Endothelial Progenitor Cells Colonies Research Articles

Microarray-Based Characterization of a Colony Assay Used to Investigate Endothelial Progenitor Cells and Relevance to Endothelial Function in Humans

An assay proposed to quantify endothelial progenitor cell (EPC) colonies in humans was investigated to determine the phenotype of recovered cells and their relevance to in vivo endothelial function. Twelve sedentary subjects participating in a worksite wellness program underwent endothelial flow-mediated dilation (FMD) testing of the brachial artery and blood sampling for EPC colony assay. Microarray-based genotypic characterization of colonies showed surface markers consistent with T lymphocyte phenotype, but not with an EPC (CD34, CD133, VEGFR-2) or endothelial (CD146) phenotype. Gene expression patterns more closely matched T lymphocytes (r=0.87) than endothelial cells (r=0.66) in our microarray database. Flow cytometry of colonies confirmed large populations of CD3+CD45+ T cells (>75%) and few CD146+CD45- endothelial cells (<1%). Further, there was no correlation between colony number and the magnitude of FMD (r=-0.1512, P=0.6389). After exercise training, subjects improved FMD, from 6.7+/-2.0 to 8.7+/-1.9% (P=0.0043). Colonies also increased (P=0.0210), but without relation to FMD (r=0.1074, P=0.7396). T lymphocyte phenotype persisted after exercise (r=0.87). Cells in a commonly used EPC colony assay have a gene expression and cell surface marker profile consistent with a predominance of T lymphocytes and have an unclear relevance to endothelial function, either before or after exercise training.

Endothelial progenitor cell number and colony-forming capacity in overweight and obese adults

ObjectiveTo investigate whether adiposity influences endothelial progenitor cell (EPC) number and colony-forming capacity.DesignCross-sectional study of normal weight, overweight and obese adult humans.SubjectsSixty-seven sedentary adults (age 45–65y): 25 normal weight (BMI ≤ 25 kg/m2; 12 males/13 females); 18 overweight (BMI = 25–29.9 kg/m2; 12 males/6 females); and 24 obese (BMI ≥ 30 kg/m2; 18 males/6 females). All subjects were non-smokers and free of overt cardiometabolic disease.MeasurementsPeripheral blood samples were collected and circulating EPC number was assessed by flow cytometry. Putative EPCs were defined as CD45−/CD34+/VEGFR-2+/CD133+ or CD45−/CD34+ cells. EPC colony-forming capacity was measured in vitro using a colony-forming unit assay.ResultsNumber of circulating putative EPCs (either CD45−/CD34+/VEGFR-2+/CD133+ or CD45−/CD34+ cells) was lower (P<0.05) in obese (0.0007±0.0001%; 0.050±0.006%) compared with overweight (0.0016±0.0004%; 0.089±0.019%) and normal weight (0.0015±0.0003%; 0.082±0.008%) adults. There were no differences in EPC number between the overweight and normal weight groups. EPC colony-formation was significantly less in the obese (6±1) and overweight (4±1) compared with normal weight (9±2) adults.ConclusionThese results indicate that: 1) the number of circulating EPCs is lower in obese compared with overweight and normal weight adults; and 2) EPC colony-forming capacity is blunted in overweight and obese adults compared with normal weight adults. Impairments in EPC number and function may contribute to adiposity-related cardiovascular risk.

Dominant Role for SDF-1 in the Vasculo-Angiogenesis Phenotype in Children with Sickle Cell Disease.

BACKGROUND: Vasculogenesis is critically important to restoring and maintaining vascular homeostasis in patients who have sickle cell disease. Emerging evidence indicates vasculo-angiogenesis is a complex process involving recruitment of endothelial progenitor cells (EPCs) from the bone marrow and homing of EPCs to sites of vascular injury. Both recruitment and homing of EPCs are intimately regulated by cytokines and growth factors that are released in response to vascular insult. Hitherto, the phenotype of growth factors and cytokines intimately involved in EPC recruitment and homing in patients who have sickle cell disease remains poorly understood.METHODS: A total of 50 children aged 6–18 years were studied, 30 with Hemoglobin SS, including 11 on chronic red blood cell transfusions, and 20 with normal hemoglobin. We measured plasma concentrations of angiogenesis growth factors/cytokines using a multiplex assay, which permitted analysis of nine factors concurrently, and determined the number and phenotype of EPC colonies derived from peripheral blood samples.RESULTS: Plasma concentrations of stromal derived growth factor (SDF-1, 1.7-fold), angiopoietin-2 (Ang-2, 2-fold) and erythropoietin (7-fold) were significantly elevated (p<0.001) in children with sickle cell disease compared to control subjects. On the contrary, plasma leptin was significantly lower (2-fold, p<0.001) in the sickle cell disease group, while no significant differences were found for several other angiogenic growth factors including VEGF, HGF, IL-8, PGF and PDGF between children with sickle cell disease and the control group. Peripheral blood-derived EPC colonies were significantly higher (almost 3-fold) in patients who have sickle cell disease compared to the control group. Interestingly, EPC colony number was lower in sickle cell patients on chronic transfusion therapy compared to those who were not on chronic transfusion. There were significant correlations between EPC colony number and plasma concentrations of SDF-1 (r=0.65, p<0.001) and Ang-2 (r=0.36, p<0.05), but no correlation between EPC colony number and plasma erythropoietin and leptin.CONCLUSION: These data demonstrate for the first time enhanced vasculogenic potential in patients with sickle cell disease at steady-state. SDF-1 enhances the homing of EPCs and is highly expressed in ischemic tissues. The association of elevated SDF-1 and Ang-2 levels with EPC colony number is consistent with the hypothesis that sickle cell disease causes ongoing ischemic vascular injury. SDF-1 may be potentially used as a marker for vascular injury and a therapeutic target that can be developed to augment vascular repair in sickle cell disease.

Abstract 402: Endothelial Specific Jagged-1/Notch Signal in Bone Marrow Niche Regulates Endothelial Progenitor Cell Development

The developmental cascade of endothelial progenitor cells (EPCs) in response to environmental signals is dynamically regulated by tight-communication between stem cells and stromal cells in bone marrow niches. We have recently reported that Jagged-1 dependent signals specifically regulated EPC-mediated vascularization using mice of Mx-cre dependent inactivation of jagged-1. However, the specific contribution of EPC development via modulating endothelial Jagged-1/Notch axis remain unclear. EPC colony forming capacity of CD34(−)/c-kit(+)/Sca-1(+)/Lin(−) stem cells in tamoxifen-treated Tie2cre-Jagged-1 null mice are drastically enhanced, although the CFU-EPC in CD34(+)/c-kit(+)/Sca-1(+)/Lin(−) progenitor cells was significantly impaired in Jagged-1 null mice. Moreover, the impaired EPC development was also confirmed in single cell-derived EPC colony forming assay. Notably, the development of primitive EPC, an early stage of EPC development, was significantly increased in stem cells, while progenitors derived from BM niche did not give rise to form definitive EPC, a late stage of EPC development. Similarly, we observed markedly decreased cell number and CFU-EPC of progenitor subset (Sca-1+/c-Kit+) in cultured EPCs. Isolated Jag-1 deficient progenitors in cultured EPCs never gave rise to definitive EPC, while WT-derived progenitor did. When co-culture with stromal cells derived from endothelial specific Jagged-1 null mice, stem cells could not develop functional EPCs. EPCs from endothelial specific Jagged-1 deficient niche showed impaired in vitro migration capacity in response to VEGF, SDF-1a, SCF or TPO, as well as the decreased incorporation ability into ischemic neo-vessel. In hind limb ischemia models, we observed the significantly reduced blood vessel regeneration with the decreased capillary density in endothelial specific Jagged-1 null mice. In conclusion, the present report clearly showed for the first time that the Jagged-1/Notch axis plays a pivotal role in EPC development in special niche, suggesting that modulation of EPC development via regulating Jagged-1/Notch axis provide a clue of therapeutic tissue regeneration.

Abstract 3747: Endothelial Specific Inactivation of CXCR4 Receptor in Adult Mice Reveals the Pivotal Role of SDF-1/CXCR4 Axis in the Development of Endothelial Progenitor Cells (EPCs) for Myocardiac Infarction

The development of endothelial progenitor cells (EPCs) is markedly modulated by vascular niche-derived factors for maintaining EPC pool in adult bone marrow. Although SDF-1a/CXCR4 axis have shown to play a critical role in maintaining hematopoietic stem cell (HSC) pool, the precise regulatory mechanism of EPC development remain unclear. In the analysis of tie-2 specific inactivation of CXCR4 in adult mice, we have found that the number of CD34(−/low)/c-kit(+)/Sca-1(+)/Lin(−) or side population (SP) stem cells is profoundly increased. In addition, EPC colony forming capacity of CD34(−)/c-kit(+)/Sca-1(+)/Lin(−) stem cells in endothelial specific CXCR4 null mice are drastically enhanced. Notably, the development of definitive EPC, a late stage of EPC development, is specifically impaired in Tie2cre-CXCR4 null mice, although the colony number of primitive EPCs, an early stage of EPC development, is profoundly increased. EPCs derived from endothelial specific CXCR4 null mice showed showed immatured EPC function, such as migration capacity, proliferation, cell survival. When cultured with SDF-1, niche-derived stem cells gave rise to promoted EPC colony forming capacity. In myocardiac infarction model, we observed the significantly reduced cardiac function,when measured by echocardiography. Furthermore, capillary density was also significantly decreased, as well as induced fibrosis in endothelial specific CXCR4 null mice. In conclusion, the present report clearly showed for the first time that the SDF-1a/CXCR4 axis plays a pivotal role in EPC maturation in special niche, suggesting that the full understanding of EPC development via regulating SDF-1-CXCR4 axis provide a key source of therapeutic tissue regeneration.

Assessing Identity, Phenotype, and Fate of Endothelial Progenitor Cells

From the paradigm shifting observations of Harvey, Malpighi, and van Leeuwenhoek, blood vessels have become recognized as distinct and dynamic tissue entities that merge with the heart to form a closed circulatory system.1 Vessel structures are comprised predominantly of a luminal layer of endothelial cells that is surrounded by some form of basement membrane, and mural cells (pericytes or vascular smooth muscle cells) that make up the vessel wall. In larger more complex vessel structures the vessel wall is composed of a complex interwoven matrix with nerve components. Understanding the cellular and molecular basis for the formation, remodeling, repair, and regeneration of the vasculature have been and continue to be popular areas for investigation. The endothelium has become a particularly scrutinized cell population with the recognition that these cells may play important roles in maintaining vascular homeostasis and in the pathogenesis of a variety of diseases.2 Although it has been known for several decades that some shed or extruded endothelial cells enter the circulation as apparent contaminants in the human blood stream,3 only more recent technologies have permitted the identification of not only senescent sloughed endothelial cells,4 but also endothelial progenitor cells (EPCs), which have been purported to represent a normal component of the formed elements of circulating blood5 and play roles in disease pathogenesis.6–9 Most citations refer to an article published in 1997 in which Asahara and colleagues isolated, characterized, and examined the in vivo function of putative EPCs from human peripheral blood as a major impetus for generating interest in the field.10 This seminal article presented some evidence to consider emergence of a new paradigm for the process of neovascularization in the form of postnatal vasculogenesis. Since publication of that article, interest in circulating endothelial cells, and particularly EPCs, has soared, …

Effects of the Antioxidative -Blocker Celiprolol on Endothelial Progenitor Cells in Hypertensive Rats

Endothelial progenitor cells (EPCs) derived from bone marrow migrate to areas of endothelial damage and repair them. EPC function is impaired by oxidative stress. We examined the effects of an antioxidative beta1-adrenoceptor blocker on the number and function of EPCs in hypertensive rats. Spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats were fed diets loaded with high salt. The SHRs were treated with celiprolol or atenolol for 2 weeks. Peripheral blood mononuclear cells (MNCs) were separated, subjected to flow cytometric analysis to determine the number of circulating EPCs, and cultured to quantify EPC colony formation. EPC migration was evaluated in migration assay chambers. EPC senescence was evaluated using beta-galactosidase assay. Oxidative stress of EPCs was evaluated using thiobarbituric acid-reactive substance (TBARS) assay. The expression of nicotinamine adenine dinucleotide phosphate (NAD(P)H) oxidase component mRNAs in the renal cortex, aorta, and heart were evaluated by real-time PCR. The number, colony formation, and migration of EPCs in SHRs were significantly lower than those in WKY rats. TBARS scores in EPCs from SHRs were significantly higher than those from WKY rats. Celiprolol increased the number of circulating EPCs and stimulated EPC colony formation and migration, while decreasing EPC senescence. Celiprolol inhibited oxidation in EPCs from SHRs, and decreased the expression of NAD(P)H oxidase component mRNAs in the renal cortex, aorta, and heart. EPCs are impaired in SHRs in response to oxidative stress. Celiprolol decreases oxidative stress in hypertension in vivo and improves EPC numbers and function. It appears, therefore, that celiprolol may exert beneficial cardiovascular effects through its antioxidative properties.

Impaired interaction of platelets with endothelial progenitor cells in patients with cardiovascular risk factors

Recent studies indicate that platelets influence endothelial progenitor cell (EPC) recruitment to sites of vascular injury and promote their differentiation to an endothelial phenotype. Patients with cardiovascular risk factors (CVRF) demonstrate a reduced number and impaired function of EPC, as well as platelet hyper-reactivity. Therefore, we investigated the interaction of platelets and EPC from patients with CVRF. Co-incubation of platelets and peripheral blood mononuclear cells, both from healthy volunteers, dose-dependently increased the number of adherent EPC. In contrast, patient-derived platelets failed to augment the number of adherent and migrating healthy and patient-derived EPC. However, co-incubation of platelets from healthy donors with mononuclear cells from patients with CVRF significantly enhanced the number of EPC, indicating that platelets from healthy volunteers are able to partially rescue the impairment of patient-derived EPC formation. Likewise, healthy donor-derived platelets augmented the impaired migration and clonal capacity of patient-derived EPC. Analysis of individual CVRF of platelet donors revealed that only diabetes mellitus inversely correlated with EPC number, colony formation and migration. The platelet supernatants from healthy volunteers that significantly increased EPC number contained IL-6, SDF-1, sCD40L and PDGF. While sCD40L and PDGF levels were comparable in platelet supernatants from healthy volunteers and patients with CVRF, the release of IL-6 and SDF-1 by patient-derived platelets was rather increased, thus, indicating that these soluble factors are not mediating the effect of platelet supernatants. Healthy volunteer-derived platelets provide a source of soluble factors to improve the number and function of EPC from patients with cardiovascular risk factors, particularly diabetes mellitus.

Predictors of Endothelial Function in Employees With Sedentary Occupations in a Worksite Exercise Program

A sedentary workforce may be at increased risk for future cardiovascular disease. Exercise at the work site has been advocated, but effects on endothelium as a biomarker of risk and relation to weight loss, lipid changes, or circulating endothelial progenitor cells (EPCs) have not been reported. Seventy-two office and laboratory employees (58 women; average age 45 years, range 22 to 62; 26 with body mass index values >30 kg/m(2)) completed 3 months of participation in the National Heart, Lung, and Blood Institute's Keep the Beat program, with the determination of vital signs, laboratory data, and peak oxygen consumption (VO(2)) during treadmill exercise. Brachial artery endothelium was tested by flow-mediated dilation (FMD), which at baseline was inversely associated with Framingham risk score (r = -0.3689, p <0.0001). EPCs were quantified by colony assay. With exercise averaging 98 +/- 47 minutes each workweek, there was improvement in FMD (from 7.8 +/- 3.4% to 8.5 +/- 3.0%, p = 0.0096) and peak VO(2) (+1.2 +/- 3.1 ml O(2)/kg/min, p = 0.0028), with reductions in diastolic blood pressure (-2 +/- 8 mm Hg, p = 0.0478), total cholesterol (-8 +/- 25 mg/dl, p = 0.0131), and low-density lipoprotein cholesterol (-7 +/- 19 mg/dl, p = 0.0044) but with a marginal reduction in weight (-0.5 +/- 2.1 kg, p = 0.0565). By multiple regression modeling, lower baseline FMD, greater age, reductions in total and low-density lipoprotein cholesterol and diastolic blood pressure, and increases in EPC colonies and peak VO(2) were jointly statistically significant predictors of change in FMD and accounted for 47% of the variability in FMD improvement with program participation. Results were similar when modeling was performed for women only. In contrast, neither adiposity at baseline nor change in weight was a predictor of improved endothelial function. In conclusion, daily exercise achievable at their work sites by employees with sedentary occupations improves endothelial function, even with the absence of weight loss, which may decrease cardiovascular risk, if sustained.

Highlights of the April 8 issue

### Decreased number and function of endothelial progenitor cells in patients with migraine Migraine carries an increased risk for cardiovascular and cerebrovascular diseases. Diminished counts and functions of endothelial progenitor cells (EPCs) have been associated with higher cardiovascular risk. The authors found that the number of peripheral blood–derived EPC colonies and their functions are reduced in patients with migraine, which suggests that dysfunctional endothelial regeneration capacity is fundamental in migraine. See p. 1510; Editorial, p. 1506 ### The NIH registry on use of the Wingspan stent for symptomatic 70–99% intracranial arterial stenosis The multicenter US registry on …

Decreased number and function of endothelial progenitor cells in patients with migraine

Migraine carries an increased risk for cardiovascular and cerebrovascular diseases that cannot be explained by traditional cardiovascular risk factors. The circulating endothelial progenitor cell (EPC) number is a surrogate biologic marker of vascular function, and diminished EPC counts are associated with higher cardiovascular risk. We investigated whether abnormalities in EPC levels and functions are present in migraine patients. Consecutive headache patients (n =166) were enrolled, including those with tension type headache (TTH; n = 74), migraine without aura (MO; n = 67), and migraine with aura (MA; n = 25). EPC colony-forming units in peripheral blood samples and migratory capacity to chemoattractants (stromal cell-derived factor 1 and vascular endothelial growth factor) and cellular senescence levels were assayed in risk factor-matched subjects (n = 6 per group). The TTH group had more cardiovascular risk factors, more headache days, and higher Framingham risk scores than the other two groups. Mean numbers of EPC colony-forming units were 47.8 +/- 24.3 in TTH, 20.4 +/-22.2 in MO, and 8.6 +/- 10.1 in MA patients (p < 0.001 in TTH vs MO; p = 0.001 in MO vs MA). EPC colony counts of normal subjects (n = 37) were not significantly different from those with TTH. Multiple linear regression models identified only MO, MA, and the presence of migraine (MO + MA) as significant predictors of EPC levels. In addition, EPCs from migraine patients (MO and MA) showed reduced migratory capacity and increased cellular senescence compared with EPCs from TTH or normal subjects. Circulating endothelial progenitor cell (EPC) numbers and functions are reduced in migraine patients, suggesting that EPCs can be an underlying link between migraine and cardiovascular risk.

Circulating Angiogenic Precursors in Idiopathic Pulmonary Arterial Hypertension

Vascular remodeling in idiopathic pulmonary arterial hypertension (IPAH) involves hyperproliferative and apoptosis-resistant pulmonary artery endothelial cells. In this study, we evaluated the relative contribution of bone marrow-derived proangiogenic precursors and tissue-resident endothelial progenitors to vascular remodeling in IPAH. Levels of circulating CD34+ CD133+ bone marrow-derived proangiogenic precursors were higher in peripheral blood from IPAH patients than in healthy controls and correlated with pulmonary artery pressure, whereas levels of resident endothelial progenitors in IPAH pulmonary arteries were comparable to those of healthy controls. Colony-forming units of endothelial-like cells (CFU-ECs) derived from CD34+ CD133+ bone marrow precursors of IPAH patients secreted high levels of matrix metalloproteinase-2, had greater affinity for angiogenic tubes, and spontaneously formed disorganized cell clusters that increased in size in the presence of transforming growth factor-beta or bone morphogenetic protein-2. Subcutaneous injection of NOD SCID mice with IPAH CFU-ECs within Matrigel plugs, but not with control CFU-ECs, produced cell clusters in the Matrigel and proliferative lesions in surrounding murine tissues. Thus, mobilization of high levels of proliferative bone marrow-derived proangiogenic precursors is a characteristic of IPAH and may participate in the pulmonary vascular remodeling process.

Effects of an ARB on Endothelial Progenitor Cell Function and Cardiovascular Oxidation in Hypertension

Angiotensin II (Ang II) receptor blocker (ARB) has been reported to have protective effects on the cardiovascular system independent of blood pressure reduction. Endothelial progenitor cells (EPCs) play a significant role in neovascularization of ischemic tissue. The average lifespan of EPCs was recently reported to be shortened by oxidative stress and regulated by anti-oxidative mechanisms. It has been reported that EPCs are present in peripheral blood and have the ability to repair cardiovascular damage. We investigated the effects of an ARB, candesartan, on EPC function and cardiovascular oxidation in salt-loaded, stroke-prone, spontaneously hypertensive rats (SHR-SP) in vivo. Salt-loaded SHR-SP were treated with candesartan (1 mg/kg/day), a diuretic (trichlormethiazide, TCM, 1.6 mg/kg/day), or an antioxidant (tempol, 5 mg/kg/day) for 2 weeks. Peripheral blood mononuclear cells (MNCs) were isolated and cultured to assay EPC colony formation and migration. Oxidative stress in EPCs was evaluated by thiobarbituric acid reactive substance (TBARS) assay. We evaluated messenger RNA (mRNA) expression of c-kit in the heart, the renin-angiotensin system (RAS) in EPC colonies, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit in cardiovascular organs. Candesartan and tempol, but not TCM, markedly increased EPC colony number in SHR-SP and reduced TBARS. Candesartan also significantly decreased mRNA expression of NADPH oxidase subunits in cardiovascular organs and increased cardiac c-kit mRNA expression. EPCs expressed mRNAs of renin, cathepsin D, chymase, and Ang II type 1 and type 2 receptors. Candesartan, an ARB, improves EPC dysfunction and increases cardiac c-kit expression through the anti-oxidative mechanism in hypertension. The local RAS induces oxidative stress and regulates the EPC functions.

Abstract 1145: Functional Heterogeneity of Angiopoietin-1, -2 on Endothelial Progenitor Cell Fate, Assessed by Single Cell culture of Cord Blood CD133+cells for Endothelial Lineage Commitment Assay

Background; Angiopoietin-1, -2 (Ang-1, Ang-2) has been demonstrated to play a pivotal role in angio-vasculogenesis, i.e. Ang-1 for vascular maturation or Ang-2 for vascular sprouting. However, the developmental mechanism of endothelial progenitor cell (EPC) fate oriented by angiopoietins for vasculogenesis remains to be elucidated. Here, we report the assessment of primitive EPC fate determined by Ang-1,-2, using in vitro endothelial lineage commitment assay of single cord blood (CB)-CD133+ cells. Methods; Single CB-CD133+cells isolated by MACS were cultured in a suspension manner for 7 days in liquid culture containing TPO, SCF, IL-6, Flt-3 ligand, and VEGF with either of Ang-1 or Ang-2 at 200 ng/ml each. The cultured cells from single CB-CD133+cells were applied to vasculogenic methylcellulose culture for EPC colony forming assay (EPC-CFA). Results; EPC-CFA disclosed the analytic potential on the degree of differentiation and commitment of single CB-CD133+cells by the assessment of two types of EPC colonies with the individual cellular sizes, i.e. primitive CFU-small and/or definitive CFU-large EPCs produced by single CB-CD133+cell. Whole EPC colony producing potential was upregulated by both Ang-1 and Ang-2 (ratio of total CFU-EPC number; 1.49 fold in Ang-1, 1.39 fold in Ang-2 vs control, p &lt; 0.05). Both the commitment and differentiation frequencies of EPC from CB-CD133+ cell were promoted by Ang-1, inversely the latter inhibited by Ang-2 (commitment= frequency of single CB-CD133+cell producing any CFU-EPC; 1.5 fold in Ang-1: differentiation= frequency ratio of single CB-CD133+cell producing definitive CFU-EPC/single CB-CD133+cell producing any CFU-EPC; 2 fold in Ang-1, 0.27 fold in Ang-2 vs control, p &lt; 0.01). On the other hand, the expansion potential of primitive CFU-EPC from single CB-CD133+ cell was augmented by Ang-2, although downregulated by Ang-1 (expansion= number of primitive CFU-EPC/ single CB-CD133+cell producing any CFU-EPC; 0.78 fold in Ang-1, 1.6 fold in Ang-2 vs control, p&lt; 0.05). Conclusion; A novel in vitro colony assay system to survey EPC fate of single CB-CD133+cells precisely provides the evidences of angiopoietin-1 mechanism for differentiation into definitive EPC and angiopoietin-2 for procurance of primitive.

The Increase of Circulating Endothelial Progenitor Cells After Acute Ischemic Stroke Is Associated With Good Outcome

Increased circulating endothelial progenitor cells (EPC) have been associated with a low cardiovascular risk and may be involved in endothelial cell regeneration. The present study was designed to evaluate the prognostic value of EPC in acute ischemic stroke. Forty-eight patients with a first-ever nonlacunar ischemic stroke were prospectively included in the study within 12 hours of symptoms onset. Stroke severity was evaluated by the National Institutes of Health Stroke Scale, and functional outcome was assessed at 3 months by the modified Rankin Scale (mRS). Infarct volume growth between admission and days 4 to 7 was measured on multiparametric MRI. EPC colonies were defined as early outgrowth colony-forming unit-endothelial cell (CFU-EC). The increment of CFU-EC was quantified during the first week and defined as the absolute difference between the number of CFU-EC at day 7 and admission. The influence of CFU-EC increase on good functional outcome (mRS <or=2) and infarct growth was analyzed by logistic regression and linear models. Patients with good outcome (n=25) showed a higher CFU-EC increment during the first week (median [quartiles], 23 [11, 36] versus -3 [-7, 1], P<0.0001) compared with patients with poor outcome. CFU-EC increment >or=4 during the first week was associated with good functional outcome at 3 months (odds ratio, 30.7; 95% CI, 2.4 to 375.7; P=0.004) after adjustment for baseline stroke severity, ischemic volume and thrombolytic treatment. For each unit increase in the CFU-EC the mean reduction in the growth of infarct volume was 0.39 (0.03 to 0.76) mL (P=0.033). The increase of circulating EPC after acute ischemic stroke is associated with good functional outcome and reduced infarct growth. These findings suggest that EPC might participate in neurorepair after ischemic stroke.

Androgens stimulate endothelial progenitor cells through an androgen receptor‐mediated pathway

Testosterone (T) treatment has recently been shown to induce an increase in the number of endothelial progenitor cells (EPCs) through a possible effect on bone marrow. Hypogonadotrophic hypogonadal (HH) men have low circulating EPCs that increase significantly after T treatment. Moreover, expression of the androgen receptor (AR) has been demonstrated by immunohistochemistry in these cells, suggesting that T might also have a direct effect on EPC function. In the present study we investigated the expression and function of the AR in human EPCs and the in vitro effect of androgens on EPC function. Design and patients EPCs obtained from healthy male anonymous blood donors were analysed after androgen stimulation with and without AR antagonist administration (flutamide). Reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence analyses demonstrated the expression of AR mRNA and protein in human EPCs. Stimulation of these cells with the synthetic androgen methyltrienolone (R1881) caused AR translocation in the nucleus, suggesting its activation. Colony forming unit (CFU), proliferation and migration assays under different doses of R1881 demonstrated a dose-dependent increase in EPC proliferation, migration and colony formation. All these effects are abolished by flutamide pretreatment. This study showed that the increase in the proliferation, migration and colony formation activity of EPCs induced by androgens is an AR-mediated pathway. Androgen exerts these effects at concentrations that are physiologically present in men and therefore further studies are needed to clarify the clinical significance of these effects in normal and pathological conditions.

Th1- and Th2-Dependent Endothelial Progenitor Cell Recruitment and Angiogenic Switch in Asthma

Increased numbers of submucosal vessels are a consistent pathologic component of asthmatic airway remodeling. However, the relationship between new vessel formation and asthmatic inflammatory response is unknown. We hypothesized that angiogenesis is a primary event during the initiation of airway inflammation and is linked to the recruitment of bone marrow-derived endothelial progenitor cells (EPC). To test this hypothesis, circulating EPC and EPC-derived endothelial cell colony formation of individuals with asthma or allergic rhinitis and health controls was evaluated. Circulating EPC were increased in asthma, highly proliferative, and exhibited enhanced incorporation into endothelial cell tubes as compared with controls. In an acute allergen challenge murine asthma model, EPC mobilization occurred within hours of challenge and mobilized EPC were selectively recruited into the challenged lungs of sensitized animals, but not into other organs. EPC recruitment was Th1 and Th2 dependent and was temporally associated with an increased microvessel density that was noted within 48 h of allergen challenge, indicating an early switch to an angiogenic lung environment. A chronic allergen challenge model provided evidence that EPC recruitment to the lung persisted and was associated with increasing microvessel density over time. Thus, a Th1- and Th2-dependent angiogenic switch with EPC mobilization, recruitment, and increased lung vessel formation occurs early but becomes a sustained and cumulative component of the allergen-induced asthmatic response.

Purification and growth of endothelial progenitor cells from murine bone marrow mononuclear cells

This study reports the culture and purification of murine bone marrow endothelial progenitor cells (EPCs) using endothelial cell-conditioned medium (EC-CM). Endothelial-like cells appeared at day 5 in culture of bone marrow mononuclear cells in the presence of EC-CM in the culture system, and these cells incorporated acetylated low-density lipoproteins (Ac-LDL) and reacted with endothelial-specific Ulex Europaeus Lectin. Continued incubation of these cells at low density with EC-CM for longer than 10 days resulted in the formation of endothelial cell colonies which gave rise to colonies of endothelial progeny and can be passed for many generations in the EC-CM culture system. Cells derived from these colonies expressed endothelial cell markers such as vWF and CD31, incorporated Dil-Ac-LDL, stained positive for Ulex Europaeus Lectin, formed capillary-like structures on Matrigel, and demonstrated a high proliferative capacity in culture. These bone marrow-derived adherent cells were identified as EPCs. The purification and the formation of EPC colonies by using EC-CM were associated with the cytokines secreted in the EC-CM. VEGF, bFGF, and GM-CSF in the EC-CM stimulated the proliferation and growth of EPCs, whereas AcSDKP (tetrapeptide NAc-Ser-Asp-Lys-Pro) in EC-CM suppressed the growth of mesenchymal stem cells (MSC) and fibroblasts. This approach is efficient for isolation/purification and outgrowth of bone marrow EPCs in vitro, a very important cell source in angiogenic therapies and regenerative medicine.

Losartan Improves the Impaired Function of Endothelial Progenitor Cells in Hypertension via an Antioxidant Effect

We evaluated the effects of the angiotensin II (Ang II) receptor blocker (ARB) losartan on the formation and number of endothelial progenitor cells (EPCs) in hypertensive rats. Wistar-Kyoto (WKY) rats and stroke-prone, spontaneously hypertensive rats (SHR-SP) were salt-loaded and then treated with losartan (10 mg/kg/day), trichlormethiazide (TCM; 1.6 mg/kg/day), or tempol (1 mmol/L) for 2 weeks. Peripheral blood mononuclear cells were isolated, subjected to flow cytometric analysis to determine the number of circulating EPCs, cultured to assay EPC colony formation, and subjected to a migration chamber assay to evaluate EPC migration. Oxidative stress in EPCs was evaluated by thiobarbituric acid reactive substance (TBARS) assay. The results showed that the number, colony formation, and migration of EPCs were markedly decreased in SHR-SP compared to those in WKY rats. The TBARS scores were significantly greater in SHR-SP than in WKY rats. Losartan and TCM decreased systolic blood pressure in SHR-SP to similar levels. Losartan and tempol increased the number of circulating EPCs and colony formation, and inhibited oxidation in SHR-SP. TCM did not affect the EPC number, colony formation, or oxidation. Both losartan and TCM stimulated EPC migration. Expression of gp91(phox), p22(phox), and p47(phox) mRNA in tissues was significantly decreased by losartan but not by TCM. These results indicate that the formation and function of EPCs are impaired by oxidative stress in SHR-SP. This is the first report to show that losartan improves the proliferation and function of EPCs in hypertension, suggesting that ARBs are useful to repair hypertensive vascular injuries.

Short-term endothelial progenitor cell colonies are composed of monocytes and do not acquire endothelial markers

The aim of this study was to identify circulating endothelial progenitor cells (EPC) with colony-forming capacity and compare them with the monocytic-macrophage lineage. Forty-two healthy donors were analyzed. EPC were cultured with VEGF and b-FGF. Sequential studies were performed on days +7 (colonies) +21 and +35. Monocytic cells were cultured using the same conditions as EPC until day +21 or alternatively by adding IGF. The number of EPC colonies was higher in BM than in mobilized or steady-state PB. Using EPC medium, monocytic cells formed cord-like structures but no colonies. However, colonies grew when IGF was added to the medium. By immunocytochemistry, colonies showed CD45, CD31 and lysozyme but no vWF. Colonies were CD4+, CD13+dim, CD14+, CD15++, CD16-/+dim, CD31+dim, CD33+dim, CD45+, CD105-/+dim, lysozyme+ and VE-cadherin+, and constantly negative for CD34, CD133 and KDR, when flow cytometry was used. The immunophenotype of pre-cultured and cultured monocytes was similar to that described for EPC. Our results suggest that the so-called 'EPC' obtained at 7 days of culture belong to the monocyte-macrophage lineage, as they share immunophenotypic and molecular features.