Characteristics Of Endothelial Progenitor Cells Research Articles

Transplantation application and mechanisms of endothelial progenitor cells

Background Recent studies have demonstrated the importance of endothelial progenitor cells(EPCs)in maintaining normal endothelial function as well as endothelial repairing after vascular injury.Objective This review summarizes transplantation therapy of EPCs and discusses the mechanisms regulating the cells-mediated neovascularization and reendothelialization.Content The text describes the biological characteristics of EPCs,summarizes the application of endothelial progenitor cell transplantation and discusses potential mechanisms of EPCs.Trend Transplantation of EPCs can be a novel cellbased,endothelium-targeted therapeutic strategy for prevention and treatment of vascular disease and inflammation injury.The clinical application of EPCs still needs to be investigated deeply. Key words: Endothelial progenitor cell ; Transplantation ;

The role of SDF-1α/Rac pathway in the regulation of endothelial progenitor cell polarity; homing and expression of Rac1, Rac2 during endothelial repair

The Stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) system is considered to be of great importance in diseases involving cardiogenesis and angiogenesis. The SDF-1α-RAC signaling pathway plays a pivotal role in a cell's polarity and serves to activate cell morphology variation and to control the direction of migration. We aimed to study whether the polarity of endothelial progenitor cells (EPCs) is changed by the induction of the SDF-1α-RAC signaling pathway, to investigate the mechanism of the effect of polarity on the homing action of EPCs, and to explore the gene and protein expression of Rac1/2 during endothelial repair. We measured the EPC characteristics of polarity induced by various final concentrations of SDF-1α; our observations included morphology variation, migration direction, and excursion. Of the dynamic variation and cytoskeleton rearrangement of EPCs induced by different final concentrations of SDF-1α, the most obvious variation was exhibited at the SDF-1α concentration of 200 ng/ml. Obvious polarity variations were also found in the EPCs and signal receptors induced by the SDF-1α concentration of 200 ng/ml. The Western blot analysis of Rac1 and Rac2 showed that the addition of AMD 3100 significantly inhibited the expression of Rac. The SDF-1α pathway potentially regulates the expression of Rac1/2. The actual excursion vector and the direction of the migration of EPCs induced by SDF-1α follows polarity, thus indicating the importance of further exploration regarding the homing induction of EPCs.

Isolation of three important types of stem cells from the same samples of banked umbilical cord blood

It is known that umbilical cord blood (UCB) is a rich source of stem cells with practical and ethical advantages. Three important types of stem cells which can be harvested from umbilical cord blood and used in disease treatment are hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs). Since these stem cells have shown enormous potential in regenerative medicine, numerous umbilical cord blood banks have been established. In this study, we examined the ability of banked UCB collected to produce three types of stem cells from the same samples with characteristics of HSCs, MSCs and EPCs. We were able to obtain homogeneous plastic rapidly-adherent cells (with characteristics of MSCs), slowly-adherent (with characteristics of EPCs) and non-adherent cells (with characteristics of HSCs) from the mononuclear cell fractions of cryopreserved UCB. Using a protocol of 48 h supernatant transferring, we successfully isolated MSCs which expressed CD13, CD44 and CD90 while CD34, CD45 and CD133 negative, had typical fibroblast-like shape, and was able to differentiate into adipocytes; EPCs which were CD34, and CD90 positive, CD13, CD44, CD45 and CD133 negative, adherent with cobble-like shape; HSCs which formed colonies when cultured in MethoCult medium.

Habitual short sleep duration and circulating endothelial progenitor cells

Chronic short sleep duration has been linked to endothelial dysfunction and increased risk of cardiovascular disease. Circulating endothelial progenitor cells (EPCs) are vital to endogenous vascular repair processes and cardiovascular health. We tested the hypothesis that habitual short sleep duration is associated with impairment in EPC number and function. Cells with phenotypic EPC characteristics were isolated from 37 healthy, sedentary adults: 20 with normal sleep duration (13M/7F; age: 59±1 years; sleep duration: 7.7±0.1 h/night) and 17 with short sleep duration (9M/8F; 56±2 years; 6.0±0.2 h/night). EPC number was assessed by flow cytometric analysis of the percentage of peripheral blood mononuclear cells negative for CD45 and positive for CD34, VEGFR-2, and CD133 antigens. EPC colony-forming capacity was determined by colony-forming unit (CFU) assay; migration by Boyden chamber; and intracellular caspase-3 concentrations by immunoassay. There were no significant differences between groups in EPC number (0.001±0.0004 vs. 0.001±0.0003 %), colony-forming capacity (6.1±1.5 vs. 5.4±1.7 CFUs), or migration to VEGF (1410.1±151.2 vs. 1334.3±111.1 AU). Furthermore, there were no group differences in basal and staurosporine-stimulated intracellular concentrations of active caspase-3 (0.3±0.03 vs. 0.5±0.1 ng/mL; and 2.9±0.4 vs. 2.7±0.3 ng/mL), a marker of apoptotic susceptibility. Taken together, these data indicate that short sleep duration is not associated with EPC dysfunction in healthy adults. Numerical and functional impairment in circulating EPCs may not contribute to the increased cardiovascular risk with habitual short sleep duration.

The characteristics of endothelial progenitor cells derived from mononuclear cells of rat bone marrow in different culture conditions

Endothelial progenitor cells (EPCs) derived from bone marrow are known to be heterogeneous. In this study, we tried to find favorable conditions that induce the differentiation of mononuclear cells (MNCs) from bone marrow into EPCs. The differentiation capacity of MNCs from rat bone marrow was investigated in different conditions, such as different media, different induction times and different culture surfaces. The cell morphology and endothelial biomarkers associated with differentiated MNCs were studied. Our results indicated that MNCs cultured in EGM-2MV (Endothelial cell basal medium-2, plus SingleQuots of growth supplements) developed a bursiform shape, a late EPC-like morphology, while MNCs cultured in complete medium (CM, M199 with 10% FBS, 20ng/mL VEGF and 10ng/mL bFGF) showed a spindle shape, an early EPC-like morphology. Cells of both morphologies were able to incorporate DiI-ac-LDL and bind lectin in vitro. MNCs cultured in EGM-2MV exhibited a higher proliferation rate and higher eNOS expression than MNCs cultured in CM. MNCs cultured in EGM-2MV had the ability to form tubes on Matrigel. Flow cytometry results indicated that CD133 expression was highest at day 12 and that the greatest number of cells positive for both FLK-1 and CD133 appeared at day 20 from cells cultured in dishes without fibronectin coating. In addition, the expression levels of CD133, CD31 and FLK-1/CD133 were not significantly different between cells of different shapes. Our experiments suggest that MNCs from bone marrow can be differentiated into late EP-like cells in EGM-2MV, which have the ability to rapidly proliferate. These MNCs can also be differentiated into early EP-like cells in CM. Additionally, fibronectin may not be necessary for the differentiation of EPCs to mature ECs after three generations. Differentiated MNCs from bone marrow in EGM-2MV have the characteristics of EPCs, although the expression levels of EPC markers were lower than previously reported.

Quantification of Circulating Endothelial Progenitor Cells Using the Modified ISHAGE Protocol

AimsCirculating endothelial progenitor cells (EPC), involved in endothelial regeneration, neovascularisation, and determination of prognosis in cardiovascular disease can be characterised with functional assays or using immunofluorescence and flow cytometry. Combinations of markers, including CD34+KDR+ or CD133+KDR+, are used. This approach, however may not consider all characteristics of EPC. The lack of a standardised protocol with regards to reagents and gating strategies may account for the widespread inter-laboratory variations in quantification of EPC. We, therefore developed a novel protocol adapted from the standardised so-called ISHAGE protocol for enumeration of haematopoietic stem cells to enable comparison of clinical and laboratory data.Methods and ResultsIn 25 control subjects, 65 patients with coronary artery disease (CAD; 40 stable CAD, 25 acute coronary syndrome/acute myocardial infarction (ACS)), EPC were quantified using the following approach: Whole blood was incubated with CD45, KDR, and CD34. The ISHAGE sequential strategy was used, and finally, CD45dimCD34+ cells were quantified for KDR. A minimum of 100 CD34+ events were collected. For comparison, CD45+CD34+ and CD45−CD34+ were analysed simultaneously. The number of CD45dimCD34+KDR+ cells only were significantly higher in healthy controls compared to patients with CAD or ACS (p = 0.005 each, p<0.001 for trend). An inverse correlation of CD45dimCD34+KDR+ with disease activity (r = −0.475, p<0.001) was confirmed. Only CD45dimCD34+KDR+ correlated inversely with the number of diseased coronaries (r = −0.344; p<0.005). In a second study, a 4-week de-novo treatment of atorvastatin in stable CAD evoked an increase only of CD45dimCD34+KDR+ EPC (p<0.05). CD45+CD34+KDR+ and CD45−CD34+KDR+ were indifferent between the three groups.ConclusionOur newly established protocol adopted from the standardised ISHAGE protocol achieved higher accuracy in EPC enumeration confirming previous findings with respect to the correlation of EPC with disease activity and the increase of EPC during statin therapy. The data of this study show the CD45dim fraction to harbour EPC.

Endothelial Progenitor Cell Function, Apoptosis, and Telomere Length in Overweight/Obese Humans

Excess adiposity is associated with increased cardiovascular morbidity and mortality. Endothelial progenitor cells (EPCs) play an important role in vascular repair. We tested the hypothesis that increased adiposity is associated with EPC dysfunction, characterized by diminished capacity to release angiogenic cytokines, increased apoptotic susceptibility, reduced cell migration, and shorter telomere length. A total of 67 middle-aged and older adults (42-67 years) were studied: 25 normal weight (normal weight; BMI: 18.5-24.9 kg/m(2)) and 42 overweight/obese (overweight/obese; BMI: 25.0-34.9 kg/m(2)). Cells with phenotypic EPC characteristics were isolated from peripheral blood. EPC release of vascular endothelial growth factor (VEGF) and granulocyte colony-stimulating factor (G-CSF) was determined in the absence and presence of phytohemagglutinin (10 microg/ml). Intracellular active caspase-3 and cytochrome c concentrations were determined by immunoassay. Migratory activity of EPCs in response to VEGF (2 ng/ml) and stromal cell-derived factor-1alpha (SDF-1alpha; 10 ng/ml) was determined by Boyden chamber. Telomere length was assessed by Southern hybridization. Phytohemagglutinin-stimulated release of VEGF (90.6 +/- 7.6 vs. 127.2 +/- 11.6 pg/ml) and G-CSF (896.1 +/- 77.4 vs. 1,176.3 +/- 126.3 pg/ml) was ~25% lower (P < 0.05) in EPCs from overweight/obese vs. normal weight subjects. Staurosporine induced a ~30% greater (P < 0.05) increase in active caspase-3 in EPCs from overweight/obese (2.8 +/- 0.2 ng/ml) compared with normal weight (2.2 +/- 0.2) subjects. There were no significant differences in EPC migration to either VEGF or SDF-1alpha. Telomere length did not differ between groups. These results indicate that increased adiposity adversely affects the ability of EPCs to release proangiogenic cytokines and resist apoptosis, potentially compromising their reparative potential.

Circulating endothelial cells in patients with chronic lymphocytic leukemia

In patients with cancer, circulating endothelial cells (CECs) are increased and are correlated with an aggressive disease course. However, the clinical and biologic significance of CECs in chronic lymphocytic leukemia (CLL) remains uncertain. In 170 patients with CLL, CEC levels were quantified by flow cytometry and were correlated with clinical and biologic data. In addition, CECs were characterized by immunophenotypic, fluorescence in situ hybridization (FISH), and gene expression profile analyses. In patients with CLL, CECs were increased compared with controls. A higher level of CECs (>20/microL) identified a subset of patients with a more aggressive disease course characterized by a shorter time to first treatment both in univariate and multivariate analyses. In FISH analysis, 7 patients had a significant proportion of CECs and presented with the same cytogenetic lesion of neoplastic lymphocytes and immunophenotypic features of endothelial progenitor cells. The gene expression profile of sorted CECs revealed a molecular pattern, suggesting a derivation from CLL leukemic cells with increased cell survival and proliferation, diminished cell adhesion to extracellular matrix, and enhanced proangiogenic function compared with their normal counterparts. The current data suggest that, in CLL, CECs may represent a biologic marker of aggressiveness and disease progression to be considered for new, targeted antiangiogenic treatments.

The Number of circulating endothelial progenitor cells is decreased in children with obesity

Circulating endothelial progenitor cells (EPCs) are thought to preserve the integrity of the vascular endothelium and to play a role in the pathogenesis of cardiovascular disease. Stromal cell-derived factor-1 (SDF-1) is involved in progenitor cell mobilization, differentiation and homing. In this study, we compared SDF-1 serum concentrations and the number and functional characteristics of EPCs between obese and lean children and assessed potential associations with endothelial dysfunction and insulin sensitivity.

Short Sleep Duration and Endothelial Progenitor Cell Function

Chronic short sleep duration is associated with increased cardiovascular (CV) morbidity. Mechanisms underlying this increased CV risk are not clear. Endothelial progenitor cells (EPCs) contribute to both reendothelialization and neovascularization. Numerical and functional impairment of EPCs has been linked to CV risk. Currently, there is no information regarding the influence of short sleep duration on EPC function. We tested the hypothesis that EPC function is impaired in adults who sleep less than 7 hrs/night (short sleep) compared with those who sleep 7 to 9 hrs/night (normal sleep). Cells with phenotypic EPC characteristics were isolated from 37 healthy, sedentary adults: 20 normal sleepers (13M/7F; age: 59±1 yr; sleep: 7.7±0.1 hrs/night) and 17 short sleepers (9M/8F; 56±2 yr; 6.0±0.2 hrs/night). There were no significant differences between groups in EPC number via FACS (0.001±0.0004 vs 0.001±0.0003 %), colony‐forming capacity (6.1±1.5 vs 5.4±1.7 CFUs), or migration to VEGF in a Boyden chamber (1410.1±151.2 vs 1334.3±111.1 AU). Furthermore, were no group differences in basal and staurosporine stimulated intracellular concentrations of active caspase‐3 (0.3±0.03 vs 0.5±0.1 ng/mL; and 2.9±0.4 vs 2.7±0.3 ng/mL) and cytochrome c (16.4±0.7 vs 14.5±1.4 ng/mL), markers of apoptotic susceptibility. Short sleep duration is not associated with EPC dysfunction in healthy adults.

The Promise of Cell-Based Therapies for Diabetic Complications

The discovery of endothelial progenitor cells (EPCs) in human peripheral blood advanced the field of cell-based therapeutics for many pathological conditions. Despite the lack of agreement about the existence and characteristics of EPCs, autologous EPC populations represent a novel treatment option for complications requiring therapeutic revascularization and vascular repair. Patients with diabetic complications represent a population of patients that may benefit from cellular therapy yet their broadly dysfunctional cells may limit the feasibility of this approach. Diabetic EPCs have decreased migratory prowess and reduced proliferative capacity and an altered cytokine/growth factor secretory profile that can accelerate deleterious repair mechanisms rather than support proper vascular repair. Furthermore, the diabetic environment poses additional challenges for the autologous transplantation of cells. The present review is focused on correcting diabetic EPC dysfunction and the challenges involved in the application of cell-based therapies for treatment of diabetic vascular complications. In addition, ex vivo and in vivo functional manipulation(s) of EPCs to overcome these hurdles are discussed.

Effect of endothelial progenitor cells on the proliferation of co-cultured vascular smooth muscle cells

To explore the effect of endothelial progenitor cells (EPCs) on the proliferation of co-cultured rat vascular smooth muscle cells (VSMCs). Mononuclear cells were isolated from fresh cord blood by 6% hydroxyethyl starch (HES) and density gradient centrifugation. Isolated mononuclear cells were cultured in EGM-2 medium supplemented with 20% fetal bovine serum (FBS), VEGF,bFGF and other growth factors. Biological features of EPCs were observed at different time points, and EPCs were identified by morphology, fluorescence double-staining and flow cytometry. Indirect immunofluorescence was performed to analyze the expression of alpha-SM-actin, calponin of VSMCs special antigen. Co-culture system of EPCs and VSMCs was established by transwell permeable support. FBS (20%) was used to stimulate the proliferation of VSMCs. In a VSMCs/EPCs co-culture system, the DNA synthesis ability, total protein level and cell cycle of VSMCs were determined by BrdU marking method,protein quantitation and flow cytometry after co-culture for 6, 12, 24,48 and 72 h. After co-culture for 12, 24, 48, and 72 h, the DNA synthesis ability and total protein level of VSMCs significantly decreased compared with the control group (P<0.05). Flow cytometry showed that the percentage of S phase of VSMCs in VSMCs/EPCs co-cultured group significantly decreased and the percentage of G1 phase increased markedly compared with the control group (P<0.05). The maximal inhibitory effect was observed at 48 h. Early EPCs could inhibit the proliferation of VSMCs.

Endothelial Nitric Oxide Synthase as A Marker for Human Endothelial Progenitor Cells

Endothelial progenitor cells (EPCs) have been proposed as a promising tool for therapeutic neovascularization, vascular repair, tumor pathology and tissue engineering, though their identification is still a subject of much discussion. EPCs consist of two different subpopulations, termed endothelial cell (EC)-like cells and endothelial outgrowth cells (EOCs). Both types of EPCs are derived from mononuclear cells, but they have different characteristics. Our aim was to characterize and compare the two types of EPCs to find reliable biological features of EPCs that can be used for identification of EPCs. In this study, human peripheral blood mononuclear cells were isolated by density gradient centrifugation and cultured on fibronectin-coated culture plates. While adherent cells were maintained, EC-like cells appeared within 4-7 days of culture, and EOCs developed after 2-3 weeks of culture. EOCs, which were characterized by high proliferation potential, were able to form capillary tubes on Matrigel, but not EC-like cells, despite the higher concentrations of three angiogenic cytokines, vascular endothelial growth factor, granulocyte colony-stimulating factor, and interleukin 8, in the conditioned medium of EC-like cells. In contrast, endothelial nitric oxide synthase (eNOS) was expressed in both types of EPCs, and both cell types could produce nitric oxide (NO), as judged by measuring the total amounts of nitrites and nitrates in culture media. In conclusion, the expression of eNOS and the production of NO could be used as common biological features to identify EPCs. These findings provide new insights into the identification of EPCs.

Effects of endothelin‐1 on endothelial progenitor cell function

Circulating endothelial progenitor cells (EPCs) play an important role in maintaining and repairing the vascular endothelium and restoring functional activity. Endothelin (ET)‐1, a potent vasoconstrictor peptide, is released by the endothelium and contributes to endothelial damage and atherogenesis. The effects of ET‐1 on EPC function are unknown. We tested the hypothesis that ET‐1 impairs EPC function. Peripheral blood samples were collected from 10 healthy adult humans. Cells with phenotypic EPC characteristics were isolated and EPC clonogenic (colony forming unit assay) and migratory (Boyden chamber) capacity as well as apoptotic susceptibility (TUNEL assay) were determined in the absence and presence and ET‐1 (100 pM). The number of EPC colony‐forming units (42±12 vs. 39±11) and migratory capacity (910±146 vs. 936±148) was not significantly different between the ET‐1 treated and untreated cells. In response to staurosporine (1 mM), EPCs treated with ET‐1 demonstrated a 20% increase (P&lt;0.05) in cellular apoptosis. The proapoptotic effect of ET‐1 was abolished with ET receptor blockade as well as with apocynin, an NADPH inhibitor. These results indicate that ET‐1 does not affect EPC colony formation or migratory capacity but does increase EPC susceptibility to apoptosis through a NADPH‐dependent mechanism. Increased EPC apoptosis may contribute to the proatherogenic effects of ET‐1.

Primary aldosteronism (PA) and endothelial progenitor cell (EPC) bioavailability

Patients with primary aldosteronism (PA) experience more cardiovascular events than patients with essential hypertension matched for risk factor profile. Endothelial progenitor cells (EPC) represent a bone marrow-derived cell population implicated in vascular healing whose number correlates to the cardiovascular risk factor profile. Aldosterone has been reported to decrease EPC proliferation in rats. We assessed (i) the growth characteristics of EPC from six PA patients and six matched normotensive controls; (ii) the growth characteristics of EPC treated with increasing doses of aldosterone. Senescence and cell-cycle analysis of EPC from PA patients and normotensive controls and of aldosterone-treated EPC from healthy volunteers. No difference was found in the senescence rate between EPC from PA patients (72.4% senescent cells) and controls (70.7%, P > 0.05). No difference was also found in the cell-cycle distribution determined by FACS (controls: 75.2% cells in G0/G1 phase; PA: 73.5%, P > 0.05). Incubation of EPC with aldosterone did not modify their senescence rate (controls: 72.4% senescent cells; aldosterone 10 nmol/l: 70.9%; aldosterone 100 nmol/l 71.6%, P > 0.05 for all comparisons) and cell-cycle distribution (controls: 73.3% cells in G0/G1 phase; aldosterone 10 nmol/l: 74.9%; aldosterone 100 nmol/l: 75.4%, P > 0.05 for all comparisons). No expression of the mineralocorticoid receptor (MR) transcript was found in EPC by RT-PCR analysis. High aldosterone levels, both in PA patients and in vitro, exert no direct or indirect effect on EPC growth characteristics.

Clinical scale ex vivo expansion of cord blood–derived outgrowth endothelial progenitor cells is associated with high incidence of karyotype aberrations

Endothelial progenitor cells (EPCs) are involved in neovessel formation. So far, therapeutic angiogenesis is hampered by the low frequency and limited proliferative potential of these cells isolated from peripheral blood. Recently, it has been shown that cord blood-derived EPCs (CB EPCs) can be ex vivo expanded on a clinical scale. In this study, we evaluated the expansion potential of CB EPCs together with their phenotypic, functional, and chromosomal stability over time. Flow cytometry, in vitro tube formation, and proliferation assays were performed to characterize CB EPC-derived cells. Chromosomal stability was evaluated by karyotype analysis. In vitro and in vivo tumorigenicity was evaluated by soft agar assay and injection into nonobese diabetic/severe combined immunodeficient mice, respectively. We showed that CB EPC-derived cells displayed phenotypic and functional features of EPCs, although a process of maturation was observed over time. Although we confirmed that CB EPCs have a greater expansion potential compared to peripheral blood EPCS, we observed a high incidence of cytogenetic alterations (71%) in the expanded endothelial cell population, even at early times of culture. In two cases, spontaneous transformation in vitro was documented, but none of the samples tested showed tumorigenic potential in vivo. Conversely, no karyotype alterations have been observed on peripheral blood EPCs-derived cells. We confirm that CB represents a good source for clinical ex vivo expansion of EPCs. However, because of high frequency of karyotype alterations, these cells cannot be considered free of risk in clinical application.

Circulating bone marrow‐derived endothelial progenitor cells: Characterization, mobilization, and therapeutic considerations in malignant disease

Until recently, tumor vascularization was thought to occur exclusively through angiogenesis. However, recent studies using different animal models of cancer suggested the importance of bone marrow-derived endothelial progenitor cells (EPCs) (i.e. postnatal vasculogenesis) in tumor vascularization and growth. EPCs are present in the peripheral blood, their levels are increased in response to certain signals/cytokines, and they home into the neovascular bed of malignant tissues. Furthermore, at the clinical level, evidence is emerging that changes in EPC levels might predict the efficacy of anticancer drug combinations that include antiangiogenic agents. On the basis of these observations, EPCs have attractive potential diagnostic and therapeutic applications for malignant diseases. In this paper, we review biological features of EPCs and speculate on the utility of these progenitor cells for medical oncology.

Frontiers in Nephrology

Cardiovascular-renal diseases are the leading causes of death and disability in the modern world. Moreover, chronic heart failure after myocardial infarction and peripheral arterial disease are predominant, devastating cardiovascular entities that limit prognosis and greatly impair quality of life in patients despite modern medical treatments. Cardiovascular diseases are also the predominant cause of death in people with renal diseases, underscoring the close relationship between these diseases. Until recently, medical efforts aimed only at prevention and slowing of functional deterioration after organ damage; however, the recent discovery of endogenous repair mechanisms involving hematopoietic stem and other progenitor cells has challenged the long-standing dogma regarding the inability to repair or regenerate terminally differentiated organs. A variety of stem and progenitor cell populations have shown properties that are potentially suited for tissue repair. After encouraging results in preclinical animal models, early clinical studies of endothelial progenitor cells (EPC) have begun. Envisioning the goal of true tissue regeneration, application of bone marrow-derived progenitors for heart and limb ischemia has provided early evidence of safety and feasibility. These studies have provided data indicating functional improvement as well. Although there is strong experimental and clinical evidence for a regenerative function of EPC even in renal disease, there has been no clinical application of this approach. After the initial flurry of activity, it is time to reconsider these approaches and attempt to optimize functional improvement and patient safety. This article briefly recapitulates biologic and functional characteristics of EPC before giving a concise overview on current therapeutic EPC applications in the field of cardiovascular-renal medicine with consideration of future challenges.

Study on culture, identification and differentiation of CD133+ endothelial progenitor cells from human umbilical cord blood

To study the isolation, culture and identification of CD133+ endothelial progenitor cells (EPCs) from human umbilical cord blood in vitro. EPC separation was performed with density gradient centrifugation and MACS separation. Purity of EPCs was determined by flow cytometry. EPC was cultured with EBM-2 to study the cultivate features of EPC. Uptake test of Dil-LDL and FITC-Lectin and immunohistochemistry were performed. According to flow cytometry, (1.13 +/- 0.10)% of mono-nuclear cells were CD133+ and the purities of CD133+ EPCs were (91.45 +/- 1.04)% on average. CD133+ EPCs became adherent, spindle-shaped and formed cluster during culture. Uptake test of Dil-LDL and FITC-Lectin were positive. (95.83 +/- 1.72)% of CD133+ cells were found positive in both uptake tests. The positive rates of immunostaining of cell markers CD34 and factor VIII were (95.83 +/- 2.23)% and (95.92 +/- 1.43)% after cultured for one week, which showed no significant differences between CD133+ EPCs and human umbilical vein endothelial cells. Capillary structures were formed by CD133+ EPCs after cultured for 4 and 7 d in vitro. High purity of CD133+ EPCs can be obtained by MACS separation. CD133+ EPCs can differentiate into mature endothelial cells with the effects of stimulating factors.

Characterization and clinical relevance of circulating and biopsy-derived endothelial progenitor cells in lymphoma patients

Endothelial progenitor cells (EPC) have been proven to be essential for tumor angiogenesis and growth in animal tumor models. However, the involvement and relevance of EPC in human cancers remain poorly studied. We, therefore, investigated the presence, differentiation potential and molecular characteristics of EPC in lymphoma patients. EPC (CD133+CD34+KDR+ cells) were detected in peripheral blood (PB) and lymph node (LN) biopsy samples of 70 lymphoma patients by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. Magnetically isolated EPC (PB and LN-derived) were tested, in vitro, for endothelial differentiation potential and RNA was collected to study their gene expression profiles by Affymetrix oligonucleotide arrays. Lymphoma patients were classified according to disease aggressiveness (indolent vs aggressive lymphoma) and their data (tumor angiogenesis, tumor stage and clinical treatment) were related to the presence or absence of EPC in the circulation or in tumor samples. Circulating EPC (CEPC) were more frequent in patients than in healthy controls and more frequent in younger patients than in older patients and in those with aggressive lymphomas. The levels of CEPC decreased in patients with complete response to treatment, but were sustained or increased in the non- or partial- responders to lymphoma therapy. Notably, EPC in the LN (LN-EPC) were more frequently detected than CEPC; LN-EPC were detected in vascular structures and also in the stroma, and after isolation differentiated into endothelial cells in vitro. The presence of LN-EPC correlated with lesion size and with increased angiogenesis in indolent lymphomas. CEPC and LN-EPC share endothelial markers but can be identified and quantified separately, since they express different CD133 isoforms. Gene expression profiling of isolated LN-EPC revealed the expression of pro-angiogenic and tumor growth factors that may influence lymphoma growth. EPC are present in the circulation and in tumor samples from patients with non-Hodgkin's lymphoma. Since there are relationships between EPC and various characteristics of lymphoma, our research has demonstrated the clinical and biological relevance of studying CEPC and LN-EPC in lymphoma patients.