Expansion Of Endothelial Progenitor Cells Research Articles

Endothelial Progenitor Cells: A Brief Update.

An enormous amount of current data has suggested involvement of endothelial progenitor cells (EPCs) in neovasculogenesis in both human and animal models. EPC level is an indicator of possible cardiovascular risk such as Alzheimer disease. EPC therapeutics requires its identification, isolation, differentiation and thus expansion. We approach here the peculiar techniques through current and previous reports available to find the most plausible and fast way of their expansion to be used in therapeutics. We discuss here the techniques for EPCs isolation from different resources like bone marrow and peripheral blood circulation. EPCs have been isolated by methods which used fibronectin plating and addition of various growth factors to culture media. Particularly, the investigations which tried to enhance EPC differentiation while inducing with growth factors and endothelial nitric oxide synthase are shared. We also include the cryopreservation and other storage methods of EPCs for a longer time. Sufficient amount of EPCs are required in transplantation and other therapeutics which signifies their in vitro expansion. We highlight the role of EPCs in transplantation which improved neurogenesis in animal models of ischemic stroke and human with acute cerebral infarct in the brain. Accumulatively, these data suggest the exhilarating route for enhancing EPC number to make their use in the clinic. Finally, we identify the expression of specific biomarkers in EPCs under the influence of growth factors. This review provides a brief overview of factors involved in EPC expansion and transplantation and raises interesting questions at every stage with constructive suggestions.

Promotion of NAD+ recycling by the hypoxia-induced shift in the lactate dehydrogenase isozyme profile reduces the senescence of human bone marrow-derived endothelial progenitor cells

Expansion of bone marrow-derived endothelial progenitor cells (EPCs) in vitro to obtain required cell numbers for therapeutic applications faces the challenge of growing cell senescence under the traditional normoxic culture condition. We previously found that 1% O2 hypoxic culture condition is favorable for reducing senescence of EPCs, but the mechanisms underlying the favorability are still unclear. Here, we found that, compared with normoxia, hypoxia induced a shift in lactate dehydrogenase (LDH) isozyme profile, which manifested as decreased LDH2 and LDH1 and increased LDH5, LDH4 and total LDHs. Moreover, under hypoxia, EPCs presented higher LDH activity, which could promote the conversion of pyruvate to lactate, as well as a higher level of NAD+, Bcl2 interacting protein 3 (BNIP3) expression and mitophagy. Additionally, under hypoxia, knock-down of the LDHA subunit increased the LDH2 and LDH1 levels and knock-down of the LDHB subunit increased the LDH5 level, while the simultaneous knock-down of LDHA and LDHB reduced total LDHs and NAD+ level. Inhibition of NAD+ recycling reduced BNIP3 expression and mitophagy and promoted cell senescence. Taken together, these data demonstrated that 1% O2 hypoxia induces a shift in the LDH isozyme profile, promotes NAD+ recycling, increases BNIP3 expression and mitophagy, and reduces EPC senescence. Our findings contribute to a better understanding of the connection between hypoxic culture conditions and the senescence of bone marrow-derived EPCs and provide a novel strategy to improve in vitro expansion of EPCs.

Prognostic, diagnostic and therapeutic potential of endothelial progenitor cells for patients with ischaemic stroke: Hype or Hope

Ischaemic stroke is a debilitating disease with immense personal, societal and economic impact. Thrombolysis with recombinant tissue plasminogen activator remains the only approved pharmacotherapy for this disease. As each year less than 1% of eligible patients receive this therapy worldwide, efficacious new therapeutics are desperately needed. Emerging evidence suggest endothelial progenitor cells (EPCs), capable of repairing damaged vasculature, as one such therapeutics. However, questions regarding their optimal dose, delivery route and in vivo survivability remain largely unanswered. Outgrowth endothelial cells, generated in large numbers by ex vivo expansion of EPCs, enable effective assessment of these issues and may eventually serve as off-the-shelf therapeutics. Correlations between circulating EPC levels and stroke outcome imply that EPCs may also serve as clinical biomarkers for stroke. This viewpoint briefly evaluates the current evidence, pinpoints the gaps in the literature and proposes new directions for research.

Insufficient production of IL-10 from M2 macrophages impairs in vitro endothelial progenitor cell differentiation in patients with Moyamoya disease

Moyamoya disease (MMD) is well known to be caused by insufficient cerebral vascular formation. However, the essential pathogenesis has not yet been identified. Using our recently developed technique of generating vasculogenic and anti-inflammatory cultures, we investigated endothelial progenitor cell (EPC) expansion and differentiation under the cytokine milieu generated by the peripheral blood mononuclear cells (PBMNCs) of the operated and non-operated MMD patients. EPC colony forming assay of the cultured PBMNCs disclosed the decline of the definitive EPC colony numbers in the both MMD patients. The level of interleukin-10 (IL-10) was lower in secretory cytokines from the cultured PBMNCs of MMD patients than that in that of controls using a cytometric bead array. The addition of human recombinant IL-10 to PBMNCs cultured from MMD patients restored the EPC colony forming potential of MMD PBMNCs. Following phorbol myristate acetate stimulation of the cultured PBMNCs, flow cytometry revealed a decrease in intracellular IL-10 storage in the main cell populations of the PBMNCs cultured from MMD patients relative to those cultured from controls. The present data provide the expected mechanism of vascular malformation in MMD pathogenesis originated from the insufficient production of IL-10 secreting cells from PBMNCs fostering EPC expansion and differentiation.

Sox7 Regulates Lineage Decisions in Cardiovascular Progenitor Cells.

Specification of the mesodermal lineages requires a complex set of morphogenetic events orchestrated by interconnected signaling pathways and gene regulatory networks. The transcription factor Sox7 has critical functions in differentiation of multiple mesodermal lineages, including cardiac, endothelial, and hematopoietic. Using a doxycycline-inducible mouse embryonic stem cell line, we have previously shown that expression of Sox7 in cardiovascular progenitor cells promotes expansion of endothelial progenitor cells (EPCs). In this study, we show that the ability of Sox7 to promote endothelial cell fate occurs at the expense of the cardiac lineage. Using ChIP-Seq coupled with ATAC-Seq we identify downstream target genes of Sox7 in cardiovascular progenitor cells and by integrating these data with transcriptomic analyses, we define Sox7-dependent gene programs specific to cardiac and EPCs. Furthermore, we demonstrate a protein-protein interaction between SOX7 and GATA4 and provide evidence that SOX7 interferes with the transcriptional activity of GATA4 on cardiac genes. In addition, we show that Sox7 modulates WNT and BMP signaling during cardiovascular differentiation. Our data represent the first genome-wide analysis of Sox7 function and reveal a critical role for Sox7 in regulating signaling pathways that affect cardiovascular progenitor cell differentiation.

Normoxia is not favorable for maintaining stemness of human endothelial progenitor cells

The in vitro expansion of endothelial progenitor cells (EPCs) is necessary for obtaining sufficient amounts of cells for clinical applications. However, EPC expansion is conventionally carried out under non-physiologic oxygen concentrations (normoxia, ~20% O2). We compared the effects of normoxic and hypoxic culture on the stemness of expanded EPCs. Human EPCs were cultured under hypoxia (1% O2) or normoxia (~20% O2), respectively. Cell proliferation, colony formation, in vitro angiogenesis, and the migration ability of the expanded EPCs were compared. To explore the underlying mechanism, whole transcriptome RNA sequencing (RNA-seq) was also performed to select differentially expressed genes (DEGs), which were then partially validated by western blotting. EPCs cultured under normoxia showed reduced proliferation, colony formation, in vitro angiogenesis, and migration abilities and a higher proportion of senescent cells compared with those cultured under hypoxia. A total of 48 DEGs were identified by transcriptome RNA-seq. Further bioinformatics analysis revealed that six pathways were enriched, among which the p53 signaling pathway. Finally, we confirmed the differential expression of the p53 pathway by Western blot analysis. Compared with hypoxia, normoxia is not favorable for maintaining the stemness of human EPCs. Several signaling pathways, including the p53 signaling pathway implicate in reducing stemness of EPCs under normoxia.

A Method to Isolate CD34+ Mononuclear Cells from Canine Peripheral Blood.

Embryonic stem cells are pluripotent whereas adult stem cells are multipotent in nature. In recent years, evidence suggests that adult stem cells not only differentiate into specific cell lineages but also transdifferentiate into multiple cell lineages. Progenitor cells are found in adult bone marrow, blood, and other organs and differentiate into numerous cell lineages regardless of origin. Identifying a subset that can differentiate into mature endothelial cells is essential. This article describes peripheral blood collection in adult beagle dogs, isolation of peripheral blood mononuclear cells (PBMNCs) from the cell fraction, separation of a subset of CD34+ cells using immunomagnetic principles, characterization of PBMNCs and CD34+ cells using flow cytometry, and evaluation of gene expression of CD34, KDR, and CD133 in CD34+ fractions. Efficient methods of isolation of endothelial progenitor cells (EPCs) will promote the ex vivo expansion and transplantation of EPCs in ischemic injury to enable neovascularization. © 2019 by John Wiley & Sons, Inc.

An effective ex-vivo approach for inducing endothelial progenitor cells from umbilical cord blood CD34+ cells

BackgroundTransplantation of endothelial progenitor cells (EPCs)/endothelial cells (ECs) has been used for the treatment of ischemic diseases and hemophilia A, due to their great capacity for producing factor VIII and for repairing vascular damage. We established an effective approach to stimulate the expansion and differentiation of EPCs for potential therapeutic applications.MethodsCD34+ cells isolated from human cord blood were cultured in a two-step system for 21 days. The generated adherent cells were characterized via flow cytometry and immunofluorescent staining. Moreover, single-cell clonogenic and tube-forming assays were carried out to evaluate their potential to proliferate and form vessel networks. Furthermore, these cells were transplanted into a mouse model of hepatic sinusoidal endothelium injury by hepatic portal vein injection to investigate their in-vivo behavior.ResultsThe two-step culture protocol promoted the expansion and differentiation of human cord blood CD34+ cells efficiently, resulting in a large number of adherent cells within 3 weeks. The generated adherent cells were identified as EPCs/ECs based on the expression of CD31, CD144, vWF, and FVIII, and cell numbers showed a 1400-fold increase compared with the initial number. Moreover, these EPCs/ECs were capable of proliferating and establishing colonies as individual cells, and forming tube-like structures. More significantly, tissue examination of mice after transplantation revealed that the injected EPCs/ECs migrated and integrated into the liver, reconstituting the sinusoidal endothelial compartment.ConclusionsWe developed an approach for the generation of cord blood-derived EPCs/ECs on a large scale, characterized them phenotypically, and demonstrated their in-vivo functional capacity. Our approach provides an excellent source of healthy EPCs/ECs for use in cell therapy in a clinical setting.

High quality in vitro expansion of human endothelial progenitor cells of human umbilical vein origin.

The limited availability of qualified endothelial progenitor cells (EPCs) is a major challenge for regenerative medicine. In the present study, we isolated human EPCs from human umbilical vein endothelial cells (HUVECs) by using magnetic micro-beads coated with an antibody against human CD34. Flow cytometric assay showed that majority of these cells expressed VEGFR2 (KDR), CD34 and CD133, three molecular markers for early EPCs. It was also found that a bioreactor micro-carrier cell culture system (bio-MCCS) was superior to dish culture for in vitro expansion of EPCs. It expanded more EPCs which were in the early stage, as shown by the expression of characteristic molecular markers and had better angiogenic potential, as shown by matrix-gel based in vitro angiogenesis assay. These results suggest that HUVECs might be a novel promising resource of EPCs for regenerative medicine and that a bio-MCCS cell culture system might be broadly used for in vitro expansion of EPCs.

DEVELOPMENT OF A COMBINATORIAL MICROASSAY TO ENGINEER BIOMIMETIC VASCULAR SCAFFOLDS

Regeneration-promoting vascular substitutes such as pro-healing stents or vascular grafts could minimise the incidence of thrombosis, restenosis and other events leading to device failure. One strategy to promote endogenous repair mechanisms is to functionalise the surfaces of the vascular graft with peptides or proteins that capture circulating endothelial progenitor cells (EPCs). EPCs are circulating CD34+ bone marrow-derived cells that could have a significant impact on endothelialisation due to high proliferative and regenerative potential. Previous studies have tested surface-immobilised antibodies to capture EPCs, as well as surface-immobilised extracellular matrix-derived peptides to promote adhesion and expansion. However, these strategies have not been tested in combination. We hypothesise that the co-immobilisation of peptides and antibodies could maximise endothelialisation by targeting both the EPC capture and expansion steps.

ECM-Dependence of Endothelial Progenitor Cell Features.

Preserving self-renewal, multipotent capacity, and large-scale expansion of highly functional progenitor cells, including endothelial progenitor cells (EPCs), is a controversial issue. These current limitations, therefore, raise the need of developing promising in vitro conditions for prolonged expansion of EPCs without loss of their stemness feature. In the current study, the possible role of three different natural extracellular substrates, including collagen, gelatin, and fibronectin, on multiple parameters of EPCs such as cell morphology, phenotype, clonogenic, and vasculogenic properties was scrutinized. Next, EPCs from GFP-positive mice were pre-expanded on each of these ECM substrates and then systemically transplanted into sublethaly irradiated mice to analyze the potency of these cells for marrow reconstitution. Our results revealed considerable promise for fibronectin for EPC expansion with maintenance of stemness characteristics, whereas gelatin and collagen matrices directed the cells toward a mature endothelial phenotype. Transplantation of EPCs pre-expanded on fibronectin resulted in widespread distribution and appropriate engraftment to various tissues with habitation in close association with the microvasculature. In addition, fibronectin pre-expanded cells were gradually enriched in the bone marrow after transplantation, resulting in marrow repopulation and hematologic recovery, leading to improved survival of recipient mice whereas gelatin- and collagen-expanded cells failed to reconstitute the bone marrow. This study demonstrated that, cell characteristics of in vitro expanded EPCs are determined by the subjacent matrix. Fibronectin-expanded EPCs are heralded as a source of great promise for bone marrow reconstitution and neo-angiogenesis in therapeutic bone marrow transplantation. J. Cell. Biochem. 117: 1934-1946, 2016. © 2016 Wiley Periodicals, Inc.

Endothelial progenitor cells proliferated via MEK-dependent p42 MAPK signaling pathway.

Endothelial progenitor cells (EPCs) clinical applications have been well reported. However, due to low number of EPCs that could be isolated, EPCs expansion study became one of the main focuses. Some optimized mediums to culture EPCs were currently available. However, the proliferation signaling pathway is not clearly disclosed yet. Peripheral blood was collected from eight healthy subjects, followed by mononuclear cells (MNCs) isolation. MNCs were then prepared and cultured for 2 days. After that, non-adherent cells were harvested and further cultured for 3 days. Resulted colony-forming unit (CFU)-Hill colonies were documented and enumerated under an inverted light microscope. To detect membrane markers, immunofluorescence was performed to detect CD34, VEGFR-2, and CD133. Cell documentation was conducted under a fluorescence microscope. To check cell proliferation, XTT Cell Proliferation Assay Kit was used according to kit insert. To detect possible activation of p44/42 MAPK, western blot was performed to detect p44/42 MAPK and phosphorylated p44/42 MAPK. All visualized bands were captured and quantified. Our results showed that EPCs markers (CD34, CD133 and VEGFR-2) were detected in 3 days culture. From XTT cell proliferation assay and CFU enumeration results, we found that EPCs proliferated significantly (p = 0.012) with addition of supplement. Phosphorylated-p42 MAPK expression of EPCs treated with supplement was significantly higher than the one of EPCs without treatment. Significant inhibition of p42 MAPK phosphorylation by U0126 was observed (p = 0.012). By pretreatment of U0126, number of viable cells and CFUs treated with supplement was significantly decreased (p = 0.012). Our results showed that MEK-dependent p42 MAPK pathway might play an important role in EPCs proliferation.

Expansion of endothelial progenitor cells in high density dot culture of rat bone marrow cells.

In vitro expansion of endothelial progenitor cells (EPCs) remains a challenge in stem cell research and its application. We hypothesize that high density culture is able to expand EPCs from bone marrow by mimicking cell-cell interactions of the bone marrow niche. To test the hypothesis, rat bone marrow cells were either cultured in high density (2×105 cells/cm2) by seeding total 9×105 cells into six high density dots or cultured in regular density (1.6×104 cells/cm2) with the same total number of cells. Flow cytometric analyses of the cells cultured for 15 days showed that high density cells exhibited smaller cell size and higher levels of marker expression related to EPCs when compared to regular density cultured cells. Functionally, these cells exhibited strong angiogenic potentials with better tubal formation in vitro and potent rescue of mouse ischemic limbs in vivo with their integration into neo-capillary structure. Global gene chip and ELISA analyses revealed up-regulated gene expression of adhesion molecules and enhanced protein release of pro-angiogenic growth factors in high density cultured cells. In summary, high density cell culture promotes expansion of bone marrow contained EPCs that are able to enhance tissue angiogenesis via paracrine growth factors and direct differentiation into endothelial cells.

Prevention of liver fibrosis by intrasplenic injection of high-density cultured bone marrow cells in a rat chronic liver injury model.

Endothelial progenitor cells (EPCs) from bone marrow have proven to be functional for the prevention of liver fibrosis in chronic liver injury. However, expansion of EPCs in culture is complicated and expansive. Previously, we have established a simple method that could enrich and expand EPCs by simple seeding bone marrow cells in high density dots. The purpose of this study is to evaluate whether cells derived from high-density (HD) culture of rat bone marrow cells could prevent the liver fibrosis in a chronic liver injury rat model, induced by carbon tetrachloride (CCl4). Flow cytometric analysis showed that cells from HD culture were enriched for EPCs, expressing high levels of EPC markers. Intrasplenic injection of HD cultured bone marrow cells in the CCl4-induced liver injury rat showed an enhanced antifibrogenic effect compared with animals treated with cells from regular-density culture. The antifibrogenic effect was demonstrated by biochemical and histological analysis 4 weeks post-transplantation. Furthermore, cells from HD culture likely worked through increasing neovascularization, stimulating liver cell proliferation, and suppressing pro-fibrogenic factor expression. HD culture, which is a simple and cost-effective procedure, could potentially be used to expand bone marrow cells for the treatment of liver fibrosis.

Whole bone marrow cell culture: A convenient protocol for the in vitro expansion of endothelial progenitor cells.

The number and function of endothelial progenitor cells (EPCs) may be a predictive factor for the severity and outcome of cardiovascular disease. However, the manipulation of bone marrow mononuclear cell (BMMC) cultures for EPCs is an elaborate and difficult procedure in small experimental animals. The present study aimed to assess the feasibility of whole bone marrow cell (WBMC) culture for expanding EPCs in small experimental animals. C57BL/6 mice (age, 3–4 weeks; weight, 9.47±0.76 g) were used as the experimental animals, and WBMCs were isolated from the femora and tibiae and cultured in endothelial cell growth medium-2. A BMMC culture for EPCs was used as a control. EPC growth, phenotype and functions were assessed in vitro and in vivo. The results demonstrated that EPCs were easily obtained from a WBMC culture in vitro. The cells exhibited similar growth and biological characteristics when compared with the EPCs derived from the traditional BMMC culture system. Thus, the cells were able to simultaneously bind to lectin and cause phagocytosis of acetylated-low density lipoproteins. In addition, the cells exhibited high expression levels of cluster of differentiation 34 and fetal liver kinase 1, and possessed similar functional properties to BMMC-derived EPCs, including vascular network formation, proliferation, adhesion and migration abilities in vitro. Thus, WBMC-derived EPCs can improve the outcome of pulmonary vascular disease when transplanted into a monocrotaline-induced pulmonary hypertension mouse model. The results of the present study indicated that the WBMC culture system is a more convenient and effective method of obtaining and expanding EPCs compared with BMMC culture, with the advantage of a simplified procedure.

Expansion of endothelial progenitor cells from bone marrow in vitro and your role in arterial thrombosis formation induced by FeCl3

Expansion of endothelial progenitor cells from bone marrow in vitro and your role in arterial thrombosis formation induced by FeCl3

Biological properties of bone marrow-derived early and late endothelial progenitor cells in different culture media

Ex vivo expansion of endothelial progenitor cells (EPCs) may be a promising strategy to overcome the clinical problem of limited cell numbers. As the culture medium is the key for the cell characteristics, the effects of different culture media on EPCs were investigated in the present study. Rat bone marrow mononuclear cells were cultured in different media, including M-199 media with 20% fetal bovine serum (FBS) and bovine pituitary extract (M1); M-199 media with 10% FBS, 20 ng/ml vascular endothelial growth factor (VEGF) and 10 ng/ml basic fibroblast growth factor (bFGF; M2) or epidermal growth medium (EGM)-2MV media. The cell morphology and biological functions, such as proliferation, adhesion, migration, tube formation and nitric oxide (NO) production were subsequently assayed in vitro. Moreover, endothelial biomarkers and apoptosis were also analyzed. The results showed that endothelial‑like cells appeared in all of the culture systems. First‑passage cells, namely early EPCs, tended to form colonies in M2 and EGM-2MV media but showed a fusiform shape in M1 media. The 3rd or 4th generation EPCs, namely late EPCs, cultured in EGM-2MV media exhibited increased adhesion, migration, tube formation and NO production as compared with EPCs in M1 or M2 media. Furthermore, late EPCs cultured in EGM-2MV expressed higher levels of endothelial cell markers, such as von Willibrand factor (vWF)and CD31, but relatively greater levels of apoptosis were observed. In conclusion, cell culture conditions, for example the medium used, affects the biological properties of bone marrow-derived early and late EPCs.

Comparison of Fibronectin and Collagen in Supporting the Isolation and Expansion of Endothelial Progenitor Cells from Human Adult Peripheral Blood

BackgroundEndothelial colony-forming cells (ECFCs), are circulating endothelial progenitor cells increasingly studied in various diseases because of their potential for clinical translation. Experimental procedures for their ex vivo culture still lack standardization. In particular two different extracellular matrix proteins, either fibronectin or collagen, are commonly used by different Authors for coating plastic plates, both allowing to obtain cells that have all the features of ECFCs. However, possible differences in the impact of each substrate on ECFCs have not been analysed, so far. Therefore, in this study we investigated whether fibronectin and collagen may differentially affect ECFC cultures.Methodology/Principal FindingsECFCs were isolated and cultured from peripheral blood mononuclear cells of healthy donors. The impact of fibronectin compared with collagen as the only variable of the experimental procedure was analysed separately in the phase of isolation of ECFC colonies and in the following phase of cell expansion. In the isolation phase, although similar frequencies of colonies were obtained on the two substrates, ECFC colonies appeared some days earlier when mononuclear cells were seeded on fibronectin rather than collagen. In the expansion phase, ECFCs cultured on collagen showed a longer lifespan and higher cell yields compared with ECFCs cultured on fibronectin, possibly related to the higher levels of IL-6 and IL-8 measured in their supernatants. ECFCs cultured on both substrates showed similar immunophenotype and ability for in vitro tube formation.Conclusions/SignificanceOverall, the results of this study indicate that, although both fibronectin and collagen efficiently sustain ECFC cultures, each of them brings some advantages within individual steps of the entire process. We suggest that colony isolation performed on fibronectin followed by cell expansion performed on collagen may represent a novel and the most efficient strategy to obtain ECFCs from adult peripheral blood samples.

Role of microenvironment in the process of expansion of late endothelial progenitor cell in vitro.

To study the role of the feeder layer cells as niche in the process of expansion of late endothelial progenitor cell in vitro. We cultured mononuclear cells (MNC)from human peripheral blood (PB)on the plate with the feeder layer cells which were irradiated late endothelial progenitor cells(EPC)or human umbilical vein endothelial cells (HUVEC) by EGM-2. After 21 days, the numbers of obtained late EPC colonies were counted separately, and their surface antigen of the late EPC was verified by fluorescence-activated cell sorter (FACS) analysis, and their ability of forming vessel structure with Matrigel in vitro. The differentiation of single stem cell on the feeder layer cell was traced by video-microscopy. After 21 days of culture,(40.0±3.9)and(39.3±3.1)late EPC colonies that MNC of a hundred milliliter PB were cultured, respectively, on the feeder layer cells of EPC and HUVEC were much more than (2.0±1.3) colonies cultured on without the feeder layer cells (all P <0.05). These cells also expressed CD31,CD34,eNOS,FLt-1,P1H12,Sendo,VE cadherin,and CD117, as shown by FACS analysis. Furthermore, they formed vessel structure with Matrigel in vitro. The video-microscopy showed the asymmetric cell division was participated by the feeder layer cell during the expansion of single stem cell. The massive expansion of late EPC can be achieved by the provision of the feeder layer cells, which may be involved in the stem cell asymmetric cell division.

Decursin inhibits vasculogenesis in early tumor progression by suppression of endothelial progenitor cell differentiation and function

Endothelial progenitor cells (EPCs) contribute to the tumor vasculature during tumor progression. Decursin isolated from the herb Angelica gigas is known to possess potent anti-inflammatory activities. Recently, we reported that decursin is a novel candidate for an angiogenesis inhibitor [Jung et al., 2009]. In this study, we investigated whether decursin regulates EPC differentiation and function to inhibit tumor vasculogenesis. We isolated AC133+ cells from human cord blood and decursin significantly decreased the number of EPC colony forming units of human cord blood-derived AC133+ cells that produce functional EPC progenies. Decursin dose-dependently decreased the cell number of EPC committing cells as demonstrated by EPC expansion studies. Decursin inhibited EPC differentiation from progenitor cells into spindle-shaped EPC colonies. Additionally, decursin inhibited proliferation and migration of early EPCs isolated from mouse bone marrow. Furthermore, decursin suppressed expression of angiopoietin-2, angiopoietin receptor Tie-2, Flk-1 (vascular endothelial growth factor receptor-2), and endothelial nitric oxide synthase in mouse BM derived EPCs in a dose-dependent manner. Decursin suppressed tube formation ability of EPCs in collaboration with HUVEC. Decursin (4 mg/kg) inhibited tumor-induced mobilization of circulating EPCs (CD34 + /VEGFR-2+ cells) from bone marrow and early incorporation of Dil-Ac-LDL-labeled or green fluorescent protein (GFP)+ EPCs into neovessels of xenograft Lewis lung carcinoma tumors in wild-type- or bone-marrow-transplanted mice. Accordingly, decursin attenuated EPC-derived endothelial cells in neovessels of Lewis lung carcinoma tumor masses grown in mice. Together, decursin likely affects EPC differentiation and function, thereby inhibiting tumor vasculogenesis in early tumorigenesis.