Published online before print November 20, 2008, doi: 10.1161/CIRCRESAHA.108.182261
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© 2009 American Heart Association, Inc.
Molecular Medicine |
Proteomics Identifies Thymidine Phosphorylase As a Key Regulator of the Angiogenic Potential of Colony-Forming Units and Endothelial Progenitor Cell Cultures
From the Cardiovascular Division, King’s College London School of Medicine, King’s College London, United Kingdom.
Correspondence to Manuel Mayr, Cardiovascular Division, BHF Centre, King’s College, London, 125 Coldharbour Lane, London SE5 9NU, UK. E-mail manuel.mayr{at}kcl.ac.uk
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Abstract |
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Endothelial progenitor cell (EPC) cultures and colony-forming units (CFUs) have been extensively studied for their therapeutic and diagnostic potential. Recent data suggest a role for EPCs in the release of proangiogenic factors. To identify factors secreted by EPCs, conditioned medium from EPC cultures and CFUs was analyzed using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer combined with offline peptide separation by nanoflow liquid chromatography. Results were verified by RT-PCR and multiplex cytokine assays and complemented by a cellular proteomic analysis of cultured EPCs and CFUs using difference in-gel electrophoresis. This extensive proteomic analysis revealed the presence of the proangiogenic factor thymidine phosphorylase (TP). Functional experiments demonstrated that inhibition of TP by 5-bromo-6-amino-uracil or gene silencing resulted in a significant increase in basal and oxidative stress-induced apoptosis, whereas supplementation with 2-deoxy-D-ribose-1-phosphate (dRP), the enzymatic product of TP, abrogated this effect. Moreover, dRP produced in EPC cultures stimulated endothelial cell migration in a paracrine manner, as demonstrated by gene-silencing experiments in transmigration and wound repair assays. RGD peptides and inhibitory antibodies to integrin
vβ3 attenuated the effect of conditioned medium from EPC cultures on endothelial migration. Finally, the effect of TP on angiogenesis was investigated by implantation of Matrigel plugs in mice. In these in vivo experiments, dRP strongly promoted neovascularization. Our data support the concept that EPCs exert their proangiogenic activity in a paracrine manner and demonstrate a key role of TP activity in their survival and proangiogenic potential.
Key Words: angiogenesis • endothelium • progenitor cells • proteomics • vascular biology
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Introduction |
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Human endothelial progenitor cells (EPCs) are attracting considerable attention in cardiovascular research,1,2 but multiple culture methods from peripheral blood mononuclear cells (PB-MNCs) have been described3–7 and studied for their clinical relevance.6,8–12 EPCs are commonly identified by cell surface antigen expression of CD133, CD34, and the vascular endothelial growth factor receptor-2 (VEGFR-2) (KDR).13 CD34 and VEGFR-2, however, are also expressed in hematopoietic stem cells14; thus, EPCs cannot yet be unambiguously defined. One alternative approach to flow cytometry has used the colony-forming unit (CFU) assay as a surrogate marker for EPCs.15 This method has been fundamental to many of the clinical studies published on EPCs to date, which predominantly reported low numbers of CFUs to be correlated to cardiovascular disease risk. Nonetheless, recent publications have cast doubts about the origin of CFUs by demonstrating that they may be clonally derived from the hematopoietic system, possess myeloid progenitor cell activity, and differentiate into phagocytic macrophages.16 Thus, there is an urgent need to provide a mechanistic underpinning for the correlation between CFUs and cardiovascular disease6,15 and for the beneficial effects of endothelial progenitor transplantation in vivo.11,16
Although it is commonly accepted that circulating progenitors may play an important role in revascularization and angiogenesis, the mechanisms by which they act remain unclear. Currently, there is little evidence of permanent engraftment of EPCs into blood vessels.8,17,18 It has therefore been suggested that EPCs stimulate endothelial repair by exerting a local paracrine effect.3,4,7,19 EPC-secreted factors, however, have not yet been fully characterized at the protein level. Although transcriptome analysis can generate a cell-specific signature,19 it cannot detail true cell phenotypes because of translational regulation and protein degradation. The addition of a proteome analysis offers an opportunity to characterize progenitor cells more comprehensively, leading to a better understanding of their role in vascular biology.20–22
In the present study, we use state-of-the-art proteomic techniques to analyze the secretome of EPC cultures and CFUs by performing a nanoflow liquid chromatography matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF) analysis on culture media conditioned by EPCs. This approach was complemented by difference in-gel electrophoresis (DIGE) and cytokine antibody multiplex array analysis. Among the angiogenic factors revealed by proteomics, was thymidine phosphorylase (TP), also known as platelet-derived endothelial cell growth factor (PD-ECGF). This growth factor and deoxyribose phosphate (dRP), the product of its enzymatic activity, were shown to be essential for EPC survival and paracrine effects on endothelial cell migration and angiogenesis.
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Materials and Methods |
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An expanded Material and Methods section is available in the online data supplement at http://circres.ahajournals.org. Key techniques involved adaptations of previously published protocols, including those for difference in-gel electrophoresis (DIGE)23 and tandem mass spectrometry,24 which are also available on our web site at http://www.vascular-proteomics.com.
CFUs and EPC Culture
Human blood was drawn from healthy volunteers. Informed consent was obtained and the study was approved by the ethics review board of King’s College London. The CFU assay was performed as described previously.15 In brief, PB-MNCs were resuspended in growth medium containing medium 199 with 20% FBS on human fibronectin-coated 6-well plates. After 48 hours, nonadherent cells were replated on human fibronectin–coated plates. For isolation of EPCs, PB-MNCs were separated onto Lymphoprep solution. EPC cultures were obtained using endothelial basal medium (EBM, Clonetics cc-3121, Lonza) containing human VEGF (10 ng/mL), as previously described.6 For secretome analysis, cells were incubated with serum-free culture medium for 24 hours before collection of the conditioned medium on day 7.
Matrix-Assisted Laser Desorption/Ionization Tandem Time-of-Flight Mass Spectrometry
For CFUs and EPCs, conditioned medium was concentrated using a Microcon Ultracel YM-10. A total volume of 1.5 mL conditioned medium was concentrated approximately 50-fold. The samples were digested overnight with trypsin, and the tryptic peptides were separated by nano liquid chromatography on a C18 column (PepMap) with a mobile phase formed from (1) high-performance liquid chromatography (HPLC)-grade water containing 5% acetonitrile and 0.1% TFA and (2) HPLC-grade acetonitrile containing 20% H2O and 0.1% TFA. The HPLC was interfaced to a spotting robot (Dionex Probot). Several hundred fractions per sample were collected on MALDI target plates and mixed with matrix.
Peptides were subsequently analyzed using a TOF/TOF analyzer (4800 ToF/ToF, Applied Biosystems). Results were filtered using ProteinPilot software (Applied Biosystems). Assignments were accepted when the total score was 
2.0 (corresponding to a 99% confidence of the protein identification). Results were further filtered for a minimum of 2 peptides per protein identification.
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Results |
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The Secretome of CFUs and EPC Cultures
CFUs consisted of a central cluster of rounded cells surrounded by multiple thin cells (Figure 1A), which showed endocytosis of acetylated LDL (red fluorescence) (Figure 1B). EPC cultures were characterized by single spindle-shaped cells positive for lectin staining and acetylated LDL uptake (Figure 1C). Both cell types express markers consistent with existing definitions of an EPC phenotype, such as von Willebrand factor, CD31 (platelet endothelial cell adhesion molecule-1), CD133, and VEGFR-2 (KDR) (Figure 1D). Analyses of the secretome of CFUs from 3 independent preparations resulted in the identification of 272 nonredundant proteins present in at least 2 samples (Table I in the online data supplement), of which 124 were also found in cultured EPCs. All identified peptides are provided in supplemental Table II. Secreted factors considered of potential relevance for the function of EPCs in the vascular system are highlighted in the Table. Although some of the factors, such as cathepsins, have previously been reported to contribute to the angiogenic potential of EPCs,25 others, such as the protein S100 family have not been implicated so far. A semiquantitative comparison based on the peptide count indicated that different members of the cathepsin family were present at similar concentrations under both culture conditions, whereas matrix metalloproteinase (MMP)-9 and interleukin (IL)-8 were secreted at higher levels by CFUs than cultured EPCs.
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To further characterize cultured EPCs and CFUs, cellular protein extracts were labeled with Cy-dyes and separated by DIGE (Figure 2A and 2B). Overall, the proteome of CFUs was similar to EPC cultures and showed surprisingly little interindividual variability (supplemental Figure I). The analysis of 53 differentially expressed protein spots (P<0.05, 1-way ANOVA) by ion trap tandem mass spectrometry resulted in the identification of 37 nonredundant proteins (supplemental Table III). Notably, the proangiogenic factor TP,26 although present in both EPC cultures and CFUs, was expressed at higher levels in cellular extracts of CFUs.
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Validation of the Proteomic Findings
Cytokine concentrations in the conditioned media of CFUs (n=15) and EPC cultures (n=13) were quantified using a multiplex assay (supplemental Table IV). IL-8, as indicated by the proteomic analysis (Table), was confirmed to be the most abundant cytokine in the secretome of CFUs (76.1±6.4 ng/mL) and cultured EPCs (1.4±0.4 ng/mL). Its expression, along with other angiogenic factors, was verified by RT-PCR (Figure 3A). TP was further investigated by immunoblotting. This proangiogenic factor was predominantly present in CFUs, but also in EPC cultures and PB-MNCs, but not in human umbilical vein endothelial cells (HUVECs) (Figure 3B and 3C). Immunofluorescence experiments revealed the presence of TP in the cytosol and nucleus (Figure 3D), possibly because of its role in nucleotide metabolism.26
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TP Is a Survival Factor
To evaluate the functional role of TP in EPCs, cells were treated with 5-bromo-6-amino-uracil (5Br-6Am-U), an inhibitor of TP. Inhibition of TP resulted in a significant increase in baseline apoptosis as quantified by histone protein release (Figure 4A) and flow cytometric analysis of annexin V/propidium iodide staining (supplemental Figure II, A). Supplementation with deoxyribose phosphate (dRP), the product of TP, abrogated this effect. dRP also protected EPCs against apoptosis in response to diethyl maleate, a sulfhydryl-reactive agent, which induces oxidative stress by depleting intracellular glutathione levels. In contrast, inhibition of TP by 5Br-6Am-U aggravated the proapoptotic effect of diethyl maleate. These findings were replicated by ablation of TP expression using small interfering (si)RNA-mediated gene silencing (Figure 4B and supplemental Figure II, B). Again, dRP reversed the effect of gene silencing of TP by siRNA on constitutive and stress-induced apoptosis. Knockdown efficiency was assessed by immunoblotting (supplemental Figure III, A). The proapoptotic effect of TP gene silencing was independent of VEGF (supplemental Figure III, B). On the other hand, inhibition of TP by 5Br-6Am-U or treatment with dRP did not alter apoptosis in HUVECs (supplemental Figure III, C), whereas prolonged inhibition (7 days, supplemental Figure III, D) or genetic ablation of TP (supplemental Figure III, E) significantly reduced EPC numbers in culture. Interestingly, dRP increased the expression of Bcl-2 in response to oxidative stress, which mitigates the proapoptotic effects of Bax but had no effect on redox-sensitive p38 mitogen-activated protein kinase (MAPK) signaling (Figure 5 and supplemental Figure IV).
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Deoxyribose Phosphate Stimulates Focal Adhesion Formation and Enhances Integrin β3 Expression
Besides its antiapoptotic effect, TP has been shown to stimulate endothelial cell motility,27,28 providing a likely explanation for its angiogenic activity. Therefore, we analyzed the formation of focal adhesions in HUVECs in response to the conditioned medium from EPCs. The conditioned medium of EPCs significantly increased the number and dimension of focal adhesions (Figure 6A and supplemental Figure V, A). Untreated HUVECs and HUVECs treated with the conditioned medium from TP knockdown EPCs (TP KD) served as controls. Supplementing the conditioned medium from TP knockdown EPCs with dRP restored its ability to enhance focal adhesion formation in HUVECs, confirming that TP-derived dRP is the active compound in the conditioned medium. Notably, treatment with dRP increased endothelial expression of integrin β3, but not integrin β1 or v, whereas other focal adhesion-associated proteins, such as vinculin and vasodilator stimulated phosphoprotein, were not affected (Figure 6B and supplemental Figure V, B).
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TP Stimulates Endothelial Cell Migration
Next, we assessed the effect of TP on endothelial cell motility in a modified Boyden chamber. The presence of EPCs in the bottom compartment stimulated the migration of HUVECs migrating from the top insert (Figure 7A), confirming the importance of paracrine factors in EPC–endothelial cell interactions. The genetic ablation of TP in EPCs by siRNA transfection significantly reduced the number of transmigrating HUVECs. The addition of 50 µmol/L dRP in the bottom chamber, but not in the insert, reversed this effect, suggesting that the product of TP in the conditioned medium of EPCs acts as a chemotactic stimulus on HUVECs. Moreover, the presence of RGD peptides or an anti-integrin vβ3 inhibitory antibody attenuated the chemotactic activity, suggesting that dRP acts via integrins, in particular vβ3 (Figure 7B).
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TP Enhances Wound Healing In Vitro and Angiogenesis In Vivo
The contribution of TP to the stimulatory activity of conditioned medium on endothelial motility was further investigated in a wound-healing assay. The process of endothelial wound-healing was attenuated in the presence of conditioned medium from TP-ablated EPC cultures compared to cultures treated with scrambled siRNA, suggesting that the activity of TP in EPCs promotes HUVEC motility in a paracrine manner (Figure 7C and supplemental Figure VI, A). The presence of an anti–integrin vβ3 inhibitory antibody (supplemental Figure VI, B) or RGD peptides (supplemental Figure VI, C) abolished the difference between conditioned medium from TP-ablated and control EPCs. Finally, the angiogenic effect of TP and its product dRP was investigated by implanting Matrigel plugs into healthy mice and assessing the vascularization of the plugs over the implantation period. Whereas both control plugs and plugs treated with 5Br-6Am-U showed cell invasion, dRP facilitated vessel formation (Figure 7D and supplemental Figure VII).
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Discussion |
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This study is the first to use state-of-the art proteomic techniques to better characterize CFUs and EPC cultures. Although it has become apparent that the markers currently used for defining EPCs are insufficient,14 EPCs have never been studied comprehensively at the protein level. By analyzing their secretome, we provide additional support to the concept that exogenously administered EPCs may augment neovascularization and endothelial repair via paracrine mechanisms.4,19
Paracrine Effects of EPCs
Despite the controversy regarding the origin of EPCs, a contribution of circulating progenitors to blood vessel growth has been shown in different animal models3,11,16,29–31 and administration of bone marrow cells appears to be beneficial in some,32–34 but not all clinical trials conducted so far.35 Although it is commonly accepted that EPCs have potential for use in angiogenic therapies, the mechanisms by which they improve revascularization remain unsettled.36 Their baseline incorporation rate is low and there is currently limited evidence of long-term engraftment of EPCs into newly formed blood vessels.37,38 Additionally, it has been shown that EPCs can cause neovascularization without physically building endothelial structures,39 supporting the concept of a paracrine effect on the vasculature. Therefore, further investigations are required to characterize paracrine factors that could mediate their proangiogenic effects. Our proteomic analysis revealed that CFUs secrete high levels of MMP-9, IL-8, and cathepsins, previously described as characteristics of EPCs.7,25 In addition, we found novel factors, in particular TP, which was further characterized for its functional relevance in the biology of EPCs.
TP Improves EPC Survival
TP is an angiogenic enzyme catalyzing the reversible phosphorolysis of 2'-deoxythymidine to dRP and thymine. The antiapoptotic role of TP has been investigated in cancer cells.40,41 The proposed mechanism of action is not clear, but it has been suggested that the protective effect of TP is mediated by 2-deoxy-D-ribose, a product of dephosphorylation of the primary TP product dRP.40 In this study, we report the presence of TP in CFUs and EPC cultures. Whereas apoptosis of mature endothelial cells was not influenced by dRP, the activity of TP correlated with levels of basal and oxidative stress-induced apoptosis in EPC cultures. Notably, dRP enhances the expression of the antiapoptotic protein Bcl-2, which heterodimerizes with Bax and thereby antagonizes its proapoptotic effect.42,43 Thus, it is likely that dRP conveys resistance to oxidative stress and apoptosis by increasing the Bcl-2/Bax ratio.
TP Mediates Paracrine Effects on Endothelial Cells
It has long been suspected that EPCs may release paracrine factors to enhance endothelial repair. Our proteomic experiments revealed TP to be involved: First, the presence of EPC cultures in the bottom of a Boyden chamber markedly stimulated migration of HUVECs across the membrane, demonstrating the former cell type is able to induce endothelial migration in a paracrine manner. Second, downregulation of TP by siRNA attenuated this paracrine effect, suggesting that TP expression in EPC cultures plays a key role in the stimulation of endothelial migration. This is in agreement with previous observations that supplementation of dRP or TP-expressing tumor cells induce HUVEC chemotaxis.27 Third, the addition of dRP to the bottom of a Boyden chamber with EPCs, but not to the top insert with HUVECs, restored the effect on HUVEC migration. Thus, the product of TP, not the enzyme itself, is the chemotactic agent that stimulates HUVEC migration in the conditioned medium of EPCs. Fourth, the expression of TP was also responsible for the enhanced regeneration of a wounded endothelial monolayer in the presence of conditioned medium from EPCs. The upregulation of integrin β3 and enhanced focal adhesion formation provide a potential mechanism of how dRP regulates endothelial cell motility.28 In summary, although the proangiogenic potential of TP and dRP has previously been established,28,44 our observations demonstrate for the first time that dRP is among the major proangiogenic factors in the conditioned medium of CFUs and EPC cultures. Therefore, the release of dRP at sites of vascular injury is likely to promote endothelial cell migration from intact neighboring regions, which could represent an important mechanism by which EPCs enhance vascular repair. In agreement with this model, the effect of dRP on angiogenesis and neovascularization was further confirmed by our in vivo experiments.
Clinical Implications
The number of CFUs is widely used as a measure of EPC function45 and has been shown to correlate negatively with cardiovascular disease risk factors and positively with vascular function. However, recent evidence casts doubts over the identity of the cells that form the colonies, suggesting they may be derived from hematopoietic rather than endothelial cell precursors.45 This raises an important question: if CFUs do not directly reflect numbers of circulating EPCs, why do they correlate with cardiovascular disease risk? Our data demonstrate that CFUs express and secrete proangiogenic factors. Thus, although CFUs may not represent endothelial precursors per se, the cellular aggregates forming the colonies could still be involved in vascular homeostasis, ie, CFUs may represent a surrogate marker for the proangiogenic potential among PB-MNCs. This alternative explanation would help to reconcile literature documenting the beneficial effects of circulating EPCs on cardiovascular function with the recent finding that CFUs may be hematopoietic rather than endothelial precursors.
Limitations of the Study
There is currently no consensus on which culture conditions are most successful in isolating the effective EPC populations. In the present study, we used 2 of the most commonly used methods, but we cannot rule out that culture conditions used by other investigators may alter protein expression and secretion. In this respect, the present proteomic dataset could serve as a reference and contribute to standardizing EPC cultures. Although mass spectrometry has proven a valuable tool to array secreted proteins, it is important to note that minor components can remain undetected, especially in the presence of intracellular proteins released during cell death in culture.
Conclusion
In summary, the proteomic analysis reported in this study identified TP to be among the main proangiogenic factors in EPC cultures and CFUs, which might facilitate the development of new therapeutic strategies.
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Acknowledgments |
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We thank Dr Dietrich Merkel (Applied Biosystems) for technical assistance.
Sources of Funding
This work was funded by grants from the British Heart Foundation and Oak Foundation. M.M. was supported by a Senior Research Fellowship of the British Heart Foundation.
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this work.
Original received June 26, 2008; revision received November 7, 2008; accepted November 12, 2008.
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