Colony-forming Unit-endothelial Cells Research Articles

Impairment of muscular endothelial cell regeneration in dermatomyositis.

Inflammatory myopathies are heterogeneous in terms of etiology, (immuno)pathology, and clinical findings. Endothelial cell injury, as it occurs in DM, is a common feature of numerous inflammatory and non-inflammatory vascular diseases. Vascular regeneration is mediated by both local and blood-derived mechanisms, such as the mobilization and activation of so-called proangiogenic cells (PACs) or early endothelial progenitor cells (eEPCs). The current study aimed to evaluate parameters of eEPC integrity in dermatomyositis (DM), compared to necrotizing myopathy (NM) and to non-myopathic controls. Blood samples from DM and NM patients were compared to non-myositis controls and analyzed for the following parameters: circulating CD133+/VEGFR-2+ cells, number of colony-forming unit endothelial cells (CFU-ECs), concentrations of angiopoietin 1, vascular endothelial growth factor (VEGF), and CXCL-16. Muscle biopsies from DM and NM subjects underwent immunofluorescence analysis for CXCR6, nestin, and CD31 (PECAM-1). Finally, myotubes, derived from healthy donors, were stimulated with serum samples from DM and NM patients, subsequently followed by RT-PCR for the following candidates: IL-1β, IL-6, nestin, and CD31. Seventeen (17) DM patients, 7 NM patients, and 40 non-myositis controls were included. CD133+/VEGFR-2+ cells did not differ between the groups. Both DM and NM patients showed lower CFU-ECs than controls. In DM, intramuscular CD31 abundances were significantly reduced, which indicated vascular rarefaction. Muscular CXCR6 was elevated in both diseases. Circulating CXCL-16 was higher in DM and NM in contrast, compared to controls. Serum from patients with DM but not NM induced a profound upregulation of mRNS expression of CD31 and IL-6 in cultured myotubes. Our study demonstrates the loss of intramuscular microvessels in DM, accompanied by endothelial activation in DM and NM. Vascular regeneration was impaired in DM and NM. The findings suggest a role for inflammation-associated vascular damage in the pathogenesis of DM.

Functional Impairment of Endothelial Colony Forming Cells (ECFC) in Patients with Severe Atherosclerotic Cardiovascular Disease (ASCVD).

Endothelial dysfunction is a key factor in atherosclerosis. However, the link between endothelial repair and severity of atherosclerotic cardiovascular disease (ASCVD) is unclear. This study investigates the relationship between ASCVD, markers of inflammation, and circulating endothelial progenitor cells, namely hematopoietic cells with paracrine angiogenic activity and endothelial colony forming cells (ECFC). Two hundred and forty-three subjects from the TELARTA study were classified according to the presence of clinical atherosclerotic disease. ASCVD severity was assessed by the number of involved vascular territories. Flow cytometry was used to numerate circulating progenitor cells (PC) expressing CD34 and those co-expressing CD45, CD34, and KDR. Peripheral blood mononuclear cells ex vivo culture methods were used to determine ECFC and Colony Forming Unit- endothelial cells (CFU-EC). The ECFC subpopulation was analyzed for proliferation, senescence, and vasculogenic properties. Plasma levels of IL-6 and VEGF-A were measured using Cytokine Array. Despite an increased number of circulating precursors in ASCVD patients, ASCVD impaired the colony forming capacity and the angiogenic properties of ECFC in a severity-dependent manner. Alteration of ECFC was associated with increased senescent phenotype and IL-6 levels. Our study demonstrates a decrease in ECFC repair capacity according to ASCVD severity in an inflammatory and senescence-associated secretory phenotype context.

Increased serum levels of fractalkine and mobilisation of CD34+CD45\u2212 endothelial progenitor cells in systemic sclerosis

BackgroundThe disruption of endothelial homeostasis is a major determinant in the pathogenesis of systemic sclerosis (SSc) and is reflected by soluble and cellular markers of activation, injury and repair. We aimed to provide a combined assessment of endothelial markers to delineate specific profiles associated with SSc disease and its severity.MethodsWe conducted an observational, single-centre study comprising 45 patients with SSc and 41 healthy control subjects. Flow cytometry was used to quantify circulating endothelial microparticles (EMPs) and CD34+ progenitor cell subsets. Colony-forming unit-endothelial cells (CFU-ECs) were counted by culture assay. Circulating endothelial cells were enumerated using anti-CD146-based immunomagnetic separation. Blood levels of endothelin-1, vascular endothelial growth factor (VEGF) and soluble fractalkine (s-Fractalkine) were evaluated by enzyme-linked immunosorbent assay. Disease-associated markers were identified using univariate, correlation and multivariate analyses.ResultsEnhanced numbers of EMPs, CFU-ECs and non-haematopoietic CD34+CD45− endothelial progenitor cells (EPCs) were observed in patients with SSc. Patients with SSc also displayed higher serum levels of VEGF, endothelin-1 and s-Fractalkine. s-Fractalkine levels positively correlated with CD34+CD45− EPC numbers. EMPs, s-Fractalkine and endothelin-1 were independent factors associated with SSc. Patients with high CD34+CD45− EPC numbers had lower forced vital capacity values. Elevated s-Fractalkine levels were associated with disease severity, a higher frequency of pulmonary fibrosis and altered carbon monoxide diffusion.ConclusionsThis study identifies the mobilisation of CD34+CD45− EPCs and high levels of s-Fractalkine as specific features of SSc-associated vascular activation and disease severity. This signature may provide novel insights linking endothelial inflammation and defective repair processes in the pathogenesis of SSc.

Cell therapy in critical limb ischemia: A comprehensive analysis of two cell therapy products

BackgroundCell therapy has been proposed as a salvage limb procedure in critical limb ischemia (CLI). In spite of the fact that clinical trials found some efficacy, the mechanism of action remains elusive. The objective of this study was to characterize two autologous cell therapy products (CTPs) obtained from patients with advanced peripheral arterial disease. MethodsBone marrow (BM-CTPs) (n = 20) and CTPs obtained by non-mobilized cytapheresis (peripheral blood [PB]-CTPs) (n = 20) were compared. CTPs were characterized by their cell composition, by the quantification of endothelial progenitor cells (EPCs) and mesenchymal stromal cells (MSCs) and by transcriptomic profiling. The angiogenic profile and the 6-month outcome of CLI patients are described. ResultsPatients presented inflammation syndrome and high levels of CXCL12, soluble stem cell factor and granulocyte colony-stimulating factor, whereas granulocyte macrophage colony-stimulating factor was low. Circulating CD34+ cells represented rare events. BM and PB-CTPs were heterogeneous. Mature cells and colony-forming unit–endothelial cells were in higher concentration in PB-CTPs, whereas CD34+ stem cells and EPCs were more abundant in BM-CTPs. MSCs were identified in both CTPs. Transcriptomic profiling revealed the strong angiogenic potential of BM-CTPs. Transcutaneous partial pressure of oxygen, C-reative protein and neutrophil content in CTPs are predictive of the clinical outcome at 6 months. DiscussionTranscriptomic allows an accurate characterization of CTPs. BM-CTPs have the richest content in terms of stem cells and transcriptome. The high content of mature cells in PB-CTPs means that they work via a paracrine mechanism. The clinical outcome indicates the deleterious influence the patients' status and the limits of an autologous approach. In this respect, MSCs may allow an allogenic strategy.

Absence of Correlation between Changes in the Number of Endothelial Progenitor Cell Subsets.

Background and ObjectivesPreviously, various methodologies were used to enumerate the endothelial progenitor cells (EPCs). We now know that these methodologies enumerate at least three different EPC subsets: circulating angiogenic cells (CACs), colony-forming unit endothelial cells (CFU-ECs), and endothelial colony-forming cells (ECFCs). It is not clear whether there is a correlation between changes in the number of these subsets. The aim of the current study is to find an answer to this question.Materials and MethodsThe number of all EPC subsets was quantified in the peripheral blood of nine pregnant women in their first and third trimesters of pregnancy. We enumerated 14 cell populations by quantitative flow-cytometry using various combinations of the markers, CD34, CD133, CD309, and CD45, to cover most of the reported phenotypes of CACs and ECFCs. Culturing technique was used to enumerate the CFU-ECs. Changes in the number of cells were calculated by subtracting the number of cells in the first trimester peripheral blood from the number of cells in the third trimester peripheral blood, and correlations between these changes were analyzed.ResultsThe number of CFU-ECs did not correlate with the number of ECFCs and CACs. Also, CACs and ECFCs showed independent behaviors. However, the number of CACs showed a strong correlation with the number of CD133+CD309+ cells (p=0.001) and a moderate correlation with the number of CD34+CD309+ cells (p=0.042). Also, the number of ECFCs was correlated with the number of CD309+CD45- cells (p=0.029) and CD34+CD45- cells (p=0.03).ConclusionOur study showed that the three commonly used methods for quantifying EPC subsets represent different cells with independent behaviors. Also, any study that measured the number of EPCs using the flow cytometry method with a marker combination that lacks CD309 may be inaccurate.

Endothelial progenitors promote hepatocarcinoma intrahepatic metastasis through monocyte chemotactic protein-1 induction of microRNA-21

ObjectivesEndothelial progenitor cells (EPCs) circulate with increased numbers in the peripheral blood of patients with highly-vascularised hepatocellular carcinoma (HCC) and contribute to angiogenesis and neovascularisation. We hypothesised that angiogenic EPCs,...

Colony forming unit endothelial cells do not exhibit telomerase alternative splicing variants and activity.

Endothelial progenitor colony forming unit-endothelial cells (CFU-EC) were first believed to be the progenitors of endothelial cells, named endothelial progenitor cells. Further studies revealed that they are monocytes regulating vasculogenesis. The main hindrance of these cells for therapeutic purposes is their low frequency and limited replicative potentials. This study was undertaken to determine telomerase activity and alternative splicing variants in CFU-EC as a potential cause of limited replicative capacity in these cells. CFU-EC were isolated from peripheral blood using a standard cell culture assay. Colonies were detached mechanically and alternative splicing variant mRNA were evaluated using real-time PCR. Telomerase enzyme activity was assessed using telomerase repeat amplification protocol. The same procedures were done on the cancer cell line Calu6 as the positive control. The cultured peripheral blood mononuclear cells formed colonies with spindle-shaped monocytic cells sprouted from the clusters. These morphological characteristics fulfill the definition of CFU-EC. Telomere length amplification protocol assay revealed no telomerase activity and real-time PCR showed no expression of telomerase enzyme mRNA in CFU-EC. Both parameters were significantly higher in the cancer cell line Calu6 taken as the positive control. The absence of telomerase activity in the CFU-EC is a result of pre-transcriptional regulation of gene expression rather than other mechanisms for controlling telomerase activity such as post-transcriptional modifications. This finding can explain the limited proliferative activity of CFU-EC cells. We propose that absence of telomerase activity in CFU-EC can be attributable to their more mature monocytic nature that needs further investigations.

Short Communication: Proangiogenic Hematopoietic Cells In Acute HIV Infection

Chronic HIV infection induces significant changes in the trafficking of circulating endothelial progenitor cells (EPCs). Specifically, it causes marked depletion of proangiogenic hematopoietic cells, the so-called colony-forming unit-endothelial cells (CFU-ECs). In this study we evaluated CFU-ECs in two subjects with acute HIV infection. We found that both patients already had a low CFU-EC level at the time of diagnosis. Nevertheless, after 6 months of antiretroviral therapy, the CFU-EC concentration reverted to normal values in both cases. HIV significantly depletes the CFU-EC compartment even in the early phase of infection, while 6-month therapy appears to be able to restore it.

Effect of antiviral therapy on pro-angiogenic hematopoietic and endothelial progenitor cells in HIV-infected people

BackgroundHIV infection is an independent risk factor for cardiovascular disease. HIV-sustained impairment of endothelial progenitor cells (EPCs) could contribute to this process, so that it is important to assess whether antiviral therapy (ART) is able to revert these abnormalities. MethodsWe quantified in 21 naïves and 34 treated patients two functionally distinct clonogenic progenitors which have been acknowledged important for vascular repair: the hematopoietic progenitor colony forming unit - endothelial cells (CFU-EC) and the true endothelial progenitor, the endothelial colony forming cells (ECFC). We correlated results obtained with conventional vascular risk factors and with HIV-related parameters. ResultsWe found that these progenitors behaved differently in naive and treated patients. In particular, CFU-EC level was significantly low in all naive patients and slowly recovered during ART. In contrast, the ECFC level was abnormally high in naive patients while it decreased upon ART. The CFU-EC level was related to conventional cardiovascular risk factors, as reported in general population, but also to inflammatory indexes and CD4 cell count. In contrast, the ECFC number was exclusively related to viral replication activity and to CD4 cell count. ConclusionsIn HIV-infected people, the levels of CFU-EC and ECFC are related to classical cardiovascular risk factors but, in addition, they are also significantly influenced by the infection itself and by antiviral therapy.

Endothelial Progenitor Cells in HIV-Positive Patients

Endothelial Progenitor Cells in HIV-Positive Patients

Authors′ Reply

Background Although in the general population circulating vascular progenitor cell levels have been implicated in the homeostasis of the vascular wall through differentiation into endothelium and/or smooth muscle cells, it has not yet been assessed in HIV-infected patients. We herein investigated the number of progenitor cell subpopulations in HIV-infected patients and its relationship to carotid intima-media thickness (c-IMT). Methods Blood samples were collected from 200 HIV-infected patients and CD34/KDR, CD34/VE-cadherin, and CD14/Endoglin progenitor cells were identified by flow cytometry. c-IMT was determined by ultrasonography. A group of 27 healthy subjects was used as control group. Results In our population (20 ART-naive patients and 180 treated patients), traditional cardiovascular risk factors were not found predictive of vascular progenitor cell levels. However, antiretroviral therapy (ART)-treatment was identified as the main predictive value for low CD34/KDR cells and high CD14/Endoglin cells after adjustment by cardiovascular risk factors (age, sex, hypertension, diabetes, and hyperlipidaemia) and HIV-related characteristics (HIV duration and ART treatment). Low levels of circulating CD34/KDR or CD34/VE-cadherin endothelial progenitor cells tended to be associated with increased c-IMT. However, a positive association was found between CD14/Endoglin cells and c-IMT. Low number of CD34/KDR cells was also associated with the longest exposure to nucleoside reverse transcriptase inhibitors and/or protease inhibitors. Conclusions ART exposure is the main predictor of circulating vascular progenitor cell levels. However, their levels are only partially associated with high c-IMT in HIV-infected patients. ART has already been found to have proatherogenic effect, but our data first describe its relationship with vascular progenitor cells and c-IMT.

Modulation of chemotactic and pro-inflammatory activities of endothelial progenitor cells by hepatocellular carcinoma

Endothelial progenitor cells (EPCs) participate in the neovascularization processes in the development of hepatocellular carcinoma (HCC). We investigated whether interactions between EPCs and HCC cells affect chemotactic and pro-inflammatory activities of EPCs. Two distinct phenotypes of circulating EPCs, i.e., myeloid-derived EPCs (colony forming unit-endothelial cells, CFU-ECs) and outgrowth EPCs (endothelial-colony forming cells, ECFCs), were co-cultured with Huh7 and Hep3B cells by using transwell chamber and IBIDITM Culture-Inserts and μ-slide plates. Transwell and horizontal migration/invasion assays and time-lapse microscopy were used to monitor and analyze the migration and invasion of EPCs induced by these HCC cells. A human cytokine antibody array was used to compare protein expression profiles in EPCs and HCC cells. Flow cytometry and electromobility shift analysis were used to detect nuclear factor-κB (NF-κB)–DNA binding activity and pro-inflammatory adhesion molecule expression in EPCs. Ectopic full-length CC chemokine receptor 6 (CCR6) plasmid was used to transfect into ECFCs to investigate the role of CCR6 in HCC-induced EPC migration and invasion. The results show that co-culture with Huh7 and Hep3B cells induces the expression of endothelial cell (EC) markers KDR, Flt1, CD31 and VE-cadherin in CFU-ECs, but down-regulates the expressions of CD31 and VE-cadherin in ECFCs. These HCC cells induce migration and invasion of CFU-ECs, but not ECFCs, and do not affect the cell cycle distribution in these EPCs. Cytokine protein array identifies macrophage inflammatory protein-3α (MIP-3α) produced by HCC cells as a critical factor responsible for the HCC-induced chemotaxis of CFU-ECs, which highly express the specific MIP-3α counterreceptor CCR6. Overexpressing CCR6 in ECFCs significantly increases their chemotaxis in response to HCC cells. Co-culturing EPCs with HCC cells results in decreases in NF-κB binding activity and hence intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expressions in EPCs. Our results indicate that HCC cells exert differential effects on CFU-ECs and ECFCs, with increased chemotaxis for CFU-ECs, but not ECFCs. This HCC-induced chemotaxis of CFU-ECs is mediated by MIP-3α produced by HCC cells, which targets to CCR6 on CFU-ECs. Tumors may provide a humoral microenvironment to attenuate the pro-inflammatory activity of EPCs, which might be associated with the tumor escape mechanism.

Insulin Resistance and Oxidative Stress Influence Colony‐Forming Unit‐Endothelial Cells Capacity in Obese Patients

The aim of this study was to investigate the relationship between a sub-population of endothelial progenitor cells (EPC), namely colony-forming unit-endothelial cells (CFU-EC), their colony-forming capacity and variable clinical parameters, including insulin resistance and oxidative stress, in obese individuals. Thirty-eight obese adults (aged 42.5 ± 12.7), with BMI 32.3 ± 4.0 and 13 normal-weight controls (aged 48.2 ± 12.9; BMI 23.2 ± 2.3) were studied. CFU-EC colony-forming capacity was impaired in the group of obese individuals compared to the normal-weight controls (P = 0.001). The inverse correlation between homeostasis model assessment-insulin resistance (HOMA(IR)) index and CFU-EC number (r = -0.558, P < 0.0001) as well as positive total antioxidant status of plasma (TAS)/CFU-EC relation were noticed during the study. Additionally, correlations between the concentration of triglycerides (TG), high-density lipoproteins (HDLs), and body composition parameters in the obese participants were established. Our results demonstrate that insulin resistance and oxidative stress have a significant impact on the CFU-EC colony formation in obesity. Moreover, in multivariate regression analysis, in both studied groups, the HOMA(IR) index and HDL concentration were independent predictors of the number of CFU-EC. Endothelium dysfunction, which can be present in obesity, may in part be caused by EPC function impairment in this condition.

Gestational diabetes mellitus alters maternal and neonatal circulating endothelial progenitor cell subsets

The purpose of this study was to examine whether women with gestational diabetes mellitus (GDM) and their offspring have reduced endothelial progenitor cell subsets and vascular reactivity. Women with GDM, healthy control subjects, and their infants participated. Maternal blood and cord blood were assessed for colony-forming unit-endothelial cells and endothelial progenitor cell subsets with the use of polychromatic flow cytometry. Cord blood endothelial colony-forming cells were enumerated. Vascular reactivity was tested by laser Doppler imaging. Women with GDM had fewer CD34, CD133, CD45, and CD31 cells (circulating progenitor cells [CPCs]) at 24-32 weeks' gestation and 1-2 days after delivery, compared with control subjects. No differences were detected in colony-forming unit-endothelial cells or colony-forming unit-endothelial cells. In control subjects, CPCs were higher in the third trimester, compared with the postpartum period. Cord blood from GDM pregnancies had reduced CPCs. Vascular reactivity was not different between GDM and control subjects. The normal physiologic increase in CPCs during pregnancy is impaired in women with GDM, which may contribute to endothelial dysfunction and GDM-associated morbidities.

Proteases and receptors in the recruitment of endothelial progenitor cells in neovascularization

Since the initial discovery of endothelial progenitor cells (EPC), and their promise in increasing angiogenesis and vasculogenesis, a myriad of papers have highlighted their potential application in experimental and clinical neovascularization and in tissue engineering. However, promising reports are contrasted by other studies that could not find a role for EPC in neovascularization. Presently, two types of endothelial progenitor cell populations are recognized. The first population provides early-outgrowth CD34+/VEGFR-2+ cells, or colony-forming unit endothelial cells (CFU-EC), which represent myeloid cells with some endothelial properties, but no ability to form endothelial colonies. They can stimulate neovascularization by paracrine means, but are not incorporated in the endothelial lining themselves. The second population generates the late-outgrowth endothelial colony-forming cells (ECFC) from a very scant blood-derived cell population. ECFC have a very high proliferative potential, can insert into the endothelial lining of new blood vessels, and can also form endothelial tubes by themselves after stimulation with the proper angiogenic stimulus. This review surveys the mobilization of progenitor cells from the bone marrow, the homing of EPC (CFU-EC) to areas of neovascularization, and the participation of EPC (ECFC) in the endothelial lining of newly formed blood vessels. Specific emphasis has been placed on the role of proteases, which include serine proteases, including urokinase, L-cathepsin, and several ADAM- and matrix metalloproteinases. The specific properties of ECFC make them a potential source of cells for tissue engineering applications, but much has to be learned about their nature, origin and properties.

The Presence of Activated CD4+ T Cells Is Essential for the Formation of Colony-Forming Unit-Endothelial Cells by CD14+ Cells

The number of colony forming unit-endothelial cells (CFU-EC) in human peripheral blood was found to be a biological marker for several vascular diseases. In this study, the heterogeneous composition of immune cells in the CFU-ECs was investigated. We confirmed that monocytes are essential for the formation of CFU-ECs. Also, however, CD4(+) T cells were found to be indispensable for the induction of CFU-EC colonies, mainly through cell-cell contact. By blocking or activating CD3 receptors on CD4(+) T cells or blocking MHC class II molecules on monocytes, it was shown that TCR-MHCII interactions are required for induction of CFU-EC colonies. Because the supernatant from preactivated T cells could also induce colony formation from purified monocytes, the T cell support turned out to be cytokine mediated. Gene expression analysis of the endothelial-like colonies formed by CD14(+) cells showed that colony formation is a proangiogenic differentiation and might reflect the ability of monocytes to facilitate vascularization. This in vitro study is the first to reveal the role of TCR-MHC class II interactions between T cells and monocytes and the subsequent inflammatory response as stimulus of monocytic properties that are associated with vascularization.

The presence of activated CD4+ T‐Cells is essential for the formation of Colony Forming Unit‐Endothelial Cells (CFU‐EC) by CD14+ Cells

The number of colony forming unit‐endothelial cells (CFU‐EC) in human peripheral blood was found to be a biological marker for several vascular diseases. In this study the heterogeneous composition of immune cells in the CFU‐EC was investigated. CFU‐EC cultures of mixtures of several populations were used to determine the cellular composition of the endothelial like colony. We confirmed that monocytes are essential for the formation of CFU‐EC. In addition, CD4+ T‐cells were found to be required for the induction of CFU‐EC. Blocking experiments showed that the interaction of MHC class II on the monocyte with the CD3/TCR complex on the T‐cell was involved in the support of colony formation by T‐cells. We showed that the contact‐induced T‐cell stimulation could be replaced by the supernatant, and thus the cytokines, of activated CD4 + T‐cells.Gene expression and protein analyses showed that colony formation represents a pro‐angiogenic differentiation of the monocytes, rather than an endothelial differentiation.In conclusion, CFU‐EC are derived from monocytes and are supported by CD4+ T‐cells. This in vitro study is the first to reveal the role of TCR/MHCII interactions between T‐cells and monocytes and the subsequent inflammatory response as stimulus of monocytic properties that are associated with vascularization.

Colony forming unit-endothelial cells (CFU-EC) are derived from CD14+ cells and are supported by CD14− cells

Colony forming unit-endothelial cells (CFU-EC) are derived from CD14+ cells and are supported by CD14− cells

Th-P15:248 Computational approaches towards prediction

Endothelial progenitor cells (EPCs) participate in the neovascularization processes in the development of hepatocellular carcinoma (HCC). We investigated whether interactions between EPCs and HCC cells affect chemotactic and pro-inflammatory activities of EPCs. Two distinct phenotypes of circulating EPCs, i.e., myeloid-derived EPCs (colony forming unit-endothelial cells, CFU-ECs) and outgrowth EPCs (endothelial-colony forming cells, ECFCs), were co-cultured with Huh7 and Hep3B cells by using transwell chamber and IBIDITM Culture-Inserts and μ-slide plates. Transwell and horizontal migration/invasion assays and time-lapse microscopy were used to monitor and analyze the migration and invasion of EPCs induced by these HCC cells. A human cytokine antibody array was used to compare protein expression profiles in EPCs and HCC cells. Flow cytometry and electromobility shift analysis were used to detect nuclear factor-κB (NF-κB)–DNA binding activity and pro-inflammatory adhesion molecule expression in EPCs. Ectopic full-length CC chemokine receptor 6 (CCR6) plasmid was used to transfect into ECFCs to investigate the role of CCR6 in HCC-induced EPC migration and invasion. The results show that co-culture with Huh7 and Hep3B cells induces the expression of endothelial cell (EC) markers KDR, Flt1, CD31 and VE-cadherin in CFU-ECs, but down-regulates the expressions of CD31 and VE-cadherin in ECFCs. These HCC cells induce migration and invasion of CFU-ECs, but not ECFCs, and do not affect the cell cycle distribution in these EPCs. Cytokine protein array identifies macrophage inflammatory protein-3α (MIP-3α) produced by HCC cells as a critical factor responsible for the HCC-induced chemotaxis of CFU-ECs, which highly express the specific MIP-3α counterreceptor CCR6. Overexpressing CCR6 in ECFCs significantly increases their chemotaxis in response to HCC cells. Co-culturing EPCs with HCC cells results in decreases in NF-κB binding activity and hence intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expressions in EPCs. Our results indicate that HCC cells exert differential effects on CFU-ECs and ECFCs, with increased chemotaxis for CFU-ECs, but not ECFCs. This HCC-induced chemotaxis of CFU-ECs is mediated by MIP-3α produced by HCC cells, which targets to CCR6 on CFU-ECs. Tumors may provide a humoral microenvironment to attenuate the pro-inflammatory activity of EPCs, which might be associated with the tumor escape mechanism.

Colony Forming Unit-Endothelial Cells (CFU-EC) Are Derived from CD14+ Cells and Need Support by CD14− Cells.

Since endothelial progenitor cells (EPC) were found in adult blood, active interferences of vascular diseases by cellular therapy with EPC were initiated. However, controversies exist on the identity of the EPC, e.g. concerning phenotype before and after culture, in vitro and in vivo behavior, as well as on quantities and differentiation. EPC can be quantified in vitro by culturing CFU-EC. In order to clarify which cells are essential and which are facilitating the formation of CFU-EC, we determined the contribution of several cell types to the formation of colonies. Peripheral blood mononuclear cells were isolated and were either positively selected or depleted for several cell populations (CD34+/KDR+/CD146+, CD3+, CD14+, CD19+ or CD56+). Subsequently the cells were labeled with PKH2 or irradiated (40Gy) to determine the contribution to colony formation. The purity of the cell populations was determined by flow cytometry. From these cell populations, CFU-EC were cultured on fibronectin-coated tissue culture plates, in endothelial selective medium EndoCultTM (Stem Cell Technologies). After 2 days of culture, non-adherent cells were subsequently cultured for an additional 3 days to form colonies. To determine the contribution of soluble factors to colony formation, transwells (0.8 μm pore size) were used to separate the cell populations during culture. CFU-EC are not derived from mature endothelial cells nor from immature EPC, as similar number of colonies per well were found when the starting population was depleted for CD146+/CD34+/KDR+ cells (18±9 vs. 20±14 in controls; n=4). In contrast, when CD14+ cells were depleted from the starting population, no CFU-EC were formed (0±0 vs. 29±23 in controls; n=7, p<0.02). After depletion of CD3+, CD19+ or CD56+ cells, CFU-EC were formed similarly as compared to controls. To investigate whether the CD14+ cells were the clonogenic cells, or whether they are facilitating the formation of colonies, monocytes were labeled with phagocyte specific PKH2 or irradiated and combined with untreated CD14− cells. Labeled cells were found in the colonies and among the spindle shaped cells surrounding the colonies. Hardly any colony formation was found after irradiating CD14+ cells (4.5±1 vs. 34±9 in controls), indicating that proliferating monocytes are necessary for CFU-EC formation. Culturing CD14+ cells alone did not result in a similar number of colonies as compared to the control (4±4 vs. 29±23, in controls; n=7, p<0.03). This indicates that CD14− cells are important in outgrowth of CFU-EC from monocytes. To test if this is due to paracrine factors, CD14− cells were cultured in a transwell insert above the >95% CD14+ fraction. This resulted in an increase in colony formation (>95% monocytes: 2±1; with insert: 10±9, n=2). In conclusion, CFU-EC are derived from CD14+ cells, but need the presence of CD14− cells, which is in part due to paracrine factors secreted by CD14− cells. This study further clarifies the identity of the EPC as determined by CFU-EC, and may give more insight to the role of several cell populations in vascular repair.