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This article was guest-edited by Ajay Shah, King's College London. Aims. It is well ...... Keller G, Kennedy M, Papayannopoulou T, Wiles MV. Hematopoietic ...
Cardiovascular Research (2010) 86, 37–44 doi:10.1093/cvr/cvp385

The 5-lipoxygenase pathway regulates vasculogenesis in differentiating mouse embryonic stem cells Andreas Finkensieper 1*, Sophia Kieser 1, Mohamed M. Bekhite 1, Madeleine Richter 1, Joerg P. Mueller 2, Rolf Graebner 3, Hans-Reiner Figulla 1, Heinrich Sauer 4, and Maria Wartenberg 1 1 Cardiology Division, Clinic of Internal Medicine I, Friedrich Schiller University, Erlanger Allee 101, Jena D-07743, Germany; 2Centre of Molecular Biomedicine, Institute of Molecular Cell Biology, Friedrich Schiller University, Jena, Germany; 3Institute for Vascular Medicine, Friedrich Schiller University, Jena, Germany; and 4Department of Physiology, Faculty of Medicine, Justus Liebig University, Giessen, Germany

Received 25 September 2009; revised 20 November 2009; accepted 2 December 2009; online publish-ahead-of-print 4 December 2009 Time for primary review: 18 days This article was guest-edited by Ajay Shah, King’s College London

Aims

It is well established that leukotrienes (LTs), products of the 5-lipoxygenase (5-LO) pathway, participate in inflammatory tissue reactions and immune responses. In the present study, the impact of the 5-LO pathway on vasculogenesis of mouse embryonic stem (ES) cells was investigated. ..................................................................................................................................................................................... Methods Immunohistochemistry studies demonstrated that 5-LO+ cells first appeared at day 6 of embryoid body (EB) formation from ES cells. 5-LO+/CD68+ as well as 5-LO+/CD45+ cells were prominent at day 10 of EB differentiation. and results Real-time PCR and western blot analysis revealed all constituents of the 5-LO pathway. High performance liquid chromatography analyses indicated the synthesis of LTB4 and LTD4 in conformity with induction of the 5-LO pathway. Furthermore, Flk-1+/CD105+ cells displayed calcium- (Ca2+) transients in response to LTB4, whereas CD11b+ cells responded to LTD4. Treatment of EBs with LTB4 and LTD4 resulted in phosphorylation of the mitogen-activated protein kinase ERK1/2. Pharmacological inhibition of the 5-LO pathway and stable shRNA targeting of 5-LO-activating protein decreased capillary cell areas positive for PECAM-1. ..................................................................................................................................................................................... Conclusion Our data demonstrate that the 5-LO pathway emerges early during ES cell differentiation into cells of the myeloid lineage and that LTs play an until now unrecognized role in vascular development of ES cells.

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Embryonic stem cells † Embryoid body † Leukotrienes † 5-Lipoxygenase † 5-Lipoxygenase activating protein

1. Introduction Previous studies have suggested a close connection between inflammatory processes and modulation of stem cell differentiation towards angiogenesis.1 Regarding embryonic development the first distinct aseptic inflammation takes place when the fertilized ovule starts to implant in the uterus.2 This process is linked to many hormonal and additional cytokine reactions on the maternal side.2 Other physiological situations which display connection between inflammation processes and angiogenesis are wound healing, neovascularization after infarction and chronic allergic reactions.3,4 The interaction between inflammatory cells, endothelial precursor cells, and endothelial cells plays a crucial role at the local inflammation site.5,6

An important group of inflammation mediators are the leukotrienes (LTs). These potent inflammatory agents are derived from arachidonic acid (AA) and are primarily produced by phospholipase A2 and 5lipoxygenase (5-LO) in inflammatory cells, e.g. monocytes, macrophages, and granulocytes, or by transcellular metabolism.7 – 9 The initial step of LT synthesis from AA is carried out by 5-LO in conjunction with 5-LO-activating protein (FLAP) to form the epoxide intermediate LTA4.10 Although FLAP does not have enzymatic activity, it transfers AA to 5-LO protein.11 Downstream in the cascade LTA4 can be hydrolyzed by LTA4 hydrolase to LTB4, or conjugated with reduced glutathione by LTC4 synthase to generate LTC4. This LT can further be modified by sequential steps to yield LTD4 and LTE4. Collectively, the LTs C4, D4, and E4 are known as cysteinyl LTs

* Corresponding author. Tel: +49 3641 932 4139; fax: +49 3641 932 5812, Email: [email protected] Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2009. For permissions please email: [email protected].

38 (CysLTs).12 They mediate their biological activity via high affinity G-protein-coupled putative seven membrane domain-type cell surface receptors, leading to an increase of intracellular Ca2+ and reduction of intracellular cAMP levels.13 In the absence of Ca2+, 5-LO only has marginal catalytic activity.14 The LT family reacts with different LT receptors (LTRs) which differ in their agonist activation capacity and affinity. LTB4 interacts with the BLT1 or BLT2 receptor. The CysLTs interact with at least three different CysLT receptors: CysLT1-R, CysLT2-R, and GPR 17.15 Until now the biological functions of 5-LO, LTs, and LTRs in vasculogenesis of ES cells have not been investigated, although there is distinct evidence in the literature showing that LTs can promote angiogenesis under certain conditions.16 During recent years, it has been shown that embryoid bodies (EBs) derived from pluripotent ES cells of murine and human origin differentiate into endothelial cells17 as well as different hematopoietic lineages including leucocyte subtypes, e.g. monocytes/macrophages,18,19 T-lymphocytes,20 NK cells,21 and dendritic cells.22 In the present study, we show that LTs generated from cells of the myeloid cell lineage regulate vasculogenesis of ES cells, thus corroborating the current opinion that a pro-inflammatory tissue micromilieu favours the differentiation of stem cells towards the endothelial cell lineage.

2. Methods 2.1 ES cell culture and EB formation The pluripotent murine embryonic stem cell line CCE (StemCell Technologies, NA) was cultured on murine irradiated fibroblast cells in basal Iscove’s medium (Biochrom, Berlin, Germany) supplemented with 15% heat-inactivated foetal bovine serum (Sigma-Aldrich, Taufkirchen, Germany), 1% non-essential amino acids, 2 M L-glutamine, 1% sodium pyruvate (all from Biochrom), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich), 100 U/mL penicillin and 100 mg/mL streptomycin (Biochrom) and 1000 U/mL leukaemia inhibitory factor (LIF, Millipore NA) in a humidified environment containing 5% CO2 at 378C. For differentiation, adherent cells were enzymatically dissociated using 0.25% Trypsin with 1 mM EDTA in HBSS (Gibco, Karlsruhe, Germany) and seeded in 250 mL siliconized spinner flasks (Integra Biosciences, Fernwald, Germany) containing medium as described above, but in the absence of LIF. The spinner flask medium was stirred at 22.5 r.p.m. using a stirrer system (Integra Biosciences, Fernwald, Germany).

2.2 Immunohistochemistry, quantitative immunohistochemistry, and confocal laser-scanning microscopy Immunohistochemistry (IHC) was mainly performed with non-adherent (quantitative IHC) EBs. For 5-LO staining, the respective tissues were fixed for 1 h at 48C in 4% paraformaldehyde in PBS and treated for 10 min with 1% Triton X-100. For other antibody reactions, the tissues were fixed in ice-cold methanol/acetone (7:3) for 1 h at 2208C, and washed with PBS containing 0.01% Triton X-100 (PBST, Sigma-Aldrich). As primary antibodies, rat anti-mouse PECAM-1 (CD31) (Millipore, Schwalbach, Germany), rat anti-mouse CD45 (Millipore, Schwalbach, Germany), rat anti-mouse CD68 (Morphosys AbD Serotec, Duesseldorf, Germany), and rabbit anti-mouse 5-LO antiserum (Cayman Chemicals, Ann Arbor, USA) were used. Blocking against unspecific binding was performed for 60 min with 10% fat-free milk powder or 5% bovine serum albumin (BSA) (Carl Roth, Karlsruhe, Germany) dissolved in PBST. The tissues were subsequently incubated overnight at 48C with primary antibodies and thereafter washed with PBST and reincubated with either a goat anti-rat Cy5-conjugated IgG (Millipore) or a Cy3-conjugated goat

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anti-rabbit IgG (Millipore) at a dilution of 1:200 in PBST containing 10% milk powder or 5% BSA. Fluorescence recordings were performed using a confocal laser scanning microscope (LSM 510, Zeiss, Germany) connected to an inverted microscope (Axiovert 135, Zeiss). Fluorescence was determined from the top to a depth of 32 mm of the tissue using sections of 8 mm. Quantitative IHC was analysed as previously described.23

2.3 Western blot analysis EBs were washed in phosphate-buffered saline (PBS, pH 7.4) and dissolved in ice-cold standard lysis buffer containing protease inhibitors and 1% phosphatase inhibitor cocktail (Sigma-Aldrich). The samples were sonicated twice and centrifuged at 12 000g for 15 min at 48C. Protein concentration was assessed by the method of Bradford. Proteins were separated in acrylamide Tris– HCl gels and transferred to a nitrocellulose membrane (Amersham, Munich, Germany). Unspecific bindings were blocked either with 20% dried fat-free milk in PBS containing 0.1% Tween 20 or with 5% BSA. Incubations with primary antibodies were carried out at 48C overnight using rabbit anti-mouse CysLT1-R, CysLT2-R (both dilution 1:500, Cayman, Hamburg, Germany), BLT1-R receptor (1:500, Imgenex, San Diego, USA), BLT2-R (1:500, Cayman), peptide-affinity polyclonal antibody to arachidonate FLAP (1:1000 Imgenex), ERK1/2, phospho ERK1/ 2, SAPK/JNK, phospho SAPK/JNK, p38 mitogen-activated protein kinase (MAPK) and p38 phospho MAPK antibodies (dilution 1:1000 all from Cell Signaling Technology, Danvers, USA). After washing, membranes were incubated with an accordingly HRP-conjugated secondary antibody (dilution 1:2000, Santa Cruz, Heidelberg, Germany) for 1 h at 48C. Protein expression was detected by ECL reagent (Amersham, Munich, Germany) and protein bands were visualized using a Digital Imaging System LAS 3000 (Fujifilm, Japan). Rabbit anti-mouse GAPDH (dilution 1:1000, Abcam, Cambridge, UK) was used as internal control.

2.4 Magnetic cell separation and transient calcium signal measurement CCE EBs were generated and cultivated as described above. EBs were dissociated by incubation for 30 min in serum-free Iscove’s medium containing collagenase type II (2 mg/mL, PAA, Coelbe, Germany) at 378C. For the cell separation, an AutoMACS (Miltenyi Biotec, Bergisch-Gladbach, Germany) was used. The separation procedure was carried out using a Biotin-conjugated rat anti-mouse CD105 antibody (dilution 1:10, SouthernBiotech, Birmingham, USA) and a PE-conjugated rat anti-mouse Flk-1 antibody (dilution 1:10, BD, Franklin Lakes, USA) as well as MicroBeadsconjugated to a rat anti-mouse CD11b antibody (dilution 1:10, Miltenyi Biotec, Bergisch-Gladbach, Germany). For labelling anti-biotin and anti-PE MicroBeads (all from Miltenyi Biotec, Bergisch-Gladbach, Germany) were used. Positive selected cells were plated on gelatinecoated glass plates and incubated at 378C and 5% CO2 for 24 h before analysing them. Cells were loaded with fluo-3/AM and Ca2+-signal measurement was performed in E1-buffer at a Zeiss cLSM 510 equipped with a 488 nm argon laser. The basal [Ca2+]i concentration was monitored for 10 s. After this time, 250 nM of the relevant LT was added to measure the increase of fluorescence as previously described.24

2.5 Real-time PCR Total RNA from CCE EBs was prepared using TRIZOL reagent (Invitrogen, Karlsruhe, Germany) according to manufacturers recommendations followed by genomic DNA digestion using DNase I (Invitrogen, Karlsruhe, Germany). cDNA synthesis was performed using 2 mg RNA and SuperScript RTase II (Invitrogen, Karlsruhe, Germany). Amplifications were performed in a Cycler Optical Module (Applied Biosystems 7500, Foster City, NA) using SYBRw Green (Qiagen, Hilden, Germany). Relative expression values were obtained by normalizing CT values of the tested gene in comparison with CT values of the housekeeping genes using the DDCT method.

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2.6 Down regulation of FLAP using shRNA technique For stable shRNA silencing, pLKO.1-puro (Sigma-Aldrich) derivative plasmids carrying an individual shRNA targeting murine FLAP were separately introduced into CCE cells using HEK293FT-based amphotropic Phoenix packaging cells (Phoenix-Ampho, Invitrogen, Karlsruhe, Germany) and a lentiviral packing mix (Sigma-Aldrich) as previously described.25 Transduction efficiency was assessed via GFPTM control vector (Sigma-Aldrich). A total of three infection rounds were carried out within 24 h. After transduction, the cells were passaged and selected with 2 mg/mL Puromycin (Sigma-Aldrich) for 10 days. FLAP down regulation was analysed by quantitative IHC and western blot.

2.7 Extraction and separation of 20:4 metabolites For the extraction of LTs, CCE EBs were taken from spinner flask and washed two times with serum free basal Iscoves medium. After that they were incubated for 15 min with 5 mM calcium ionophor A23187 and 50 mM AA (Sigma-Aldrich) for a maximum yield. The 20:4 metabolites were purified and separated by reverse-phase high performance liquid chromatography (HPLC) using a C-18 column (Merck, Darmstadt, Germany) with a mixture of 70:30 (vol/vol) methanol:ammonium acetate. The metabolites were extracted and further purified as previously described.26,27 For LTB4 separation, the solvent (70:30 methanol:ammonium acetate) pH 6.8 was adjusted and the absorbance of column effluents was monitored at 269 nm. Separation of LTD4 was performed with solvent pH 5.6 and absorbance at 280.

2.8 Statistical analyses Data are presented as weighted mean value + SEM with n denoting the number of experiments unless otherwise indicated. In each experiment, at least 50 – 80 EBs were analyzed. t-Test for unpaired data was applied as appropriate. A value of P , 0.05 was considered significant and marked with an asterisk.

3. Results 3.1 5-LO and FLAP are expressed during differentiation of ES cells To examine whether the 5-LO pathway plays a role during ES cellderived EB differentiation, first the relative mRNA expression level of 5-LO and FLAP was examined using semi-quantitative real-time PCR. These analyses were performed from day 2 to day 16 of EB culture (Figure 1). EBs did not express 5-LO or FLAP before day 5 of culture. In contrast, a significant increase of 5-LO and FLAP mRNA expression was observed in a time frame between day 6 and day 10 of culture. IHC studies showed that 5-LO positive cells appear first in 6-day-old EBs (Figure 2A). The relative mRNA level of 5-LO and FLAP decreased after day 10 of cell culture. In contrast, 5-LO positive cells were observed until day 16 of EB differentiation.

3.2 Macrophages and leucocytes express 5-LO in EBs To study the cellular localization of 5-LO within EBs, and to detect cells which potentially produce and secrete LTs, representative IHC double staining against the 5-LO antigen in combination with a specific marker of macrophages (CD68) (Figure 2A and B) and leucocytes (CD45) (Supplementary material online, Figure S1) was performed. It was evident that CD68+ cells first appear at day 8 of EB

Figure 1 Representative relative mRNA expression of 5-LO and FLAP using real-time PCR analysis and polymerase as an internal control in EBs derived from the ES cell line CCE cultured in spinner flasks from day 2 to 16. Asterisk indicates significance (P , 0.05) against day 2 of EB culture (n ¼ 3). Each RNA sample included 50 – 80 EBs.

differentiation. These data were consistent with real-time PCR data of CD68.18 CD68/5-LO double positive cells were detected first at day 10 of culture (Figure 2B). At this time, 20% of differentiated CD68+ cells carried the 5-LO antigen. Single cells positive for CD45 were found from day 9 of EB differentiation. CD45+/5-LO+ cells were detected from day 10 of culture (Supplementary material online, Figure S1). In an early timeframe between day 6 and 8 which included the first appearance of 5-LO, no correlation of 5-LO staining with the examined antigens was found (Figure 2A).

3.3 CCE EBs secrete LTs in a timeframe between day 6 and 10 of differentiation To correlate leucocyte differentiation with LT synthesis, the relative mRNA expression of LTA4 hydrolase and LTC4 synthase was examined (Figure 3). mRNA expression of LTA4 hydrolase, the enzyme which converts LTA4 to LTB4 was hardly expressed until day 5 of EB development. In the following days, the mRNA level increased until day 7. During subsequent cultivation, the expression level declined and remained constant over the time. The LTC4 synthase mRNA level was upregulated from day 6 of culture and remained highly expressed from day 8 to 11. The released amount of LTs reflects the activity of the corresponding enzyme. Therefore, secretion of LTs was measured using HPLC. Highest secretion of LTB4 and LTD4 was observed by HPLC analysis in a timeframe between day 6 and 10 (Figure 4). Notably, the amount of released LTB4 was higher when compared with LTD4. At day 12, LT production decreased sharply and remained low until day 16 of EB differentiation.

3.4 Differentiating ES cells express CysLT1/ 2 as well as BLT1/2 receptors To investigate the time-dependent expression level of the CysLT1/ 2-Rs as well as BLT1/2-Rs, real-time PCR, and western blot analyses were performed (Supplementary material online, Figure S2 and S3). Differentiation dependent protein analysis of the BLT1-R showed an expression in a timeframe between day 6 and 12 (Supplementary

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Figure 3 Relative mRNA expression of LTA4 hydrolase and LTC4 synthase using real-time PCR analysis and polymerase as an internal control in EBs derived from the ES cell line CCE cultured in spinner flasks from day 2 to 16. Asterisk indicates significance (P , 0.05) against day 2 of EB culture (n ¼ 3). Each RNA sample included 50 – 80 EBs.

Figure 2 Representative cryo-sections of 6-day-old (A) and 12-day-old (B) EBs derived from the ES cell line CCE which are stained for 5-LO (red), CD68 (yellow), and DAPI as nuclear control. The white arrow (A) assigns a group of cells in a 6-day-old EB which is positive for 5-LO as well as DAPI. In contrast, the white arrows (B) assign cells which are triple positive for 5-LO, CD68, and DAPI in 12-day-old EBs. Furthermore, there are some cells detectable which express either 5-LO or CD68. Overview (A) 20-fold magnification and detailed view (B) 40-fold magnification.

material online, Figure S2 and S3B). These findings were consistent with the mRNA data. The BLT2-R was detectable first at day 8 of EB differentiation and subsequently continuously expressed until day 16 of differentiation. CysLT1-R and CysLT2-R differ in the rank order of agonist activation. Protein analysis as well as mRNA data showed a temporary higher expression of CysLT1-R in a timeframe between day 6 and 16 during EB development, whereas it was marginally detectable during early stages of differentiation. In contrast, the CysLT2-R, which was hardly detectable during early stages of differentiation, showed a higher protein expression between day 8 and 12 of EB development (Supplementary material online, Figure S2 and S3A).

Figure 4 HPLC analysis of LTB4 and LTD4 using the serum free supernatant of EBs derived from the ES cell line CCE cultured in spinner flasks in a timeframe between day 2 and 16. LTs were extracted 15 min after stimulation with arachidonic acid and Ca2+ionophor A23187. The extraction was carried out following the protocol of Unger et al.26

3.5 A subfraction of CD1051/Flk-11 cells as well as CD11b1 cells express LTRs When cells derived from 8-day-old EBs were treated with either LTB4 or LTD4, a transient Ca2+ response was observed in 3 – 5% of total cells, thus suggesting functionality of the LT signalling pathway in differentiating ES cells (Figure 5A). To study whether macrophages and hematopoietic precursor cells (HPC) react on the different type of LTs, cells were purified using magnetic cell separation technique. After this procedure and a 24 h rest, cells were exposed to different LTs and Ca2+ transients were measured. Approximately 65% of total single cells from differentiating EBs sorted for Flk-1/CD105, i.e. a marker for HPCs implicated in angiogenesis and heart development, showed Ca2+ transients in response

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Figure 5 Ca2+ transients in response to either stimulation with LTB4 (250 nM) or LTD4 (250 nM) of cells (A) derived from 8-day-old EBs. Additional

Flk-1+/CD105+ cells isolated and sorted from 10-day-old EBs showed Ca2+ transients after stimulation with LTB4 (250 nM) (B). mRNA analysis of 5-LO and FLAP transcripts in the Flk-1+/Flk-12 fraction assessed at day 8 of culture (C) demonstrated that Flk-1+ cells displayed only minor 5-LO and FLAP expression. The Flk2 fraction was adjusted to 100%. Asterisk indicates significance (P , 0.05). In contrast, CD11b positive cells isolated and sorted from 10-day-old EBs showed Ca2+ transients after stimulation with LTD4 (250 nM) (D).

to LTB4 (Figure 5B). These Flk-1+/CD105+ cells displayed an elongated endothelial-like phenotype (see false colour images, Figure 5B). These findings indicated that some Flk-1+/CD105+ cells carried at least one of the BLT-Rs and that these cells responded towards LTs. In mRNA analyses of Flk-1 positive cells, it was found that these cells express transcripts of 5-LO and FLAP only on a negligible level (Figure 5C). Notably, a distinct fraction of single cells sorted positive for CD11b, i.e. a marker for granulocytes and macrophages and displaying a round phenotype with multiple small lamellipodiae showed calcium transients in response to LTD4, indicating sensitivity for CysLTs and consecutive expression of either CysLT1-R or CysLT2-R (Figure 5D).

3.6 LTB4 and LTD4 stimulation of 8-day-old EBs is mediated through phosphorylation of ERK1/2 To investigate which type of MAPK was involved in the 5-LO pathway a functional analysis of the 5-LO signalling pathway after stimulation with either 100 nM LTB4 or LTD4 was performed (Supplementary material online, Figure S4). Using western blot analysis, the phosphorylation level of ERK1/2, SAPK/JNK, and p38 was investigated. Stimulation with LTB4 as well as LTD4 showed an involvement of the ERK1/2 pathway in 8-day-old EBs. A phosphorylation of ERK1/2 was observed in a timeframe between 15 and 60 min after stimulation (Supplementary material online, Figure S4A). Notably EKR1/2 phosphorylation after stimulation with LTB4 was not affected by a VEGF-R2 blockade using SU5614 (Supplementary material online,

Figure S4D). No involvement of p38 phospho and SAPK/JNK phospho could be detected. Therefore, it can be concluded that the SAPK/JNK or the p38 pathway was not involved after stimulation with LTB4 or LTD4 when 8-day-old EBs were analysed.

3.7 Pharmacological inhibition of FLAP exerts anti-angiogenic effects in differentiating ES cells According to the working hypothesis of the present study, LT synthesis and signalling play a role in blood vessel differentiation of ES cells. To verify a physiological function of LT secretion on vasculogenesis during EB differentiation, cells were incubated in a timeframe between day 6 and 16 with BAY-u9773 (CysLT-R antagonist with equal affinity to CysLT1-R and CysLT2-R) and MK591 (FLAP inhibitor) at concentrations between 0.5 and 2 mM (Supplementary material online, Figure S5 and S6). Analyses were carried out using western blot, mRNA analysis, and quantitative IHC. MK591 (Supplementary material online, Figure S6) and BAY-u9773 (Supplementary material online, Figure S5) both decreased the endothelial cell marker PECAM-1. Using MK591, the PECAM-1 expression as well as PECAM-1-positive cell area measurement was significantly decreased in 10-day-old EBs, thus indicating that pharmacological inhibition of LT signalling significantly inhibited vasculogenesis of ES cells. To exclude toxic effects of the inhibitors, western blot analysis of caspase-3/ cleaved caspase-3 was performed which, however, did not reveal increased apoptosis (data not shown).

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3.8 Stable shRNA silencing of FLAP in EBs decreases capillary-like areas in EBs during late angiogenesis To exclude potential pharmacological side effects of the used inhibitors, shRNA targeting of FLAP (Supplementary material online, Figure S7) was performed to analyse the effect of gene inactivation of the activator protein on vasculogenesis of ES cells (Figure 6). shRNA targeting of FLAP significantly reduced the cell areas positive for PECAM-1 (Figure 6A and B). In addition to that mRNA transcripts of CD34, CD144 and endothelial nitric oxide synthase (eNOS) were significantly down regulated at day 12 after shRNA targeting of FLAP (Figure 6C). Interestingly, the strongest effects were observed in late stages of vasculogenesis, i.e. between day 8 and 12 of cell culture. In contrast, only minor effects were observed on day 6 where angioblasts are formed and capillary sprouting is a rare event. Taken together these data clearly demonstrate that vasculogenesis of ES cells is controlled by LT-mediated signalling pathways.

4. Discussion Although the relation between inflammation, the cellular immune response, and angiogenesis is well established,28 the potential involvement of stem cells in these processes and the underlying signalling pathways remain elusive. Since stem cells are a potential source for pro-angiogenic therapies in inflamed, ischaemic tissues, the present study was undertaken to investigate the impact of the proinflammatory 5-LO pathway on vasculogenesis of ES cells. Our data show that differentiating ES cells within EBs express all necessary enzymes of the 5-LO pathway and acquire the ability to synthesize all types

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of LTs. Although undifferentiated ES cells were devoid of components of the 5-LO pathway, its initialization was observed around day 6 of spontaneous EB differentiation. First, a more nuclear localization was observed which could be an explanation for an initially higher LTB4 secretion. In later stages, a more cytosolic expression pattern was found. Interestingly, the establishment of LT signalling parallels endothelial differentiation and maturation, early hematopoietic as well as cardiac differentiation processes,17,18,29 thus suggesting mutual interaction of independent differentiation pathways. Around day 6 of EB differentiation, a fraction of cells expressing 5-LO was detectable but was devoid of markers characteristic for specific leucocyte subtypes (e.g. CD68 and CD45). It may be assumed that these early 5-LO+ cells belong to a population of cells which direct differentiation processes in autocrine and paracrine manner through secretion of LTs. After day 12 of differentiation, a sudden drop of LT synthesis was observed which may correlate with the spherical growth limitation of EBs. The time course and physiological significance of LT expression in differentiating ES cells are largely unknown. From studies on adult cells, it is known that 5-LO expression is present in mature macrophages, dendritic cells, foam cells, mast cells, and neutrophilic granulocytes which are located in the asthmatic lung as well as in human atherosclerotic plaques.30 – 32 Our results showed that around day 10 of EB differentiation a subfraction of cells in EBs expresses 5-LO. These cells could be identified as neutrophilic granulocytes, macrophages, or leucocytes. We found an obviously earlier expression and higher expression level of the BLT1-R in comparison to BLT2R. These findings indicate that the BLT1-R is expressed on a larger population of cells in the EB and that some of the LTB activity is mainly mediated through this receptor.

Figure 6 PECAM-1 protein (A and B) as well as CD34, CD144, and eNOS mRNA expression (C) of untreated control EBs in comparison with EBs containing a stable shRNA targeting FLAP (shRNA target 3). (A) Blood vessel-like structures were assessed by quantifying CD31-positive cell areas at day 6, 8, 10, and 12 of EB differentiation (shRNA target 3, n ¼ 5; control, n ¼ 6). Asterisk indicates significance (P , 0.05). Representative PECAM-1 IHC of untreated control EBs in comparison with EBs which include a stable shRNA targeting of FLAP (B) (shRNA target 3). (a – d) Control EBs, (e –h) EBs with shRNA target 3; a + e (day 6), b + f (day 8), c + g (day 10), d + h (day 12).

The 5-lipoxygenase pathway regulates vasculogenesis in differentiating mouse embryonic stem cells

To assess functionality of LT signalling intracellular Ca2+ responses were recorded upon stimulation of specific subtypes of cells with different LTs. Furthermore, the activation of MAPK pathways upon stimulation with LTs was assessed. We found a population of Flk-1+/CD105+ cells responding with a transient Ca2+-signal after stimulation with LTB4, suggesting that Flk-1+/CD105+ cells isolated from murine EBs either express the BLT1-R or the BLT2-R. Comparing the time-dependent protein expression of these two receptors, a predominant BLT1-R expression on early endothelial cells or HSC cells would be feasible, and an influence of LTB4 on HSC differentiation may be assumed. Furthermore, CD11b+ cells responded towards LTD4 with a transient Ca2+ signal. From these data, it can be concluded that a population of monocytes, granulocytes, or macrophages express at least one of the CysLTRs, most likely CysLT1-R, which is prominently expressed during ES cell differentiation. CysLT1-R signalling has been previously shown to be involved in degranulation of eosinophil granulocytes.33 Furthermore, it has been demonstrated that CysLT1-R on monocytes and macrophages may be activated through IL-13 and IL-4.34 CysLT1-R mediated signalling appears to be involved in inflammatory processes, because CysLT1-R2/2 mice showed a reduction in inflammation intensity and vascular permeability.35 Our findings indicate that CysLT2-R protein expression in the murine EBs is only present in a defined timeframe around day 8– 13 of differentiation. Currently, little is known about the function of this receptor. The CysLT2-R as well as the BLT2-R is expressed on HUVEC cells.36,37 Micro-array data suggest after CysLT2-R stimulation an upregulation of inflammatory genes.37 We found that Flk-1+/ CD105+ cells only showed Ca2+ transients after stimulation with LTB4 but not with LTD4. These data suggest that the isolated cells do not hold a functional CysLT2-R. Intracellular Ca2+ signals may be associated to the activation of signalling cascades. Our data provide evidence that LT action in differentiating ES cells is mediated through the ERK1/2 pathway. This signal transduction seems to be independent from VEGF-R2 signalling. Consistent with our findings, a recent report demonstrated that in cultured monocytic cells LTB4 induced a rapid phosphorylation of MAPKs ERK1/2 and SAPK/ JNK.38 ERK1/2 has previously been shown by us to be involved in ES cell-derived vasculogenesis.23 To investigate whether LTs are involved in the regulation of vasculogenesis, EBs were either incubated with pharmacological inhibitors of LT signalling or FLAP was downregulated by a shRNA approach. Under these conditions, PECAM-1 expression and capillary areas, positive for PECAM-1, were significantly decreased. In parallel CD34, CD144 and eNOS transcripts were significantly downregulated in EBs using shRNA targeting FLAP. This demonstrates that the 5-LO/LT system co-regulates late phases of inflammation mediator triggered ES cell-derived vasculogenesis, although 5LO2/2 mice showed a normal embryonic development and fertility.39 Our data suggest that during ongoing differentiation and maturation of hematopoietic progenitor cells, e.g. macrophages, paracrine effects mediated by cytokines or other inflammation mediators may modulate differentiation processes in the ES cells. We hypothesize a model of paracrine interaction of 5-LO positive cells with endothelial cells or endothelial precursor cells in differentiating EBs. This interaction may gain in vivo relevance during embryogenesis or in pathological inflammatory processes where leucocytes including macrophages may blaze the trail for neovascularization or invading endothelial cells through LT signalling. Taking our findings together, the present study provides evidence

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that all components of LT signalling are expressed in differentiating ES cells, and LT signalling pathways are involved in processes of blood vessel formation. Thus, the ES cell system represents an outstanding model for the study of tissue inflammation, for the assessment of the impact of inflammatory processes for neo-angiogenesis and tissue perfusion as well as for the preclinical testing of newly developed anti-inflammatory drugs.

Supplementary material Supplementary material is available at Cardiovascular Research online.

Acknowledgements We thank Prof. Dr A. Habenicht (IVM Jena) for his expert advice. Conflict of interest: none declared.

Funding The work was supported by ‘Interdisziplina¨res Zentrum fu¨r Klinische Forschung (IZKF) an der Medizinischen Fakulta¨t der Friedrich-Schiller-Universita¨t Jena—Rotationsstellen fu¨r Assistenza¨rzte 2008– 2009’ and the ‘Deutsche Stiftung Herzforschung’ project F/08/07 (BIOCARD).

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