stem cells tissue specific stem cells - tasmc.org.illibrary.tasmc.org.il/ · hif-1α and hif-2 α...

Author contributions: J.B.-S.: Conception and design, Collection and assembly of data, Data analysis and interpretation, Manuscript writing; S.S.: Collection and assembly of data, Data analysis and interpretation; G.L.: Collection and assembly of data, Data analysis and interpretation; S.M.-A.: Collection and assembly of data, Data analysis and interpretation; A.Barzelay: Collection and assembly of data; S.P.-C.: Data analysis and interpretation; E.T.: Conception and design, Provision of study material; I.B.: Provision of study material Data analysis and interpretation; A.Barak: Provision of study material Data analysis and interpretation; H.L.-V.: Provision of study material, Data analysis and interpretation; G.K.: Conception and design, Provision of study material, Data analysis and interpretation; J.G.: Conception and design, Data analysis and interpretation, Manuscript writing, Final approval of manuscript. Correspondence: Jacob George, Department of Cardiology, Tel Aviv Sourasky Medical Center, 6 Weizmann Street, Tel Aviv 64239. Tel: 972-3-6974250, Fax: 972-3-6974808, E-mail: [email protected]. *This work was performed as a part of the Ph.D. thesis of Jeremy Ben-Shoshan. 8These authors contributed equally to this work. Received April 18, 2008; accepted for publication July 28, 2008; first published online in Stem Cells Express August 7, 2008. ©AlphaMed Press 1066-5099/2008/$30.00/0 doi: 10.1634/stemcells.2008-0369

STEM CELLS® TISSUE-SPECIFIC STEM CELLS Constitutive Expression of HIF-1α and HIF-2α in Bone Marrow Stromal Cells Differentially Promote their Pro-angiogenic Properties.* Jeremy Ben-Shoshan1,2, Shulamit Schwartz2,7,8, Galia Luboshits1,8, Sofia Maysel-Auslender1, Aya Barzelay1,2, Sylvie Polak-Charcon4, Eldad Tzahor5, Iris Barshack2,4, Adiel Barak2,3, Hani Levkovitch-Verbin2,6, Gad Keren1,2, Jacob George1,2. 1 Department of Cardiology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel; 2 Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; 3 Department of Ophthalmology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel; 4 Institute of Pathology, Sheba Medical Center, Tel Hashomer, Israel; 5 Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel; 6 Institute of Ophthalmology, Sheba Medical Center, Tel Hashomer, Israel; 7 Department of Ophthalmology, Assaf Harofeh Medical Center, Zrifin, Israel Key Words. Bone marrow stromal cells • hypoxia inducible factor • angiogenesis • paracrine effects • cell-therapy. ABSTRACT Aims - Bone marrow stromal cells (BMSCs) contain progenitors capable to participate in postnatal angiogenesis. Hypoxia inducible factors (HIF) mediate endothelial activation by driving the expression of multiple angiogenic factors. We explored the potential of HIF-1α and HIF-2α modification in BMSCs, as a tool to improve cell-based angiogenic therapy. Methods and Results- - BMSCs were retrovirally transduced to express stable forms of HIF-1α and HIF-2α. HIF-1α and, to a greater extent, HIF-2α overexpression promoted differentiation of BMSCs to the endothelial lineage, evident by CD31 and Tie-2 expression and improved adhesive properties. Whereas chemotaxis towards SDF-1 was higher in both HIF-α expressing BMSCs, enhanced migration towards VEGF was found only following

overexpression of HIF-2α, supported by a robust expression of its receptor- Flk-1. HIF-α expression was associated with up-regulation of angiogenic proteins and improved tube formation. Cytokine arrays of endothelial cells stimulated by medium collected from HIF-α expressing BMSCs revealed further angiogenic activation and improved adhesive capacity. Eventually, delivery of HIF-2α transduced BMSCs induced a more robust angiogenic response, compared to sham-transduced or HIF-1α-transduced BMSCs in the corneal micropocket angiogenesis model. Conclusions- Our results support the use of HIF-α genes, particularly HIF-2α, to augment the efficacy of future cell-based therapy.

Stem Cells Express, published online August 7, 2008; doi:10.1634/stemcells.2008-0369

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HIF-1α and HIF-2 α promote BMSCs mediated angiogenesis

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INTRODUCTION

Bone marrow stromal cells (BMSCs) contains endothelial progenitors capable of migrating towards ischemic tissues, proliferating and differentiating into mature endothelial cells (ECs) [1]. Numerous studies have documented the contribution of endothelial progenitors to new vessel formation [1] [2], however, their effectiveness as a therapeutic tool remains to be further supported. Indeed, several preliminary clinical trials that tested administration of bone marrow (BM) progenitors in ischemic diseases yielded equivocal results [3]. Consequently, different groups have suggested gene modifications to improve the therapeutic potential of BM-derived cells [4] [5]. Hypoxia-inducible factor (HIF) plays key roles in controlling glycolysis, angiogenesis, erythropoiesis, vascular tone and cell survival [6]. Under normoxia, HIF-α subunits degradation is driven by hydroxylation of specific proline sites that allow VHL protein binding and subsequent ubiquitination [6] [7]. In the absence of oxygen, HIF-α hydroxylation is abrogated and HIF α and β subunits heterodimerize to activate target genes. The gene spectrum induced by HIF includes multiple angiogenic factors and receptors [7]. The main HIF-α subunits, HIF-1α, which is ubiquitously expressed, and HIF-2α, mainly expressed in ECs, both plays requisite roles in vascular development and homeostasis [8] [9]. Despite their high degree of homology, HIF-1α and HIF-2α fulfill distinct roles in gene regulation [10]. This study aims to evaluate the differential effects of constitutive expression of HIF-1α and HIF-2α on the differentiation, functional properties and therapeutic potential of BM-derived progenitor cells.

MATERIALS AND METHODS Animals. Male Wistar rats (220-240g; Harlan) care conformed to the local Review Board for Animal Trials. Cell culture. BM-mononuclear cells were separated using density gradient centrifugation (Axis-Shield) and grown on fibronectin in M199 medium (Biological Industries) 10% FCS, 50 ng/ml rhVEGF165 (R&D), 10ng/ml rhbFGF (Calbiochem), 10IU/mL rhEPO (R&D) and 100ng/ml rhGM-CSF (Calbiochem) in 37°C and 8% CO2. After 72h non-adherent cells were washed and medium change every 72h for 16 days. H5V murine heart ECs were maintained in 37°C and 8% CO2 in DMEM/F12 (Biological Industries) 10% FCS. Plasmids and Antibodies. pLZRS-IRES-eGFP constructs were kindly provided by P.Ratcliff. pCDNA3-hHIF-1αP564A/hHIF-2αP531A were a gift from M.Safran. Mutant forms were inserted in BamH1/NotI sites of pLZRS-IRES-eGFP. Constructs were

confirmed by DNA sequencing (Hylabs). Antibodies: anti-HIF-1α (R&D), mouse anti-HIF-1α (Abcam), polyclonal rabbit anti-HIF-2α (Novus Biologicals), PE anti-mouse/rat CD29, PE anti-rat CD45, FITC anti-rat CD11b and FITC anti-rat CD90/mouse CD90.1 (Biolegend), polyclonal rabbit anti-Tie-2, polyclonal goat anti-CD31 and monoclonal mouse anti-Flk-1 (Santa Cruz), polyclonal rabbit anti-GFP (Invitrogen), FITC rat IgG2b Isotype Control, FITC anti-mouse VCAM-1 and FITC anti-mouse ICAM-1 (eBioscience). Immunofluorescence. BMSCs were grown on 18X18 glass slides, fixed and incubated with primary antibodies at 2μg/ml for 2hrs and with secondary antibodies (Jackson labs) for 1hr. Incubation with secondary antibodies only served as negative controls. Slides were mounted on cover-slips and analyzed by fluorescent microscopy (x40-x80). Flow Cytometry Analysis. BMSCs were incubated with DiI-labeled ac-LDL

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(Biomedical Technologies) 1μg/ml for 4hrs and with FITC-UE lectin (Sigma-Aldrich) 2μg/ml for 30mins, washed and analyzed by flow cytometry (FACSCalibur, Becton Dickinson). Alternatively, double staining was performed on glass slides and analyzed by fluorescence microscopy. For VCAM-1/ICAM-1 staining cells were incubated with FITC- isotype or specific antibodies for 30mins, washed and analyzed. Retroviral Transduction of BMSCs. pLZRS retroviral plasmids were transfected into Phoenix packaging cells (ATCC) using standard Ca2PO4 protocol. Medium was changed after 24hrs. Cells were grown for additional 24hrs and virus collected and centrifuge 5mins/1500 x rpm/4°C. Viral titer was determined as >6 x 10 [6] IU/ml using NIH 3T3 cells. Day 12 BMSCs were incubated with viral supernatant for 8hrs in the presence of 5μg/ml polybrene (Sigma-Aldrich). Infection was repeated after 24hrs. 72hrs post-transfection, infection efficiency was determined by GFP flow cytometry analysis and routinely reached 80-85% in all groups. Active HIF-α ELISA. Human/Mouse Active HIF-1α DuoSet® Intracellular ELISA (R&D) was used according to manufacturer's instructions. Briefly, a biotinylated oligonucleotide containing the consensus HRE HIF binding site was incubated with BMSCs nuclear extracts. HIF-ds oligonucleotide complexes were captured by immobilized HIF-1α/HIF-2α antibodies. Unlabeled oligonucleotide was used to test assay's specificity. Detection was performed utilizing Streptavidin-HRP and ELISA reader. Cell-based ELISA of Tie-2, CD31 and Flk-1. 2*10^4 BMSCs/well were seeded in 96 wells tissue culture plates coated with 10 µg/ml poly-L-lysine for 30min at 37 °C. Cells were then fixed with 4%, PFA washed with PBS-Tween 0.1%, blocked with 5% BSA and incubated for 2hrs with specific antibodies (1μg/ml). After washing, cells were incubated 1hr with HRP-secondary antibodies (Jackson labs). Detection was performed using TMB substrate and ELISA reader.

Reverse Transcription-Polymerase Chain Reaction. Total RNA was extracted from cultured 3*10^6 BMSCs by phenol/chloroform/isoamyl alcohol (Biological Industries). RNA was treated with DNase I (Ambion). RNA (2µg/reaction) was transcribed using AMV-RT (Promega). Amplification reactions were carried out using REDTaq ReadyMix (Sigma-Aldrich). Reaction conditions were calibrated for each gene and primers (sequences supply by request). Quantification was assessed using Image J software. Adhesion Assays. For adhesion to fibronectin, 10^4 BMSCs/well were seeded for 30mins on 96wells immunoplates pre-coated with fibronectin (Chemicon). For adhesion to H5V cells, H5V were seeded in 96 wells plates and allowed to adhere. 3*10^4 BMSCs were added for 30mins. Fibronectin-coated wells and H5V-only seeded wells served as background, respectively. Non-adherent cells were washed away and adherent cells quantified by XTT based colorimetry (Biological Industries). Migration Assay. 100ng/ml SDF-1 (R&D) or 10ng/ml VEGF165 (R&D) was loaded on the lower chamber of a modified Boyden chamber

(5µm polycarbonate filter; Neuroprobe). 5x10^4 BMSCs in 50µl serum free media were loaded into the top chamber. Migrating cells were fixed and stained with Diff-Quick Stain (Dade Behring AG), and counted in 4 high-power fields (x20). For CXCR4 and Flk-1blockade, BMSCs were pre-incubated 1hr at with 5μg/ml AMD3100 or 50nM SU5416, (Sigma-Aldrich), respectively. Matrigel Tube Formation Assay. 2x10^5 BMSCs or, alternatively, 10^5 BMSCs mixed with 10^5 H5V cells/well were seeded on 24well plates coated with 200μl Matrigel (BD). Tube formation was scored after 6hrs by microscopy (x40) as follow; 1, individual cells, well separated; 2, cells migrate and attach; 3, cells begin to form incomplete round structures; 4, one closed, delineated, ring-shaped structure appears; 5, multiple closed circular structures formed.

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Antibody Array. RayBio® Mouse Cytokine Array C series 1000 assay was performed according to manufacturer's instructions. Briefly, H5V whole cell extract proteins (350µg) were incubated with arrayed antibody membranes, which were exposed to specific biotin-antibody cocktail. Signals were detected using labeled-streptavidin and exposure on X-OMAT AR films (see supplemental data). Data analysis was performed using ImageJ. Significance threshold was set as 2 fold ± SD (p<0.05). In Vivo Corneal Micropocket Angiogenesis Assay. Elvax (ethylene vinyl acetate copolymer; Aldrich) was dissolved in ethylene chloride (100mg/ml) and dried in a laminar air flow hood. 1x1x0.1mm polymere pellets were embedded 50mins in 50ng/μl rhbFGF (Calbiochem), dried and stored at -20ºc. Rats were anesthetized using Xylazine 10mg\kg and Ketamine 90mg\kg. A linear keratotomy 1-1.5mm centrally to the temporal limbus was made. Stromal micropocket (1.2mm²; average depth 200μm) was dissected and pellet was incorporated. Immediately after pellet implantation rats (n=7) were injected with BMSCs 3.5x10^6/0.5ml in NS or NS only via the femoral vein. Vascular Density and Cell homing Assessment. At day 8, vascular index was obtained by the multiplication of two measurements: 1. Maximal vessel length extending from limbus towards the micropocket 2. Circumferential neovascularization zone, as clock hours. Animals were sacrificed, eyes enucleated and 5μm thick paraffin sections prepared. Vascular density was evaluated using isolectin B4 (Sigma-Aldrich) and H&E staining. Quantification was performed in high-power fields (x20; n=10), employing ImageJ (vessels number x mean vessels area). For homing assesment, sections were incubated with anti-GFP polyclonal antibody, 2μg/ml, for 1hrs. After washing, FITC-secondary antibody was added for 1hr. Incubation with secondary antibody only served as negative control. Slides were washed, mounted on cover-slips and analyzed by fluorescent microscopy (x60).

Statistical analysis - Comparison between groups was performed employing the One way ANOVA test. Level of significance was set at p<0.05(*) (** = p<0.005). Results are expressed as mean ± SD.

RESULTS Culture and characterization of BMSCs. Rat adherent BM mononuclear cells were cultured under endothelial selective conditions. Colonies appear after 5-6 days and after 9-10 days cells exhibited an endothelial-like morphology and about half of the cells incorporated DiI-ac-LDL and bind FITC-lectin Ulex europaeus. At day 16, flow cytomerryt analysis revealed high capacity of ac-LDL uptake and UE lectin binding and cells formed cord-like structure on Matrigel (Figure 1.A,B). Additionally, cells showed no expression of the hematopeitic surface markers CD45 and CD11b, whereas marked expression of CD90 and CD29 was detected. The cells expressed abundantly the endothelial markers CD31, Tie-2 and Flk-1 by immunofluorescence (Figure 1.C) and cell-based ELISA (see below, Figure 2.A). Retrovirus-mediated constitutive expression of HIF-1α and HIF-2α in BMSCs. In order to induce HIF-α constitutive expression in day 12 BMSCs, we employed retroviruses encoding mutant forms HIF-1αP564A and HIF-2αP531A, in which mutation of proline 564 and 531 to alanine leads to enhanced HIF-α stability [11]. Efficiency of transduction was detected by GFP expression, and routinely attained 80-85% in all groups (Figure 1.D). Whereas endogenous rat HIF-α mRNA did not show altered expression following transductions, human HIF-α was strongly induced (Figure 1.E). BMSCs nuclear extracts were then analyzed for their ability to bind a double stranded oligonucleotide containing HRE DNA binding site of HIF (Figure 1.F). An increased activity of HIF-1 and HIF-2 was measured following transduction, which was significantly elevated following infections with HIF-1αP564A and HIF-2αP531A mutants, compared to HIF-α wild type forms.

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HIF-α promote differentiation of BMSCs. The effect of HIF-1α versus HIF-2α on the phenotypic differentiation of BMSCs was assessed by cell-based ELISA of endothelial markers (Figure 2.A). Compared to H5V cells, wild type BMSC's relative expression of Tie-2 and CD31, was 34±2% and 40±3% after 8 days in culture, and reached 55±3% and 71±6% at day 16, respectively. HIF-1α transduction on day 12 contributed to Tie-2 and CD31 expression on day 16, showing 73±2% and 86±9%, respectively (p<0.005). A prominent increase in Tie-2 and CD31 was measured following HIF-2α transduction, showing 82±3% and 96±5% relative expression, respectively, compared to H5V cells (p<0.005). The functional aspect of BMSCs differentiation was determined by assessing BMSC's adherence to fibronectin and H5V cells (Figure 2.B). An increase of 2.15±0.63 and 2.76±0.84 fold in the adhesive capacity of BMSCs to fibronectin was evident following HIF-1α and HIF-2α overexpression, respectively (p<0.005). Likewise, adhesion to ECs was 3.85±0.F31 and 5.17±0.38 fold higher in BMSCs transduced with HIF-1α and HIF-2α, respectively (p<0.005). HIF-1α and HIF-2α improve migration of BMSCs trough SDF-1/CXCR-4, whereas only HIF-2α induces VEGF/Flk-1 migration pathway. We next investigated HIF-α effect on two well established migration pathways of progenitor and ECs: SDF-1/CXCR-4 and VEGF/Flk-1 pathways [12] [13]. Migrating BMSCs towards SDF-1 (100ng/ml) increased by 4.87±0.68 and 3.72±0.76 fold following HIF-1α and HIF-2α transduction, respectively (p>0.005). The increased migration correlated with CXCR-4 mRNA levels (Figure 3.A) and was abolished by the CXCR-4 blocker AMD3100 (5μg/ml) (Figure 3.B). Interestingly, the novel SDF-1 receptor CXCR-7 (RDS) was induced in BMSCs by both HIF-1α and HIF-2α. Migration towards VEGF (10ng/ml) was significantly improved in HIF-2α-BMSCs (7.5±1.2), but not HIF-1α-BMSCs (Figure 3.C). Analysis of mRNA levels of VEGF receptors exhibited an increase in Flt-1 in both HIF-1α and HIF-2α groups. However, Flk-1

transcription was higher only in BMSCs expressing HIF-2α. Pre-incubation with the Flk-1 specific inhibitor SU5416 reduced HIF-2α-induced migration to baseline. Flk-1 cell-based ELISA (Figure 3.D) showed no significant up-regulation during the culture period or following HIF-1α stabilization. However, HIF-2α strongly promoted Flk-1 expression, comparable to that of H5V cells. HIF-1α and HIF-2α constitutive expression differentially intensify the paracrine properties of BMSCs. Following migration to ischemic sites, BMSCs activate resident ECs by local secretion of angiogenic factors [14]. We thus investigated the differential effects of HIF-1α versus HIF-2α on VEGF, SDF-1, bFGF, PDGF-B and IGF-1 transcription in BMSCs (Figure 4.A). VEGF and SDF-1 were up regulated at distinct levels by both HIF-1α and HIF-2α. bFGF was strongly expressed in BMSCs, however, it was not affected by HIF-α expression. PDGF-B and IGF-1 showed increased expression only in HIF-2α-BMSCs. In order to study the potential consequences of the above paracrine alterations, we performed a matrigel tube formation assay (Figure 4.B). In this assay, BMSCs (10^5) and H5V cells (10^5) were mixed and plated on matrigel coated dishes. In addition, serum free media collected from BMSCs at day 16 was applied on matrigel co-culture. This assay allowed us to examine the ability of BMSCs to incorporate into growing vessels and their paracrine effects on the vasculogenic capacity of endothelial cells. The relative contribution of BMSCs to tube formation was considerable, as assessed by GFP-positive cells detection, but did not differ significantly between cell groups. However, the total tube formation ability was markedly increased in cultures containing HIF-1α and, to a greater extent, HIF-2α-BMSCs. We next performed a cytokine array of H5V ECs stimulated with conditioned media collected from BMSCs (see supplemental data). Among 96 analyzed proteins, H5V cells subjected to HIF-α-BMSCs medium differentially expressed 17 secreted proteins, membrane receptors and adhesion molecules [≥2 fold modification set as threshold (p<0.05);

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Figure 4.C]. The expression of several angiogenic proteins was induced by both HIF-1α and HIF-2α-BMSCs, such as Flt-1, Flk-1, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1). While HIF-1α-BMSCs uniquely promote the expression of VEGFR-3 and KC, proteins induced only by HIF-2α-BMSCs included VEGF, IGF-1, Eotaxin-2, LIX, M-CSF and LeptinR. HIF-α transduced BMSCs paracrinely promote the adhesion capacities of mature ECs. Focusing on adhesion molecules induced by HIF-α-BMSCs, flow cytometry analysis of VCAM-1 and ICAM-1 expression on H5V grown 72hrs in medium of HIF-α-BMSCs was performed (Figure 5.A). Both HIF-α-BMSCs dose dependently induced the expression of VCAM-1 and ICAM-1. Consequently, we tested the capacity of H5V cells, stimulated by medium of BMSCs, to attach wild type BMSCs (Figure 5.B). An improvement of 2.8±0.48 and 5.47±1.06 fold in adhesion capacity was evident in H5V grown in medium of HIF-1α and HIF-2α-BMSCs, respectively (p<0.005). Delivery of HIF-2α-transduced BMSCs more potently promotes neovascularization compared to HIF-1α. We next assessed the effects of delivery of HIF-α-BMSCs on angiogenesis in a corneal micropocket assay. On day 0, bFGF containing Elvax pellets were implanted in rat corneas to initialize local neovascularization and systemic delivery of 3.5x10^6 BMSCs was performed immediately. After 8 days the vascular zone was measured and revealed a 1.3±0.44, 2.9±0.57 and 3.9±0.55 fold increase in neovascularization in rats injected with BMSCs transduced with control vector, HIF-1α and HIF-2α (p<0.005), respectively, compared to animals implanted with pellets only (Figure 6.A). These observations were further confirmed by vascular density evaluation employing H&E (not shown) and isolectin B4 staining (Figure 6.B). GFP detection in corneal sections (Figure 6.C) revealed an increased number of HIF-1α and HIF-2α-BMSCs, compared to controls (control vector: 3±0.82, HIF-1α: 4.50±0.58, HIF-2α: 5.25±0.96 cells/per x60 field, p<0.05)

both in perivascular zone and incorporated within vessel wall. The percentage of vessels containing GFP+ cells per x60 field was found to be increased in sections of animals transferred with HIF-1α and HIF-2α-BMSCs (LZRS: 9±3.16%, HIF-1α: 13.75±3.4% and HIF-2α: 15.5±3.87%, p<0.05).

DISCUSSION Several studies have demonstrated virus-based genetic modifications to augment the therapeutic potential of bone marrow-derived progenitors [4] [5]. Here, we show for the first time retrovirus-mediated constitutive expression of transcription factors, in BM-derived BMSCs. Such an approach has the advantage of inducing a broad pro-angiogenic program instead of focusing on a single gene, thus potently affecting both the transduced and neighboring cells. HIF transcriptional activity has been previously shown to play a role in embryonic [15] [10] and adult [16] [17] stem cell differentiation and function. We observed basal levels of HIF-1α and HIF-2α in BMSCs after 10-16 days in culture. Nevertheless, our findings show that HIF-2α is more potent in promoting differentiation and vasculogenic properties of BMSCs. Indeed, HIF-2α was shown to be highly expressed in the endothelial lineage and to regulate key functional genes [18] [19]. HIF-2α may thus play a role in the differentiation of BMSCs into mature ECs. The migratory capacity of BMSCs is of considerable importance in the application of systemic cell therapy for local neovascularization. Both HIF-1α and HIF-2α improved migration towards SDF-1 via up regulation of CXCR-4, previously described as HIF-target in cancer cells [20], while its inhibition abolished the induced migration. Of note, SDF-1 up-regulation in HIF-α-BMSCs did not reverse migration through the SDF-1 gradient applied in the Boyden chamber, implying for the magnitude of CXCR-4 up-regulation, driving BMSCs toward SDF-1 higher concentration. A marked increase in mRNA levels of CXCR-7, recently associated with SDF-1-induced chemotactic signals [21],

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was also noticed, raising interesting questions about HIF involvement in CXCR-7 transcription. Increased Flk-1 expression was induced mainly by HIF-2α, probably accounting for the increased chemotaxis towards VEGF in this group. Flk-1 inhibition resulted in a striking decrease in HIF-2α-induced migration. VEGF/Flk-1 is a well established migration pathway of mature ECs [12], previously described as regulated by HIF-2α [18, 19]. Up-regulation of this pathway corresponds with the early maturation observed in HIF-2α-BMSCs. Apparently, the incorporation of stem cells into growing vessels does not solely account for the enhanced angiogenic response induced by BMSCs. Consistent with the notion that BMSCs and ECs work in concert during vasculogenesis, we investigated the crosstalk between those two cell populations. The angiogenic paracrine activity of BMSCs noticeably accounts for their contribution to vasculogenesis [14]. We found that HIF-1α and HIF-2α potently promote the transcription of angiogenic factors in BMSCs. Whereas VEGF and SDF-1 were similarly up regulated by HIF-1α and HIF-2α, PDGF-B and IGF-1 showed increased transcription only in HIF-2α-BMSCs. Consistently, conditioned media from HIF-1α and HIF-2α-BMSCs induced differential angiogenic response when applied on ECs. Compared to HIF-1α, HIF-2α drives a more potent paracrine activity, resulting in enhanced expression of angiogenic receptors and cytokines. Consequently, advanced tubular organization in co-cultures of HIF-α-BMSCs and H5V was evident. Importantly, upregulated proteins in ECs exposed to HIF-α-BMSCs media comprised of adhesion molecules VCAM-1, ICAM-1 and E-Selectin, pivotal in recruitment of BMSCs [22] [23] [24], accompanied by an increased capacity to attach to BMSCs.

Eventually, we employed the corneal micropocket assay for the assessment of neovascularization following transduced-BMSCs administration. In contrary to other angiogenesis models, the cornea, which is physiologically a-vascular, allowed us to perform an accurate and clear evaluation of the newly-formed blood vessels during the experiment time course. The initiation of the angiogenic process was achieved by bFGF release from the implanted pellet, since no variation in bFGF expression was observed in the RT-PCR analysis, allowing an equal chemotactic gradient between cell groups. Following local angiogenic activation, ECs secrete multiple angiogenic factors which drive BMSCs to migrate toward the angiogenic site [1] [2] [3]. Consistent with our previous findings, we found a significantly improved capacity of HIF-1α and, particularly, HIF-2α transduced-BMSCs to migrate, incorporate and induce vessel formation. Importantly, the relatively low incorporation of BMSCs in the growing vessels suggests that physical assembly per se was not a major contributor to neovascularization obtained by the transfer of BMSCs. This finding along with our in vitro data regarding the paracrine effects of HIF transduced BMSCs may point out these indirect effects as being influential in neovessel growth. In conclusion, we suggest HIF-α constitutive expression as a tool to improve the contribution of transferred BMSCs to vessel formation. The main aspects shown to be improved by HIF-α activity in BMSCs-mediated angiogenesis are summarized in Figure 7. We also demonstrate here, for the first time, the superiority of HIF-2α over HIF-1α in the regulation of differentiation and function of BMSCs.

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Figure 1. Characterization and retroviral transduction of BMSCs. BMSCs morphology was analyzed by microscopy at days 0, 6, 12, 16. On day 16, cord-like structures on Matrigel was assessed (A). DiI-ac-LDL uptake and FITC-UE-lectin binding capacities were assessed by flow cytometry at days 8 and 16 as well as the expression of CD45, CD11b, CD90 and CD29 at day 16 (B). Additionally, cells were submitted to ac-LDL/UE-lectin, CD31/Tie-2 or CD31/Flk-1 co-staining by immunofluorescence (C). HIF-α transduction: Day 12-BMSCs were infected with LZRS-hHIF-1αP564A/hHIF-2αP531A -IRES-eGFP or empty virus. Transduction efficiency was determined by GFP fluorescence and flow cytometry analysis (D). Levels of rat (r) and human (h) HIF-1α and HIF-2α were assessed by RT-PCR using corresponding primers (E). GA3PDH - house keeping gene. hHIF-1/2α DNA binding activity was assessed by ELISA (F).

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Figure 2. HIF-α promotes BMSCs differentiation. On day 16, BMSCs were analyzed for endothelial marker expression and adhesion capacity. For CD31 and Tie-2 expression, BMSCs were submitted to cell-based ELISA. H5V served as positive control (A). For adhesion assays, BMSCs were allowed to adhere to fibronectin-coated and H5V-seeded wells, respectively (B). Non-adherent cells were then washed away and quantified by XTT. Fibronectin-coated wells and H5V-only seeded wells served as background, respectively.

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Figure 3. The effect of HIF-1α and HIF-2α on BMSCs migration capacities. Following HIF-α transduction, SDF-1/CXCR-4 and VEGF/Flk-1 migration pathway were assessed. The mRNA expression of CXCR-4, CXCR-7, Flt-1 and Fk-1 was detected in each group by RT-PCR (A; right panel). GA3PDH - house keeping gene. OD ± SD, assessed using Image J, was standardized compared to wild type BMSCs (A; chart). For migration assays, BMSCs were allowed to migrate towards SDF-1 (100ng/ml) (B) or VEGF (10ng/ml) (C) in a modified Boyden chamber. Migrating cells were counted in 4 high-power fields (x20). For inhibition of CXCR4 and Flk-1, BMSCs were pre-incubated 1hr at 37°C with 5μg/ml AMD3100 or 50nM SU5416, respectively. Flk-1 protein expression was assessed by cell-based ELISA on day 8 and 16 (D). H5V served as positive control.

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Figure 4. HIF-1α and HIF-2α differentially contribute to BMSCs paracrine activity. HIF-1α and HIF-2α effect on the transcription of VEGF, SDF-1, bFGF, PDGF-B and IGF-1 in BMSCs was assessed by RT-PCR (A; right panel). GA3PDH - house keeping gene. OD ± SD, assessed using Image J, was standardized to wild type BMSCs (A; chart). For tube formation, H5V cells and BMSCs were seeded on Matrigel (B). Wells were examined after 6hrs by phase-contrast and fluorescent microscopy (x40) and scored 1-5 (1- separated cells, 5- closed polygons). For cytokine expression analysis H5V were grown for 72hrs in HIF-α-BMSCs or control media and arrays performed (C). Areas in the array in which modifications occurred appear in white boxes. Data analysis was performed using Image J. Proteins showing an induction/repression of ≥2 fold in one or two analyzed groups (HIF-1α and HIF-2α) are presented in the chart. Values are presented as fold of LZRS empty vector ± SD (p<0.05) (representative protein arrays membranes appear in supplemental data).

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Figure 5. Conditioned medium of HIF-α expressing BMSCs induce EC adhesion. H5V cells were subjected to media collected from HIF-α-BMSCs or control for 72hrs and seeded until confluence. BMSCs were added to wells for 30mins. Non-adherent cells were washed away and adherent cells quantified by XTT colorimetry (A). H5V-only seeded wells served as background. VCAM-1 and ICAM-1 expression in each H5V cells group was analyzed by flow cytometry (B). HIF-α-BMSCs media were diluted 1:0, 1:1, 1:2 and 1:4 with serum free medium before applied on H5V cells.

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Figure 6. HIF-1α and HIF-2α promote BMSCs-mediated angiogenesis in vivo- corneal micropoket assay. On day 0, bFGF (50ng/μl) containing Elvax pellets were implanted in rats cornea (n=7). Simultaneously, systemic delivery of 3.5x10^6 BMSCs was performed. On day 8, corneas were analyzed (A; right panel) and 'vascular index' was calculated as described in methods (A; chart). Paraffin sections were stained with isolectin B4 for vascular density assessment (B; right panel). Quantitative analysis was performed in high-power fields (x20; n=10), employing Image J [identifiable vessels number x mean vessels area (MVA)] (B; chart). Following GFP immunostaining (C; right panel), total GFP+ cells homing (C; upper chart) and the percentage of vessels containing GFP+ cell/s (C; lower chart) was assessed per x60 field by examination of corneal sections (p<0.05).

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Figure 7. HIF-α contribution to BMSCs-mediated angiogenesis. Described are the different stages in BMSCs function improved by HIF-α constitutive expression; 1. Migration to site of neovascularization; 2. Adhesion to ECs and extracellular matrix; 3. Paracrine activity- resulting in increased angiogenic activation of resident ECs; 4. EC activation results in increased expression of adhesion molecule and improved attachment and homing of BMSCs.

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DOI: 10.1634/stemcells.2008-0369 published online Aug 7, 2008; Stem Cells

Levkovitch-Verbin, Gad Keren and Jacob George Barzelay, Sylvie Polak-Charcon, Eldad Tzahor, Iris Barshack, Adiel Barak, Hani

Jeremy Ben-Shoshan, Shulamit Schwartz, Galia Luboshits, Sofia Maysel-Auslender, Aya Cells Differentially Promote their Pro-angiogenic Properties

Constitutive Expression of HIF-1{alpha} and HIF-2{alpha} in Bone Marrow Stromal

This information is current as of August 13, 2008

& ServicesUpdated Information

http://www.StemCells.comincluding high-resolution figures, can be found at:

Supplementary Material http://www.StemCells.com/cgi/content/full/2008-0369/DC1

Supplementary material can be found at:

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http://www.StemCells.com

http://www.StemCells.com/cgi/content/full/2008-0369/DC1


stem cells tissue specific stem cells - tasmc.org.illibrary.tasmc.org.il/ · hif-1α and hif-2 α...

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