Journal of Molecular and Cellular Cardiology 42 (2007) 1036–1044www.elsevier.com/locate/yjmcc

Original article

HIF-1α induced-VEGF overexpression in bone marrow stem cells protectscardiomyocytes against ischemia

Ying Dai a,b, Meifeng Xu a,⁎, Yigang Wang a, Zeeshan Pasha a,c,Tingyu Li b, Muhammad Ashraf a

a Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, OH 45267, USAb Chongqing University of Medical Science, P.R. Chinac Center of Excellence in Molecular Biology, Pakistan

Received 26 January 2007; received in revised form 26 March 2007; accepted 2 April 2007Available online 6 April 2007

Abstract

Hypoxia inducible factor-1α (HIF-1α) is a proangiogenic transcription factor stabilized and activated under hypoxia. It regulates the expression ofnumerous target genes, including vascular endothelial growth factor (VEGF) and other cytoprotective proteins. In this study, we hypothesized thatbone marrow stem cells (BMSCs) secrete growth factors which protect cardiomyocytes via HIF-1α pathway. BMSCs were obtained from transgenicmice overexpressing green fluorescent protein (GFP). The study was carried out in vitro using co-culture of BMSCs with cardiomyocytes. LDHrelease, MTT uptake, DNA fragmentation and annexin-V positive cells were used as cell injury markers. The level of HIF-1α protein as well as itsactivated form and VEGFwere measured by ELISA. The expression of HIF-1α and VEGF in BMSCswas analyzed by quantitative PCR and cellularlocalization was determined by immunohistochemistry. LDH release was increased andMTTuptake was decreased after exposure of cardiomyocytesto hypoxia for 30 h, which were prevented by co-culturing cardiomyocytes with BMSCs. Cardiomyocyte apoptosis induced by hypoxia and H2O2

was also reduced by co-culture with BMSCs. VEGF release fromBMSCswas significantly increased in parallel with high level of HIF-1α in BMSCsfollowing anoxia or hypoxia in a time-dependent manner. Although no significant up-regulation could be seen in HIF-1αmRNA,HIF-1α protein andits activated form were markedly increased and translocated to the nucleus or peri-nuclear area. The increase and translocation of HIF-1α in BMSCswere completely blocked by 2-methoxyestradiol (2-ME2; 5 μmol), a HIF-1α inhibitor. Moreover, the protection of cardiomyocytes by BMSC andVEGF secretion was abolished by neutralizing HIF-1α antibody in a concentration dependent manner (200–3200 ng/ml). Bone marrow stem cellsprotect cardiomyocytes by up-regulation of VEGF via activating HIF-1α.© 2007 Elsevier Inc. All rights reserved.

Keywords: Bone marrow stem cells; HIF-1α; VEGF; Cardiomyocytes; Hypoxia; HIF-1α neutralizing antibody

1. Introduction

Myocardial infarction is a leading cause of heart failure. Cardiacstem cells [1] and human embryonic stem cells [2] and bonemarrow stromal cells (BMSCs) [3–5] have been shown toparticipate inmyocardial repair process and repopulate the infarctedmyocardium. Under normal conditions, BMSCs are rarely seen intissue and organs. However, acute myocardial infarction (AMI)enhances their mobilization into blood circulation and their lodgingin the damaged tissue to repair ischemic myocardium [6–9].

⁎ Corresponding author. Tel.: +1 513 558 4725; fax: +1 513 558 0807.E-mail address: Meifeng.Xu@uc.edu (M. Xu).

0022-2828/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.yjmcc.2007.04.001

Moreover, BMSCs can be directly transplanted into infarcted areaby intra-coronary infusion or catheter based intra-myocardialinjection or direct intra-myocardial injection [10]. The mobilizedor transplanted BMSCs significantly improve left ventricularfunction [3–5] which might be due to better regional perfusionand systolic function following acute coronary artery occlusion[10,11] and transdifferentiation into functional active cardiomyo-cytes [3,4,12]. However, some studies reported that transdiffer-entiated cardiomyocytes from BMSCs were not detected in therepaired tissue [13].

It is well known that the loss of cardiac myocytes is the majorproblem in heart failure; thus, it is important to protect nativecardiac myocytes and preserve myocardial tissues against cell

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death besides regeneration of injured heart. A growing body ofevidence has shown that VEGF, a endothelial cell-specificangiogenic factor, induces expression of Bcl-2 which eventuallyfunctions to enhance cell survival in the anoxic and oxygen-deficient environment [14] and activates the myocardial PI-3Kpathway to decrease myocardial infarct size [15]. Low oxygentension is a common phenomenon in ischemic cardiomyopathichearts. Our previous study has shown that BMSCs secreteVEGF, basic fibroblast growth factor (b-FGF), insulin-likegrowth factor (IGF) and stromal cell–derived factor-1 (SDF-1),which were increased from 30% to 150% after BMSCs wereexposed to anoxia for 4 h [4]. It is unclear which pathway isactivated in BMSCs under lower oxygen tension to induce up-regulation of these cytoprotective proteins. Some studiesreported that hypoxia triggers hypoxia-inducible factor-1(HIF-1) signaling pathway. HIF-1 consists of a constitutivelyexpressed subunit HIF-1β and an oxygen-regulated subunitHIF-1α [16,17]. HIF-1α protein is ubiquitously expressed,whereas its homologues HIF-2α and HIF-3α have morerestricted expression patterns. Under lower oxygen tension,hydroxylation is inhibited because of substrate (O2) deprivation,and HIF-1α accumulates, dimerizes with HIF-1β, and mediatesprofound changes in hypoxia-inducible genes (HIGs) expres-sion [18,19]. To our knowledge, there are no reports on the roleof HIF-1α on the ischemic heart repair by BMSCs. Wehypothesize that BMSCs directly protect cardiomyocytes by up-regulation of cardioprotective protein, VEGF via activatingHIF-1α under lower oxygen tension environment.

2. Method

2.1. Cell culture

BMSCs were isolated according to the method describedby us previously [20]. In brief, femurs and tibias from greenfluorescent protein (GFP)-transgenic mice developed byHadjantonakis et al. [21] were removed. Muscle andextraossial tissue were trimmed. Bone marrow cells wereflushed and cultured with Iscove's Modified Dulbecco'sMedium (Gibco) supplemented with 20% FBS and penicillin(100 U/ml)/streptomycin (100 μg/ml) at 37 °C in humid airwith 5% CO2. After being seeded for 2 days, BMSCs adheredto the bottom of culture plates, and hematopoietic cellsremained suspended in the medium. The non-adherent cellswere removed by a medium change at 48 h and every 4 daysthereafter. Myocytes were isolated and cultured fromventricles of 2-day-old neonatal Sprague–Dawley rats (Har-lan, Indianapolis, IN) using the neonatal cardiomyocyteisolation kit (Worthington biochemical Co. NJ) as previouslydescribed [20].

2.2. In vitro ischemic model

To mimic the ischemic injury in vitro, cells were incubatedunder hypoxia or anoxia for various periods after the mediumwas replaced with a new serum free medium for 16 h. Foranoxia, cells were exposed to anaerobic glucose-free medium

and placed into the anoxic chamber (Forma 1025 anaerobicsystem). For hypoxia, cells were exposed to low glucose (1 g/L)DMEM and placed in hypoxic incubator (Sanyo, O2/CO2

incubator-MCO-18M) and oxygen was adjusted to 1.0% andCO2 to 5%. Normal culture (serum free regular medium under21% oxygen and 5% CO2) served as a control. LDH release andMTT intake were used as cell injury parameters. Apoptosis wasdetermined by annexin-V (MBL International) binding test andDNA fragmentation (Biovision) following manufacture'sinstructions. In some experiments, cells were exposed to200 μmol H2O2 for 2 h to induce cell apoptosis. To confirmthe role of HIF-1α in the protection by BMSCs oncardiomyocytes, cells were pretreated with different concentra-tions of HIF-1α neutralizing antibody or 2-methoxyestradiol (2-ME2), a HIF-1α inhibitor for 1 or 16 h, respectively. Theexperiments were carried out in triplicate and each experimentwas repeated three times unless otherwise mentioned.

2.3. Measurement of VEGF and HIF-1α protein by ELISA

VEGF release from BMSCs into culture medium was directlymeasured by ELISA kit according to manufacturer's instructions(R&D Systems). As a control, basal media were also analyzed.The absorbance was measured at 450 nm and 570 nm.

Total HIF-1α protein was extracted from whole cells. The cellswere washed twice with ice-cold PBS and lysed with lysis buffer(pH 7.4) including (mM) Tris 50; EDTA 3; MgCl2 1; β-glycerophosphate 20; NaF 25; NaCl 300; 10% (w/v) glycerol; 1%Triton X-100 and protease inhibitor cocktail (Roche). Whole cellextract was obtained by centrifuging the lysates at 16,000×g at4 °C for 10min followed by sonication. Total HIF-1αwas detectedby ELISA mouse total HIF-1α kit (R&D Systems) according tothe manufacturer's instructions. The concentration of HIF-1α wascalibrated with HIF-1α standard curve. Activated HIF-1α wasmeasured from nuclear extract which was obtained by solubilizingnuclear pellet in freshly prepared cold lysis buffer (mM)containing: HEPES (pH 7.9) 20; MgCl2 1.5; NaCl 420; DTT0.5, Na3VO4 2; NaF 5; 25% glycerol; 25 μg/ml Chymostatinand protease inhibitor cocktail. After centrifugation the lysates at16,000×g at 4 °C for 10 min, the supernatant was collected. Thevalues were corrected for total protein. To measure the active HIF-1α, 50 μg/well nuclear extracts were incubated with biotinylateddouble stranded (ds) oligonucleotide containing a consensus HIF-1α binding site from Duo-set ELISA mouse active HIF-1α kit(R&D Systems). The activity of HIF-1α was expressed by OD(450 nm–570 nm).

2.4. RNA preparation and quantitative PCR

Total RNA was isolated and purified from cell pellet usingthe RNeasy mini kit as recommended by the manufacturer(Qiagen). Using SuperScript™ III first-strand synthesis kit(Invitrogen), 1 μg of total RNA was reverse transcribed tosynthesize first-strand cDNA (total 20 μl). Two microliters ofthe reverse transcription reaction was mixed with iQ SYBRGreen Supermix (Bio-Rad) and amplified by iQ5 real-timesystem (Bio-Rad). The product was quantified using a

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standard curve that calculated each cycle number at which theamplification of the product was in the linear phase. Toensure the fidelity of the mRNA extraction and reversetranscription, this value was normalized to the value of theinternal standard glyceraldehyde phosphate dehydrogenase(GAPDH) for each analysis. Primers for amplification ofVEGF, HIF-1α and GAPDH are listed below.

HIF-1α: Sense primer 5′-CTGCTGTCTTACTGGTCCTT-3′;Anti-sense primer 5′-GTC GCT TCT CCA ATT CTT AC-3′VEGF: Sense 5′-ATG AAC TTT CTG CTC TCT GG-3′;Antisense: 5′-TCA TCT CTC CTA TGT GCT GGC-3′GAPDH: Sense 5′-TGC AGT GGC AAA GTG GAG-3′;Anti-sense 5-ACA TAC TCA GCA CCG GCC TC-3′The expression of each target mRNA relative to GAPDH underexperimental and control conditions was calculated based onthe threshold cycle (CT) as r=2−Δ(ΔCT), where ΔCT=CT

target−CT GAPDH and Δ(ΔCT)=ΔCT experimental−ΔCT control.

2.5. Immunofluorescence and histological analysis

For HIF-1α staining, cells cultured on glass coverslips werefixed in 4% paraformaldehyde and incubated with mousemonoclonal anti-HIF-1α (Sigma). After washing thoroughly,the secondary antibodies of goat anti-mouse IgG conjugatedwith Alexa 546 (Molecular Probes, Eugene, OR) were applied.Nuclei were stained with 4′,6-diamino-2-phenylindole (DAPI)(Vector Laboratories). Myocyte staining was similar to ourprevious report [22]. Fluorescent images were processed usingOlympus BX41 microscope equipped with an Olympus U-TV0.5XC digital camera (Olympus, Japan).

2.6. Statistical analysis

All data were presented as mean±SEM. Statistical sig-nificance was evaluated with an unpaired Student's t test for

Fig. 1. BMSCs were obtained from transgenic mice expressing GFP and cardiomyocySame as “A”, but the nuclei of BMSCs were stained with DAPI. (C) Cultured myocymyocytes. The nuclei were stained with DAPI.

comparison between 2 groups. A probability value of b0.05was considered significant.

3. Results

3.1. BMSCs protected cardiomyocytes against ischemic injury

Previous studies indicated that the improvement of cardiacfunction by transplanted stem cells might partially be due tothe direct protection of native cardiomyocytes by stem cells[23,24]. To assess cardiomyocyte protection, BMSCs were co-cultured with myocytes at a ratio of 1:20. BMSCs had thetendency to grow in clusters (Fig. 1A). The nucleus of eachBMSC had more than one nucleolus (Fig. 1B). Cardiomyocytesbegan to beat spontaneously after being cultured for 24 h.Immunostaining showed that myocytes were positive for α-actinin and myofibers were seen with clear Z-lines insarcomeres. Myocytes had physical contacts with neighboringmyocytes via connexin 43 (Fig. 1C).

To determine that BMSCs provided protection to myocytesunder lower oxygen environment, a series of parameters were usedto determine cell injury. After exposure to hypoxia for 30 h, MTTuptake was significantly decreased and LDH release frommyocytes was significantly increased. A significant reduction inLDH release and an increase of MTT uptake were observed inmyocytes which were co-cultured with BMSCs (Fig. 2). DNAfragmentation was seen in myocytes after exposure to hypoxia for48 h (Fig. 3A), which could be prevented by co-culturing withBMSC. However, co-culture with BMSCs was ineffective toprevent DNA fragmentation if hypoxia was prolonged to 72 h (Fig.3B). To quantify myocyte apoptosis, cells were labeled withannexin V-PE following hypoxia. The cells were examined underfluorescence microscope and counted by FACS. Co-culture withBMSCs significantly reduced annexin V positive cardiomyocytesand were ineffective in the presence of HIF-1α antibody (Fig. 4A).To further demonstrate the protection of cardiomyocytes by

tes from neonatal rat ventricles. (A) Primary cultured GFP-positive BMSCs. (B)tes were positive for α-actinin (green). Connexin 43 (red) was observed between

Fig. 2. LDH release and MTT uptake by cultured cardiomyocytes underdifferent treatments. #pb0.05 vs. normal control; *pb0.05 vs. hypoxicmyocytes alone; †pb0.05 vs. myocytes co-cultured with BMSCs.

Fig. 3. Effect of BMSCs on DNA fragmentation under hypoxic conditions. (A)Myocytes alone, (B) Myocytes co-cultured with BMSCs (Lane 1: marker; Lane2: Normal; Lane 3: Hypoxia 24 h; Lane 4: Hypoxia 48 h; Lane 5: Hypoxia 72 h;Lane 6: Positive control). (C) Myocytes co-cultured with BMSCs with orwithout anti-HIF-1α antibody treatment.

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BMSCs against oxidative stress, H2O2 was used to mimicoxidative stress. The number of apoptotic cells was significantlyincreased after myocytes exposure to H2O2 (200 μmol) for 2 h(32.5±3.3% vs. 8.4±1.1% in normal control, pb0.05). TheAnnexin V positive cells were significantly reduced in co-culturecells as compared to cardiomyocytes culture alone (6.1±0.7% vs.32.5±3.3%, pb0.05). However, the protection by BMSCs wasabolished by pretreating cells with HIF-1α neutralizing antibodies(200–3200 ng/ml) or 2-ME2 (1–10 μmol) in a concentration–dependent manner (Figs. 4A, H).

3.2. Hypoxia up-regulated HIF-1α

It is generally known that HIF-1α pathway is activatedwhen cells are exposed to lower oxygen tension. We askedwhether HIF-1α pathway was also activated in BMSCsduring hypoxia. HIF-1α protein was significantly increased inBMSCs which were exposed to hypoxia as compared toBMSCs cultured under normal conditions (Fig. 5A). Hypoxianot only increased HIF-1α in whole cell lysate, but alsoincreased activated HIF-1α in nuclei (Fig. 5B). However,hypoxia did not increase HIF-1α RNA in cultured bonemarrow stem cells (Fig. 5C). In normal conditions, HIF-1αwas mainly localized in the cytosol (Fig. 5D). Hypoxiainduced the translocation of HIF-1α into the nuclei or to peri-nucleus areas (Fig. 5E, arrows). The up-regulation and

translocation of HIF-1α protein were completely abolishedby pre-treating cells with 2-ME2 (5 μmol) (Fig. 5F).

3.3. BMSCs secreted VEGF and protected cardiomyocytes

VEGF is one of the downstream genes of HIF-1α. Secretionof VEGF from BMSCs was assayed by ELISA. Fig. 6A showed

Fig. 4. Apoptosis assay after cells were exposed to hypoxia for 24 h and H2O2 (200 μmol) for 2 h. (A) Hypoxic treatment. (B–G) H2O2 treatment. (B–D) Annexin Vpositive cells are shown in red color; (E–G) flowcytometry assay. (B and E) Normal cultured myocytes; (C and F) H2O2 treated myocytes; (D and G) H2O2 treatedcardiomyocytes co-cultured with BMSCs; (H) percentage of annexin V positive cells after H2O2 treatments. #pb0.05 vs. normal cultured myocytes; *pb0.05 vs.hypoxia- or H2O2-treated myocytes alone. †pb0.05 vs. myocytes co-cultured with BMSCs.

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Fig. 5. Activity and distribution of HIF-1α in BMSCs. (A) Total HIF-1α (n=10) and (B) Activated HIF-1α (n=8) in the BMSCs under hypoxia for 30 h. It was inhibitedby 2-ME2. #pb0.05 vs. normal culture and *pb0.05 vs. hypoxia alone. (C) Quantitative PCR for HIF-1α mRNA (n=4). There was no significant difference amongvarious treatments. (D) HIF-1αwas scattered in cytosol of normal BMSCs; (E) HIF-1 was highly concentrated in peri-nucleus and in nuclei (arrows) after BMSCs wereexposed to hypoxia for 30 h. (F) Pretreatment of cells with 2-ME2 (5 μmol) for 16 h significantly abolished the translocation of HIF-1α. Red: HIF-1α; Blue: DAPI(nuclei).

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that VEGF release was significantly increased in BMSCs afterexposure to hypoxia compared to cells cultured in normalcondition and was decreased when cells were exposed tohypoxia for 72 h. Also the release of VEGF from BMSCs wasdecreased by pretreatment with 2-ME2 (Fig. 6B). To furtherconfirm the relationship of VEGF release with the HIF-1αactivity, specific HIF-1α neutralizing antibodies were used.Quantitative PCR data showed that VEGF mRNA wassignificantly up-regulated in BMSCs after exposure to hypoxiafor 10 h. The overexpression of VEGF by hypoxia wasabolished by HIF-1α neutralizing antibodies and 2-ME2(Fig. 6C).

To duplicate the protective effect of VEGF on cardiomyo-cytes, cardiomyocytes were pretreated with exogenous VEGF(0–50 ng/ml) for 16 h. Indeed, VEGF (12.5–100 ng/ml)prevented DNA fragmentation of cultured myocytes following48 h hypoxia and reduced the number of annexin-V positive

Fig. 6. Secretion of VEGF by BMSCs. (A) VEGF release from BMSCs.*pb0.05 vs. normal culture, respectively. (B) Release of VEGF from BMSCs byexposure to 30 h hypoxia was partially inhibited by 2-ME2 (5 μmol). #pb0.05vs. normal culture. (C) The expression of VEGF in BMSCs. Bars represent thefold increase in VEGF concentrations measured in hypoxic cultured BMSCsrelative to normoxic conditions. The expression of VEGF was significantlyreduced by specific HIF-1α neutralizing antibodies (3200 ng/ml) and HIF-1αinhibitor 2-ME2 (5 μmol). #pb0.05 vs. normal culture; *pb0.05 vs. cellsexposed to hypoxia.

Fig. 7. Effect of VEGF on cardiomyocyte apoptosis. (A) VEGF reduced theDNA fragmentation induced by hypoxia. (B) VEGF also prevented apoptosis ofmyocytes exposed to H2O2 (200 μmol/L) for 2 h. #pb0.05 vs. normal culture;*pb0.05 vs. myocytes exposed to H2O2 without VEGF treatment.

cells after exposure to H2O2 (200 μmol) in a concentration-dependent manner (Fig. 7).

4. Discussion

We have demonstrated for the first time that bone marrowstem cells protected cardiomyocytes against ischemic injurythrough secreting VEGF via activating HIF-1α pathway. Thecell apoptosis was prevented by VEGF released from BMSCsunder lower oxygen tension environment via activation of HIF-1α pathway. The secretion of VEGF from BMSCs waspositively co-related with HIF-1α activity which was abolishedby a HIF-1α inhibitor, 2-ME2 and specific HIF-1α neutralizingantibody in a concentration-dependent manner.

4.1. BMSCs protected cultured cardiomyocytes via secretion ofVEGF

It is generally agreed that protection of cardiac myocytes iscritical in the repair of ischemic heart since the loss of cardiacmyocytes is the major cause of heart failure. The repair processincludes protection of native cardiomyocytes in early stage andmyogenesis of new cardiomyocytes from stem cells at laterstage. Therefore, preservation of host myocardial tissues is oneof the benefits of stem cell based strategies after MI. The presentin vitro study provided direct evidence that BMSCs preservednative myocytes by preventing cell apoptosis induced byoxidative stress.

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Various growth factors, including IGF and VEGF, have beenshown to protect the heart against oxidative stress [25–27]. Therelease of VEGF from cultured cardiomyocytes was signifi-cantly increased when they were exposed to anoxia for 15 h(375.29±79.1 vs. 160.54±50.24 pg/ml in normal control,pb0.05) (our unpublished data). However, the release ofVEGF from cardiomyocytes does not appear to be enough toprotect cardiomyocytes against oxidative stress. Our previousstudy has shown that BMSCs secreted a number of growthfactors, which were enhanced by exposure to mild anoxia [4].Here, we further demonstrated a significant increase in VEGF,but not IGF (data not shown) in BMSCs under hypoxicconditions both at protein and mRNA levels. The protectionby VEGF was further duplicated by exogenous VEGF givenduring hypoxia. It suggests that BMSCs protect myocytes viasecretion of VEGF. Tang et al. have reported that implantation ofmesenchymal stem cells significantly elevatedVEGF expressionwhich was accompanied by increased vascular density andregional blood flow in the infarct zone [28]. Moreover, it hasbeen reported that implantation of bone marrow stem cellstransfected with phVEGF165 can increase the survival ofimplanted cells, and enhance the cardiac function after acutemyocardial infarction [29]. The latter study also showed thatVEGF protected cells against apoptosis. VEGF has directneurotrophic and neuroprotective as well as angiogenic proper-ties. It exerts neuroprotective actions directly through theinhibition of apoptosis and the stimulation of neurogenesis[30]. The mechanism of VEGF in the prevention of apoptosis isnot clear yet. VEGF may bind to its receptors and trigger thephosphatidylinositol 3-kinase (PI3K)/Akt signal transductionsystem thus resulting in the inhibition of apoptosis by activatingantiapoptotic proteins through the transcription factor NFκB andby suppressing proapoptotic signaling by Bad, caspase-9,caspase-3 and other effectors [30].

4.2. Activation of HIF-1α pathway induced VEGF release

The cellular mechanism by which BMSCs secrete VEGF inresponse to low oxygen environment is unknown. In this study, nosignificant changes in HIF-1α mRNA were observed in BMSCafter exposure to hypoxia for 10–30 h. However, HIF-1α proteinand its active form were significantly increased in lower oxygentension environment (Fig. 5). HIF-1α was further translocated tothe nucleus and peri-nuclear areas following hypoxia. HIF-1 is atranscription factor which facilitates the adaptation of cells andtissues to low O2 concentrations [16,17]. It has been demonstratedthat the transcription and synthesis of HIF-1α are constitutive andregulated by its degradation rate [31,32].

Regulation of HIF-1α function primarily occurs at theposttranslational level. It has been reported that 2-ME2 down-regulates HIF-1α at the post-transcriptional level and inhibitsHIF-1α-induced transcriptional activation of VEGF expressionin human breast and prostate cancer [33]. Our data show that 2-ME2 blocked the protection by BMSCs in a concentration-dependent manner in parallel with down-regulation of the HIF-1α protein levels and activity, but has no significant effect onHIF-1α mRNA expression. Moreover, 2-ME2 partially atte-

nuated the release of VEGF from BMSCs. This suggests that 2-ME2 abrogated the protection of BMSCs by partially abolishingVEGF release from BMSCs via HIF-1α pathway. Mabjeesh etal. [34] reported that 2-ME2 inhibited HIF-1α protein synthesisand proposed that this effect was a consequence of microtubuledisruption by 2-ME2. 2-ME2 also inhibited HIF-1α activity in adose-dependent manner, as determined by using a hypoxia-responsive reporter construct [35].

Inhibition of VEGF expression and loss of protection byspecific HIF-1α neutralizing antibody support our hypothesisthat HIF-1α plays a very important role in secretion of VEGFfrom bone marrow stem cells under hypoxia. HIF-1α protein issteady [31,32] under low O2 levels (b5%) and translocates fromthe cytoplasm to the nucleus, where it dimerizes with HIF-1β,and forms a dimer with aryl hydrocarbon receptor nucleartranslocator protein (ARNT). The heterodimer HIF-1α-ARNTis a transcriptional activator of genes and results in expressionof several hypoxia-inducible genes, including erythropoietin,VEGF, glucose transporters and glycolytic enzymes, etc.[16,36]. HIF-1α heterodimerizes with HIF-1β and binds tohypoxia response elements, thereby activating the transcriptionof numerous genes important for adaptation and survival underhypoxia [37,38]. Kimbro and Simons reviewed [33] that HIF-1α signaling induced VEGF release by activating the proteintyrosine kinase c-Src and/or its downstream mediator phospha-tidylinositol 3-kinase (PI3K). Src and PI3K activation appearsto increase the stability of HIF-1α, thereby, increasing VEGFlevels [39]. Several investigators have demonstrated thatoptimal transcriptional control of the VEGF promoter requiresbinding of both HIF-1α and STAT3 [40–42].

In summary, VEGF secretion by BMSCs is regulated by theactivation of HIF-1α under hypoxic conditions and protectscardiomyocytes against injury.

Acknowledgment

This work was supported by National Institutes of Healthgrants R37-HL074272, HL087246, HL080686, HL70062 (M.Ashraf), HL083236 (M. Xu) and HL081859 (Y. Wang).

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