achieved by exposing cells to 30 min of hypoxiaand cell deathwas assessed usingMTT assay. (B). Hypoxic preconditioning (P) significantly attenuated OGD-induced cell injuryin PC12 and EC cells. (C). The line chart illustrates the hypoxic preconditioning protocols used in the present study. Data areexpressed as mean±SEM of five to six independent experiments, performed in triplicate. * P<0.05 as compared with controlgroup; # P<0.05 as compared with OGD group.
Fig. 2 – Hypoxic preconditioning reduced OGD-induced protein oxidization and mitochondrial ROS generation. Proteinoxidization and mitochondrial ROS were assessed using Oxyblot and MitoSOX dye, respectively (A) and (B). Representativeblots show that hypoxic preconditioning reduced OGD-induced protein oxidization in endothelial cells (EC) and neuronal cells(PC12) (C). The bar chart shows relative band densities of blots (D). The bar chart shows that hypoxic preconditioning reducedOGD-induced mitochondrial ROS generation. Data are expressed as mean±SEM of five to six independent experiments,performed in triplicate. * P<0.05 as compared with control group; # P<0.05 as compared with OGD group.
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Fig. 3 – Hypoxic preconditioning enhanced HIF-1α, PGC-1α and VEGF expression.(A) and (B). Representative blots showhypoxic preconditioning enhanced OGD-induced expression levels of HIF-1α, PGC-1α and VEGF in endothelial cells (EC) and inneuronal cells (PC12). (C). The bar chart shows relative band densities of blots. Data are expressed as mean±SEM of five to sixindependent experiments, performed in triplicate. * P<0.05 as compared with control group; # P<0.05 as compared with OGDgroup.
Fig. 4 – Expression pattern of HIF-1α, PGC-1α and VEGF proteins and cell viability changes at different time intervals afterhypoxic preconditioning. (A) and (B). Representative blots show that expression pattern of HIF-1α, PGC-1α and VEGF proteinsat 0 (P0h), 24 (P24h), 48 (P48h) and 72 (P72h) following hypoxic preconditioning in endothelial cells (EC) and neuronal cells(PC12) (C) and (D). The bar charts show that OGD-induced cell damaged was significantly reduced at 0 (P0h), 24 (P24h), 48 (P48h)and 72 (P72h) following hypoxic preconditioning in endothelial cells and PC12 cells. Data are expressed asmean±SEM of five tosix independent experiments, performed in triplicate. * P<0.05 as compared with control group; # P<0.05 as compared withOGD group.
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Fig. 5 – Gene knockdown of HIF-1α or PGC-1α diminished the protection of hypoxic preconditioning on OGD-induced cell damage.(A) and (B). Bar charts show that the protection of hypoxic preconditioning was diminished in HIF-1α or PGC-1α knockdownendothelial cells (A) and PC12 neuronal cells (B). (C) and (D). Representative blots show VEGF expression levels in HIF-1α or PGC-1αknockdown endothelial cells (C) and PC12 cells (D) under different conditions. Data are expressed as mean±SEM of five to sixindependent experiments, performed in triplicate. * P<0.05 as comparedwith control group; # P<0.05 as comparedwith OGD group.
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knocked-down endothelial cells, and, to a greater extent, inPGC-1α knocked-down cells in both preconditioning OGDgroups (Fig. 6A). In PGC-1α knocked-down PC12 cells, VEGF
Fig. 6 – hypoxic preconditioning-induced VEGF expression and aknocked-down cells. (A) and (B). The bar charts show VEGF proteknocked-down endothelial cells (A) and PC12 neuronal cells (B) uprotein oxidation levels in HIF-1α knocked-down cells and PGC-cells (D) under different conditions. Data are expressed as meantriplicate. * P<0.05 as compared with control group; # P<0.05 as
significantly decreased in cells under normal condition anddecreased further to barely detectable level after OGD. More-over, VEGF could not be induced by hypoxic preconditioning
nti-oxidization effects were attenuated in HIF-1α or PGC-1αin levels in HIF-1α knocked-down cells and PGC-1αnder different conditions. (C) and (D). The bar charts show the1α knocked-down endothelial cells (C) and PC12 neuronal±SEM of five to six independent experiments, performed incompared with OGD group.
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in HIF-1α knocked-down cells and PGC-1α knocked-downcells (Fig. 6B).
2.4. Oxidized-Protein detoxification level reduced in PGC-1α knocked-down endothelial cells
In PGC-1α knocked-down endothelial cells, a noticeably highlevel of oxidized protein was found in both OGD and hypoxicpreconditioning group, indicating the involvement of PGC-1αin hypoxic preconditioning-mediated protection (Fig. 6C). InPGC-1α knocked-down PC12 cells, protein oxidization was evi-dent in OGD group. In contrast, OGD-induced oxidized-proteinwas significantly reduced in hypoxic preconditioning group,suggesting that the involvement of non-PGC-1α pathways inhypoxic preconditioning mediated protection (Fig. 6D).
3. Discussion
In this study, we found that hypoxic preconditioning attenu-ated cell damage, increased the expression of protective fac-tors, and decreased oxidative stress in both PC12 andendothelial cells in OGDmodel. Importantly, the protective ef-fects of preconditioning were significantly attenuated by geneknocked-down of either HIF-1α or PGC-1α.
Most of the preconditioning studies focus primarily on neu-rons and the role of endothelial cells in preconditioning has re-ceived less attention (Andjelkovic et al., 2003; Deplanque et al.,2000; Kunz et al., 2007; Zhang et al., 2007). However, cerebrovas-cular dysfunction plays a critical role in the pathophysiology ofischemia stroke. Therefore, it is necessary to investigate therole of endothelial cells in preconditioning study. As weexpected, hypoxic preconditioning also provided profound pro-tection against OGD-induced cell damaged in endothelial cells.Noticeably, hypoxic preconditioning-induced protection wascomparable in both neuronal and endothelial cells, suggestingthat hypoxic preconditioning-mediated effects may be univer-sal among different cell types.
To further explore the molecular mechanisms underlyinghypoxic preconditioning, we focused on HIF-1α and PGC-1α,two important molecules responsible for preconditioning-mediated protection. Consistent with previous reports, pre-conditioning increased HIF-1α and PGC-1α protein levelswhereas either HIF-1α or PGC-1α knocked-down diminishedthe protective effects of hypoxic preconditioning in both en-dothelial and PC12 cells. Interestingly, HIF-1α and PGC-1αme-diated protection through regulation of VEGF. VEGF is a strongneuroprotective molecular and has been regarded as a keyplayer in preconditioning-mediated protection. HIF-1α is awell-known VEGF regulator in a gene-transcription-controlmanner. PGC-1α also can regulate VEGF expression. A recentstudy has demonstrated that PGC-1α can activate VEGF byco-activating one of its downstream transcription factors ina manner independent of HIF-1 (Arany et al., 2008). Our obser-vation suggested that VEGF might coordinate protective ef-fects between neurons and endothelial cells in ischemicbrain. Indeed, a previous study has demonstrated that cross-talk between neurons and endothelial cells is mediated main-ly via VEGF (Li et al., 2006). Furthermore, VEGF has alsobeen reported to mediate a shared pathway underlying the
preconditioning-induced protective effect in neurons and vas-cular endothelial cells (Lee et al., 2009).
Oxidative stress has been implicated in the pathophysio-logical change that occurred during and after cerebral ische-mia (Abbott et al., 2006). Consistently, OGD inducedexcessive ROS in both PC12 and endothelial cells. PGC-1α is amajor transcriptional regulator of the mitochondrial detoxifi-cation system. It has been shown to be involved in defensesagainst ROS by directly promoting MnSOD expression (Valleet al., 2005). We found that PGC-1α knockdown almostcompletely diminished hypoxic-preconditioning-mediatedanti-oxidative effects, suggesting a pivotal role of PGC-1α inhypoxic preconditioning-induced ROS suppression. Recently,HIF-1α has also been shown to reduce excessive ROS in hyp-oxic cells (Guo et al., 2009; Semenza, 2007). However, wefound that HIF-1α knockdown slightly reduced hypoxicpreconditioning-induced ROS suppression in endothelialcells but not in PC12 cells, indicating a minor role of HIF-1αin preconditioning-mediated anti-oxidative effects.
In summary, both HIF-1α and PGC-1α participated in hyp-oxic preconditioning-mediated protection in which HIF-1αwas crucial for cell viability whereas PGC-1α played a centralrole in ROS scavenging system and VEGF modification. In ad-dition, PGC-1α and HIF-1α may also play different roles in dif-ferent cell types. PGC-1a-induced intracellular antioxidantand up-regulation of VEGFmay be themajor player in hypoxiapreconditioning in endothelial cells. On the other hand,enhanced VEGF expression by PGC-1α and HIF-1α may beresponsible for hypoxic preconditioning in PC12.
The present study has clinical implications. Stroke has beenproved to be a poor candidate for a single approach treatment(Fisher and Ratan, 2003). In the present study, PGC-1α, togetherwithHIF-1α, modulates the complex network of hypoxic precon-ditioning. Therefore, combinational therapy of HIF-1α and PGC-1α aimed at multi-cell targets of the CNS and multi-factorsmight benefit the ischemia-damaged brain better. Further stud-ies are needed to address the roles of HIF-1α and PGC-1α in theinteractions between neurons and endothelial cells.
4. Experimental procedures
4.1. Cell culture
Two immortalized cell lines used in this experiment wereobtained from BioMark Cell Bank (China). Rat artery endothe-lial cells were cultured in RPMI-1640 (GIBCO, USA) supplemen-ted with 10% heat-inactivated fetal calf serum (GIBCO, USA) ina humidified incubator (Thermo electron corporation, USA)containing 5% CO2 at 37 °C. And PC12 (rat pheochromocyto-ma) cells were cultured in RPMI-1640 supplemented with10% heat-inactivated horse serum (GIBCO, USA) and 5%heat-inactivated fetal calf serum in the same condition.Experiments were conducted on cells from passages 5 to 15.
4.2. Hypoxic preconditioning and oxygen–glucosedeprivation (OGD)
Hypoxic preconditioning were achieved by replacing the cellsinto an anaerobic chamber that was flushed with a gas
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mixture of 5% CO2 and 95% N2 (v/v) at 37 °C for 30 min withoutchanging the culture medium. For OGD, the culture mediumwas changed to the glucose-free RPMI-1640 and cells wereplaced into an anaerobic chamber. The following reperfusionprocess with normal oxygen and glucose re-supply lasted for24 h before anymeasurements were performed. In the normalcontrol groups, the cell cultures were subjected to the sameexperimental procedures without exposure to the glucose-free medium or anoxia.
4.3. Measurement of cellular viability
In order to assess the viability of the cultured cells, the 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide (MTT,Sigma, USA) assay was used. Cell viability was also evaluatedby using DAPI (Sigma, USA). Briefly, cells were fixed in 4%polylysine on ice for 15 min, and then incubated with DAPI(2.5 μg/ml) for 20 min at room temperature. Nuclear morphol-ogy stained by DAPI was visualized under a fluorescence mi-croscope (Olympus DP70, Japan) at 20× objective. Condensed,shrunken, fragmented or irregular nuclei were considered tobe the sign of cell death. To quantitative analysis the changesof nuclear morphology, 10 different pictures were selectedrandomly and more than 600 cells were observed in differentgroups. Ratio of dead cells to total cells was comparedamong groups.
4.4. Western blot
Protein was extracted by using Mammalian Cell extraction kit(BioVision, USA). Following electrophoresis and transfer topolyvinylidene difluoride PVDF membranes (Millipore, USA),membranes were blocked for 1 h and incubated with appro-priately diluted antibodies (1:1000 rabbit monoclonal antibodyPGC-1α, Cell signaling technology, USA; 1:1000 mouse mono-clonal antibody HIF-1α, R&D systems, USA; 1:200 rabbit poly-clonal antibody VEGF, Millipore, USA) for 16–18 h at 4 °C.Then the membrane was washed and incubated with eitherhorseradish peroxidase (HRP)-conjugated anti-mouse IgG oranti-rabbit IgG (R&D systems, USA) for 1 h at room tempera-ture. The intensity (INT×mm2) of each band was measuredand analyzed with a Chemi Doc XRS imaging system (BIO-RAD, USA). Data are given as a percentage of vehicle controland β-actin was used as an internal control.
4.5. Measurement of mitochondrial ROS production andprotein oxidation
Estimation of mitochondrial ROS generation was performedby using MitoSOX (Invitrogen, USA). Briefly, cells were loadedwith the MitoSOX Red (2.5 μM) in the dark for 10 min. Quanti-tative measurements of fluorescence readings weredetermined at 510/580 nm (excitation/emission) using a Spec-traMax GEMINI EM fluorescent plate reader (Molecular De-vices, USA). Superoxide production was calculated as theaverage fluorescence intensity over six coordinate wells andexpressed relative to vehicle control (Jones et al., 2008). Oxy-blot offered a convenient measurement for detecting andquantifying protein carbonyl formation levels, an importantmarker of protein oxidative modification induced by ROS
(Canton et al., 2004). According to the manufacturer's proto-col, 5 μl protein sample was added with 5 μl of 12% SDS and10 μl of 1× 2,4-dinitrophenylhydrazine (DNPH) solution into atube. Tubes were incubated at room temperature for 15 min.7.5 μl of neutralization solution was added to each tube,and one mixed sample per lane was loaded onto 12% SDSpolyacrylamide gel. The following procedure was similar toWestern blot assay mentioned above.
4.6. RNA interference
Knockdown of PGC-1α or HIF-1α was established by small in-terfering RNA (siRNA) transfection, the process of which wasconducted by Bochuang Biotech Ltd. (Guangzhou, China).SiRNA sequences of PGC-1α and HIF-1α were designed andsynthesized by GeneChem Co., Ltd. (Shanghai, China). ThePGC-1α gene-specific sequences were: sense, 5′-caC TCA GCTCAG CTA CAA TGA and antisense, TCA TTG TAG CTG AGCTGA Gtg-3′. The HIF-1α gene-specific sequences were: sense,5′-aaC CAG TTG AAT CTT CAG ATA and antisense, TAT CTGAAG ATT CAA CTG Gtt-3′. The silencer negative controlsiRNA was used as a negative control.
4.7. Statistics
All of the experimentswere repeated three times independently.Data are shown at the form of mean±SEM. The comparisons ofmultiple quantitative variables were analyzed using one wayanalysis of variance (ANOVA) with LSD as the post-hoc test orusing Kruskal–Wallis test when the data were not normallydistributed or variance unhomogeneity. Chi-square test wasused to analyze categorical variables. The analyses were per-formed using SPSS software. P<0.05 was considered to be statis-tically significant unless otherwise stated.
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (NSFC) (No. 81071069) and also supportedby the Science and Technology Planning Project of GuangdongProvince, China (No. 2008B080703029 and No. 2010B080701008).
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