Vascular Endothelial Progenitor Research Articles

Role of vascular endothelial progenitor cells in construction of new vascular loop

Since bone marrow-derived endothelial progenitor cells (EPCs) have been detected in adult peripheral blood, the mode of vasculogenesis in the adult tissue has caught attention in field of vascularization research. To confirm the role of EPCs in construction of new vascular loop, we took the biomaterial scaffold implanted into adult rat as an experimental model to observe and examine the actions of the EPCs in neovascularization of the material by immunohistochemistry and transmission electron microscopy. Additionally, by establishing a chemotactic migration model for vascular endothelial cells (ECs) and EPCs, the migrations of ECs and EPCs were explored in simulations. The results of 20,000 simulations showed that the number of the vascular loops assisted by the EPCs was 2–5 times that of the vascular sprouts being naturally joined. Based on the results of experiments and simulations, we conclude that the EPCs are able to assist the angiogenic sprouts in joining under the condition of plenty of the EPCs being mobilized, which aggregate at sites close to sprout tips, forming a cell cord and differentiating to ECs in situ, and become vessel segments between neighboring sprouts. This suggests that there is a difference between the adult and embryo in the manner of vasculogenesis and that a small number of EPCs can play an important role to make the new blood vessels achieve rapid functionalization.

Wnt/β-catenin signalling regulates Sox17 expression and is essential for organizer and endoderm formation in the mouse

Several signalling cascades are implicated in the formation and patterning of the three principal germ layers, but their precise temporal-spatial mode of action in progenitor populations remains undefined. We have used conditional gene deletion of mouse β-catenin in Sox17-positive embryonic and extra-embryonic endoderm as well as vascular endothelial progenitors to address the function of canonical Wnt signalling in cell lineage formation and patterning. Conditional mutants fail to form anterior brain structures and exhibit posterior body axis truncations, whereas initial blood vessel formation appears normal. Tetraploid rescue experiments reveal that lack of β-catenin in the anterior visceral endoderm results in defects in head organizer formation. Sox17 lineage tracing in the definitive endoderm (DE) shows a cell-autonomous requirement for β-catenin in midgut and hindgut formation. Surprisingly, wild-type posterior visceral endoderm (PVE) in midgut- and hindgut-deficient tetraploid chimera rescues the posterior body axis truncation, indicating that the PVE is important for tail organizer formation. Upon loss of β-catenin in the visceral endoderm and DE lineages, but not in the vascular endothelial lineage, Sox17 expression is not maintained, suggesting downstream regulation by canonical Wnt signalling. Strikingly, Tcf4/β-catenin transactivation complexes accumulated on Sox17 cis-regulatory elements specifically upon endoderm induction in an embryonic stem cell differentiation system. Together, these results indicate that the Wnt/β-catenin signalling pathway regulates Sox17 expression for visceral endoderm pattering and DE formation and provide the first functional evidence that the PVE is necessary for gastrula organizer gene induction and posterior axis development.

Abstract 230: Effect of aerobic exercise on Endothelial Progenitor Cells (EPCs) in patients with prediabetes

Introduction At least 79 million Americans have pre-diabetes. Though life-style modification prevents progression from Pre-diabetes to overt diabetes, the effect on vascular endothelial progenitor cells (EPC) stem cells is unknown. Methods This is a crossover study of 16-week duration, using exercise-naive pre-diabetes patients, n=12, aged 45-65 yrs with a BMI of 25-34.9. We studied flow mediated dilatation (FMD), EPC function and gene expression and serum endothelial inflammatory levels after 6 weeks each of aerobic exercise (150min/week) and non exercise phase, in a cross over design with 4 week wash-out period between the 2 phases. EPC migration was noted in response to chemotactic factors such as Vascular Endothelial Growth Factor (VEGF-A, 0- 50ng/ml) and Stromal Cell-Derived Factor-1(SDF1α, 0-100 ng/ml). Exercise compliance was monitored by regular phone calls and downloadable accelerometers. Results FMD studies (undertaken by 3 observers) showed mean FMD in non-exercise group of 5.7+0.6% which improved to 11.2+0.9% post-exercise. No significant weight loss noted between the 2 groups, however, significant reduction in leptin, IL-6, TNFα, hs-CRP, Triglyceride, and ApoB levels was noted. EPC gene expression analysis showed decrease of eNOS (5 fold) and increase of PECAM-1 (4-fold), Endothelin-1 (3-fold), IL-6 (2-fold), TNFα (3-fold) in non-exercise which improved following exercise. EPC migration improved post exercise, particularly in response to SDF-1α as did CD34+ cell number and EPC colony formation improved significantly also. Conclusion We demonstrate that pre-diabetic state is associated with poor vascular reactivity and impaired EPC stem cell function. Pre-diabetes may be a clinical window of therapeutic intervention opportunity when short course of aerobic exercise may allow improvement of vascular reactivity and EPC function despite no statistically significant weight loss. This study also indicates EPCs may act as an important biomarker for vascular function.

Arterial and Venous Progenitors of the Major Axial Vessels Originate at Distinct Locations

Currently, it remains controversial how vascular endothelial progenitor cells (angioblasts) establish their arterial or venous fates. We show using zebrafish that the arterial progenitors of the major axial vessels originate earlier and closer to the midline than the venous progenitors. Both medial and lateral progenitor populations migrate to distinct arterial and venous positions and not into a common precursor vessel as previously suggested. Overexpression of VEGF or Hedgehog (Hh) homologs results in the partially randomized distribution of arterial and venous progenitors within the axial vessels. We further demonstrate that the function of the Etv2 transcription factor is required at earlier stages for arterial development than for venous. Our results argue that the medial angioblasts undergo arterial differentiation because they receive higher concentration of Vegf and Hh morphogens than the lateral angioblasts. We propose a revised model of arterial-venous differentiation that explains how angioblasts choose between an arterial and venous fate.

Detection of vascular endothelial progenitor cells in patients with systemic lupus erythematosus

Objective To characterize the alteration in peripheral blood endothelial progenitor cells (EPCs) of patients with systemic lupus erythematosus (SLE).Methods Venous blood samples were obtained from 82 female patients with SLE aged (35 ± 10) years and 50 healthy female controls aged (35 ± 13) years.ACL 9000 automated coagulation analyzer was used to determine the level of Von Willebrand factor antigen (vWF Ag).Flow cytometry was performed to detect peripheral blood EPCs and circulating endothelial cells (CECs).Analysis of variance was performed to assess the differences in these parameters between patients with active and stable SLE and the controls,and Pearson correlation analysis was conducted to evaluate the relationship between these parameters.Results The number of CD34+ cells,CD133+ cells and CD34+CD133+cells per 200 000 peripheral blood mononuclear cells was 35.4 ± 16.7,86.5 ± 32.1 and 361.3 ± 176.4 in patients with active SLE,significantly higher than that in the patients with stable SLE (17.1 ± 10.9,28.7 ± 21.5,107.2 ±44.3,respectively,all P ＜ 0.01)) and the controls (13.8 ± 9.6,11.2 ± 5.5,92.3 ± 50.5,respectively,all P ＜0.01).The patients with active SLE exhibited an elevated level of vWF Ag (438.9％ ± 205.3％ vs.130.2％ ±51.5％,P ＜ 0.01),an increased number of EPCs (361.3 ± 176.4 vs.107.2 ± 44.3,P ＜ 0.01) but a similar number of CECs (127±51 vs.118 ± 39,P ＞ 0.05) per 200 000 peripheral blood mononuclear cells compared with the healthy controls.No significant differences were observed in these parameters between the patients with stable SLE and the controls (all P ＞ 0.05).The number of EPCs was positively correlated with the level of vWF Ag (r =0.67,P ＜ 0.01),but uncorrelated with the number of CECs (P ＞ 0.05) in patients with active SLE.Conclusions The quantity of EPCs in peripheral blood is closely correlated with the level of the vascular injury marker vWF Ag,hinting that the number of EPCs can serve as a useful marker of disease severity.

Nestin: A novel angiogenesis marker and possible target for tumor angiogenesis

Abnormal vasculature, termed tumor vessels, is a hallmark of solid tumors. The degree of angiogenesis is associated with tumor aggressiveness and clinical outcome. Therefore, exact quantification of tumor vessels is useful to evaluate prognosis. Furthermore, selective detection of newly formed tumor vessels within cancer tissues using specific markers raises the possibility of molecular targeted therapy via the inhibition of tumor angiogenesis. Nestin, an intermediate filament protein, is reportedly expressed in repair processes, various neoplasms, and proliferating vascular endothelial cells. Nestin expression is detected in endothelial cells of embryonic capillaries, capillaries of the corpus luteum, which replenishes itself by angiogenesis, and proliferating endothelial progenitor cells, but not in mature endothelial cells. Therefore, expression of nestin is relatively limited to proliferating vascular endothelial cells and endothelial progenitor cells. Nestin expression is also reported in blood vessels within glioblastoma, prostate cancer, colorectal cancer, and pancreatic cancer, and its expression is more specific for newly formed blood vessels than other endothelial cell markers. Nestin-positive blood vessels form smaller vessels with high proliferation activity in tumors. Knockdown of nestin in vascular endothelial cells suppresses endothelial cell growth and tumor formation ability of pancreatic cancers in vivo. Using nestin to more accurately evaluate microvessel density in cancer specimens may be a novel prognostic indicator. Furthermore, nestin-targeted therapy may suppress tumor proliferation via inhibition of angiogenesis in numerous malignancies, including pancreatic cancer. In this review article, we focus on nestin as a novel angiogenesis marker and possible therapeutic target via inhibition of tumor angiogenesis.

Human adipose stromal vascular cell delivery in a fibrin spray.

Adipose tissue represents a practical source of autologous mesenchymal stromal cells (MSCs) and vascular-endothelial progenitor cells, available for regenerative therapy without in vitro expansion. One of the problems confronting the therapeutic application of such cells is how to immobilize them at the wound site. We evaluated in vitro the growth and differentiation of human adipose stromal vascular fraction (SVF) cells after delivery through the use of a fibrin spray system. SVF cells were harvested from four human adult patients undergoing elective abdominoplasty, through the use of the LipiVage system. After collagenase digestion, mesenchymal and endothelial progenitor cells (pericytes, supra-adventitial stromal cells, endothelial progenitors) were quantified by flow cytometry before culture. SVF cells were applied to culture vessels by means of the Tisseel fibrin spray system. SVF cell growth and differentiation were documented by immunofluorescence staining and photomicrography. SVF cells remained viable after application and were expanded up to 3 weeks, when they reached confluence and adipogenic differentiation. Under angiogenic conditions, SVF cells formed endothelial (vWF+, CD31+ and CD34+) tubules surrounded by CD146+ and α-smooth muscle actin+ perivascular/stromal cells. Human adipose tissue is a rich source of autologous stem cells, which are readily available for regenerative applications such as wound healing, without in vitro expansion. Our results indicate that mesenchymal and endothelial progenitor cells, prepared in a closed system from unpassaged lipoaspirate samples, retain their growth and differentiation capacity when applied and immobilized on a substrate using a clinically approved fibrin sealant spray system.

Abstract 9633: Human Blood and Cardiac Stem Cells Synergize to Enhance Cardiac Repair When Co-transplanted Into Ischemic Myocardium

Blood-derived circulatory angiogenic cells (CACs) and resident cardiac stem cells (CSCs) have both been shown to improve cardiac function after myocardial infarction (MI). However, whether one cell type is superior has long been an area of speculation with no head-to-head trial. In this study, we compared the effect of human CACs and CSCs, alone or in combination, on myocardial function in an immunodeficient mouse model of MI. Methods and Results- CACs and CSCs were cultured from left atrial appendages and blood samples obtained from patients undergoing clinically-indicated heart surgery. In hypoxic culture conditions designed to mirror ischemic myocardium, protein arrays demonstrated that CACs expressed a broader cytokine profile than CSCs (36 vs. 5, respectively; p<0.05) with 3 cytokines in common. Co-culture of CACs and CSCs further enhanced the production of stromal cell derived factor-1α and vascular endothelial growth factor (p<0.05). Conditioned media promoted equivalent vascular networks and endothelial progenitors recruitment with superior effects using co-cultured conditioned media (p<0.05 vs. single cell cultures). Intramyocardial injection of CACs or CSCs alone improved left ventricular ejection fraction (LVEF) when injected one week after MI (p<0.05 vs. vehicle (PBS) or normal human dermal fibroblasts (NHDF); Figure). Co-transplantation of CACs and CSCs together improved LVEF to a greater extent than either stem cell therapy alone (p<0.05 vs. CAC or CSC injection alone). Conclusions- CACs and CSCs provide unique paracrine repertoires with equivalent effects on angiogenesis, stem cell migration and myocardial repair. Combination therapy with both cell types synergistically improves post infarct myocardial function greater than either therapy alone. This synergy is likely mediated by the complimentary paracrine signatures that promote revascularization and the growth of new myocardium.

Transfer of Monocrotaline-Induced Pulmonary Hypertension to Healthy Mice Via Microparticles

Abstract 5190 Rationale:Circulating endothelial cell-derived microparticles (MP) are altered in pulmonary arterial hypertension (PAH) but whether they are biomarkers of cellular injury or participants in disease pathogenesis is unknown. We have shown that lung-derived MP induce marrow cells to express lung-specific mRNA, protein. Objectives:Determine if lung, plasma-derived MP (LMP, PMP) alter pulmonary vascular endothelial or marrow progenitor cell phenotype to induce pulmonary vascular remodeling in PAH. Methods and Main Results:LMP, PMP isolated from monocrotaline (MCT)- or vehicle-treated mice were injected into healthy mice. Right ventricular (RV) hypertrophy and pulmonary vascular remodeling were assessed by RV-to-body weight (RV/BW), blood vessel wall thickness-to-diameter (WT/D) ratios. RV/BW, WT/D ratios were elevated in MCT vs. vehicle-injected mice (1. 99+0. 09vs. 1. 04+0. 09mg/g; 0. 159+0. 002vs. 0. 062+0. 009%). MP from MCT and vehicle-treated mice were quantitatively similar; however, MCT-LMP had higher endothelial cell mRNA expression and higher expression of mRNAs of proteins known to be abnormally expressed in PAH vs. vehicle-LMP. RV/BW, WT/D ratios were higher in mice injected with MCT-MP vs. mice injected with vehicle-MP (1. 63+0. 09vs. 1. 08+0. 09mg/g; 0. 113+0. 02vs. 0. 056+0. 01%). Lineage-depleted bone marrow cells co-cultured with MCT-MP and bone marrow cells isolated from MCT-MP infused mice had higher endothelial progenitor cell gene expression vs. cells co-cultured with vehicle-MP or cells isolated from vehicle MP infused mice. Conclusions:MP from MCT-injured mice have increased expression of growth factors implicated in PAH pathogenesis and induce RV hypertrophy, pulmonary vascular remodeling in healthy mice. Circulating MP may contribute to the development of MCT-induced pulmonary hypertension by inducing a pathologic phenotype in pulmonary vascular endothelial and endothelial progenitor cells. Disclosures:No relevant conflicts of interest to declare.

Investigation of ETV2/Etsrp Role in Lymphangiogenesis: A Potential Therapeutic Target in Lymphatic Disease

Abstract 614Lymphangiogenesis, defined as novel growth of lymphatic vessels to constitute a functional vascular network, captures great clinical interest because of its essential role such pathologic settings as inflammation, vascular malformations, and tumor metastasis. Previous observational hurdles are now overcome by use of the optically clear and genetically modifiable zebrafish embryo, which provides a vertebrate model for understanding lymphangiogenesis and for revealing putative therapeutic targets for metastatic disease and vascular/lymphatic malformations that currently have no cure.The zebrafish ets1-related protein, Etsrp, encodes a novel ETS domain transcription factor and is an early marker for vascular endothelial progenitors. Its function is necessary and sufficient to initiate vasculogenesis and is conserved during vertebrate development. Human and murine Er71/Etv2 proteins are functional orthologs of Etsrp. However, its role in lymphatic development is unknown. Our data show that both vascular and lymphatic vessels express etsrp driven GFP transgene. To analyze Etsrp role in lymphatic development, we performed functional Etsrp knockdown by photoactivatable morpholino oligonucleotide (MO) injection into fli1:GFP; flk1:mCherry transgenic reporter zebrafish embryos whose fluorescent background allowed distinction of lymphatic from vascular vessels.Inhibition of etsrp function at early stages resulted in the absence of both lymphatic and vascular endothelial development. Inhibition of etsrp function at 1 day-post-fertilization, after the major axial vessels have already formed, specifically affected lymphangiogenesis without affecting vascular development. The major lymphatic vessel, the thoracic duct, was absent or discontinuous in Etsrp-depleted embryos, while the major axial and secondary intersegmental blood vessels were not affected. Further analysis demonstrated that the lymphatic progenitors, parachordal lymphangioblasts, failed to migrate from the posterior cardinal vein in the absence of Etsrp function. Control morpholino-injected but not photoactivated embryos did not show significant defects in the lymphatic development, demonstrating the specificity of the observed defects. Because expression of the major VegfC receptor, VegfR3 was strongly down regulated in Etsrp MO-injected embryos, our results suggest that Etsrp initiates lymphangiogenesis by regulating VegfR3 expression. In the absence of Etsrp function, lymphangioblasts would fail to respond to VegfC signaling.We report for the first time the requirement of Etsrp for normal lymphangioblast migration and thoracic duct development in zebrafish. Additional studies are in process to determine the mechanism of Etsrp role in lymphangiogenesis. Our results suggest that Etsrp may represent a potential novel target for chemical modulation in multiple human lymphatic disorders. Disclosures:No relevant conflicts of interest to declare.

Erythropoietin Treatment Enhanced Cell Surface CXCR4 Expression On Neonatal CD34-Positive Cells

Abstract 4724Erythropoietin (EPO), which is used to treat anemic immature neonates, is thought to be effective for treating neonatal hypoxic encephalopathy by stimulating nervous system cells or vascular stem cells. On stem cells, CXCR4 is an important chemokine receptor for cell migration and proliferation. Previously, we reported that increased expression of CXCR4 on cord blood (CB)-derived CD34-positive cells, caused by low oxygen tension, enhanced cell homing activity and engraftment (Ohno et al., ASH. 2010). In human endothelial stem cells, CXCR4 expression is controlled by hypoxia-inducible factor-1α (HIF-1α), and EPO expression is enhanced by hypoxia via HIF-1α. In this study, we examined the effect of EPO treatment on CXCR4 expression on premature neonatal endothelial stem cells and cultured CB-derived CD34-positive cells.First, we examined the cell surface markers CD34, CD133, and CXCR4 in preterm infants diagnosed with anemia and treated with EPO. The 10 enrolled neonates were first administered EPO when they were over 32 weeks old and were not given oxygen therapy. Blood samples were collected twice before and once 24 h after EPO administration. Mononuclear cells were isolated from peripheral blood and stained for the hematopoietic stem cell marker CD34, vascular endothelial progenitor cell marker CD133, and surface CXCR4. The cells were analyzed by flow cytometry. No change in the proportions of CD34- and CD133-positive cells were found after the treatment (CD34 positive: 0.59%→0.57%; CD133 positive: 0.52%→0.49%). The proportion of cell surface CXCR4 on the CD133-positive cells did not change after EPO administration, whereas the proportion of cell surface CXCR4 on the CD34-positive cells increased significantly after EPO was administered (average: 7.6%→11.0%, p < 0.01). In particular, slightly CD34-positive cells showed enhanced cell surface CXCR4.Next, we examined CXCR4 enhancement in EPO-treated CB-derived CD34-positive enriched samples. Samples were divided into three aliquots and cultured. The first aliquot was incubated for 24 h in RPMI-1640 medium alone as a control; the second, for 24 h in RPMI-1640 medium with EPO (10 U/ml); and the third, for 24 h in RPMI-1640 medium with EPO (10 U/ml) containing the specific HIF-1 antagonist rapamycin. Flow cytometry revealed significantly increased surface CXCR4 expression on CD34-positive cells after incubation with EPO compared with the control expression (average: 51.7% vs. 30.3%, p< 0.05). This enhancement was inhibited completely by the addition of rapamycin. Intracellular HIF-1 was enhanced significantly in the EPO-treated cells compared with the control expression on flow cytometry.The enhanced CXCR4 expression on CD34-positive cells reflects an amplification of the chemotactic and homing abilities of stem cells. The CXCR4 enhancement caused by EPO may increase tissue migration of CD34-positive cells, although this is probably a result of an active HIF-1 pathway. Disclosures:No relevant conflicts of interest to declare.

Endothelial impairment and bone marrow-derived CD34(+)/133(+) cells in diabetic patients with erectile dysfunction.

The present study was undertaken to determine vascular endothelial impairment and endothelial progenitor cells (EPCs) in patients with type 2 diabetes mellitus and erectile dysfunction (ED). A total of 100 type 2 diabetic men were enrolled. Flow-mediated dilatation (FMD) and anaerobic threshold (AT) were measured. Also, EPCs in the peripheral blood were determined by flow cytometry. In the 42 ED diabetic patients, FMD and AT were significantly less than those in the 58 patients with normal erectile function (FMD 2.84 vs 3.82%, P=0.038, and AT 11.2 vs 12.7mL/kg/min, P=0.022). Exercise tolerance significantly increased the number of EPCs in the patients with and without ED (49-60 cells/100μL, P=0.015, and 72-99 cells/100μL, P=0.003). In the diabetic patients without autonomic neuropathy, FMD was significantly reduced in the patients with ED than those without ED (P=0.015). In response to exercise tolerance, the number of EPCs increased in both the diabetic patients with ED (P=0.003) and without ED (P=0.007). In contrast, in the diabetic patients with autonomic neuropathy, there was no difference in FMD between the patients with and without ED. The exercise tolerance increased the number of EPCs in the patients without ED (P=0.023), but it disappeared in those with ED. ED diabetic patients have endothelial impairment during the early period of diabetic complications, whose deranged endothelial function is concomitantly repaired by promoting bone marrow-derived EPCs.

Endothelialization and altered hematopoiesis by persistent Etv2 expression in mice

Etv2 is a master gene for the commitment of hematopoietic/endothelial cells and is a potent inducer of endothelial/hematopoietic cells from embryonic stem cells. Etv2 is highly expressed in endothelial/hematopoietic precursors but is downregulated when they are differentiated, indicating that Etv2 should have transient but not constitutive function. However, relatively little attention has been paid to the importance of transient Etv2 expression. To determine whether transient Etv2 expression is essential to normal development and cell differentiation, we generated mice that constitutively express Etv2 from a Cre-activatable ROSA26 locus in endothelial/hematopoietic, somite, or neuronal lineages. Constitutive Etv2 expression caused profound phenotypes in hematopoietic/endothelial cells, with little effect on somite or neuronal lineages. In hematopoietic/endothelial lineages, constitutive Etv2 expression induced by Tie-2 Cre transgene caused abnormal yolk sac vasculature. Prolonged vascular endothelial cadherin expression and decreased B lymphopoiesis were observed in Etv2 expressing vascular endothelial cadherin(+)/CD45(+) cells, indicating that Etv2 forces endothelial program on hematopoietic cells. Etv2 expression in adult hematopoietic cells by Vav-iCre transgene also conferred an endothelial phenotype on hematopoietic stem cells and suppressed hematopoiesis, with erythropoiesis severely affected. We conclude that constitutive Etv2 expression perturbs vascular development and hematopoiesis. While promoting hematopoiesis/vasculogenesis, Etv2 expression should be tightly regulated to achieve normal vascular development and hematopoiesis.

Isolation of primitive endoderm, mesoderm, vascular endothelial and trophoblast progenitors from human pluripotent stem cells.

To identify early populations of committed progenitors derived from human embryonic stem cells (hESCs), we screened self-renewing, BMP4-treated and retinoic acid-treated cultures with >400 antibodies recognizing cell-surface antigens. Sorting of >30 subpopulations followed by transcriptional analysis of developmental genes identified four distinct candidate progenitor groups. Subsets detected in self-renewing cultures, including CXCR4(+) cells, expressed primitive endoderm genes. Expression of Cxcr4 in primitive endoderm was confirmed in visceral endoderm of mouse embryos. BMP4-induced progenitors exhibited gene signatures of mesoderm, trophoblast and vascular endothelium, suggesting correspondence to gastrulation-stage primitive streak, chorion and allantois precursors, respectively. Functional studies in vitro and in vivo confirmed that ROR2(+) cells produce mesoderm progeny, APA(+) cells generate syncytiotrophoblasts and CD87(+) cells give rise to vasculature. The same progenitor classes emerged during the differentiation of human induced pluripotent stem cells (hiPSCs). These markers and progenitors provide tools for purifying human tissue-regenerating progenitors and for studying the commitment of pluripotent stem cells to lineage progenitors.

Characterization of vascular endothelial progenitor cells from chicken bone marrow

BackgroundEndothelial progenitor cells (EPC) are a type of stem cell used in the treatment of atherosclerosis, vascular injury and regeneration. At present, most of the EPCs studied are from human and mouse, whereas the study of poultry-derived EPCs has rarely been reported. In the present study, chicken bone marrow-derived EPCs were isolated and studied at the cellular level using immunofluorescence and RT-PCR.ResultsWe found that the majority of chicken EPCs were spindle shaped. The growth-curves of chicken EPCs at passages (P) 1, -5 and -9 were typically “S”-shaped. The viability of chicken EPCs, before and after cryopreservation was 92.2% and 81.1%, respectively. Thus, cryopreservation had no obvious effects on the viability of chicken EPCs. Dil-ac-LDL and FITC-UAE-1 uptake assays and immunofluorescent detection of the cell surface markers CD34, CD133, VEGFR-2 confirmed that the cells obtained in vitro were EPCs. Observation of endothelial-specific Weibel-Palade bodies using transmission electron microscopy further confirmed that the cells were of endothelial lineage. In addition, chicken EPCs differentiated into endothelial cells and smooth muscle cells upon induction with VEGF and PDGF-BB, respectively, suggesting that the chicken EPCs retained multipotency in vitro.ConclusionsThese results suggest that chicken EPCs not only have strong self-renewal capacity, but also the potential to differentiate into endothelial and smooth muscle cells. This research provides theoretical basis and experimental evidence for potential therapeutic application of endothelial progenitor cells in the treatment of atherosclerosis, vascular injury and diabetic complications.

Identification of CD133-, CD34- and KDR-positive cells in the bovine ovary: A new site of vascular wall resident endothelial progenitor cells

Intense angiogenesis, vascular remodelling as well as regression of its vasculature are prerequisites for ovarian function with its cyclically developing and regressing follicles and corpora lutea. So far neither a stringent explanation for the enormous angiogenic potential of the ovary nor its cellular origins have been suggested. In an earlier study of our work group, endothelial cells were isolated from the bovine corpus luteum and cultivated in vitro. They performed vasulogenesis in vitro and showed properties of progenitor cells. The present study aimed at in situ identification of endothelial progenitor cells (EPCs) in the bovine ovary. Immunohistochemical examinations, based on the detection of KDR and CD34 co-labelled cells - a marker combination that amongst others is commonly accepted as typical for EPC identification - were performed. Hormonal cycle dependent expression varieties were analysed by the measurement of mRNA amounts of CD34 and KDR as well as the stem cell marker CD133 (Prominin-1). Ovarian samples comprising corpora lutea of varying stages (developing and mature corpus luteum, corpus luteum in regression, corpus luteum of pregnancy) from 17 adult cows were examined. Results show that specific mRNA of CD133, CD34 and KDR was expressed in ovaries of all luteal stages. Expression data analysis revealed significant differences in CD133 and CD34 expression levels between the luteal stages but no significant differences in KDR expression. CD34/KDR co-immunoreactive cells were predominantly situated within the media of arterial vessel wall. The detection of ovarian EPCs represents an important step towards further understanding of the mechanisms involved in the reproductive biology and pathophysiology of the ovary.

A review of the evidence for the effects of total dietary fat, saturated, monounsaturated and n-6 polyunsaturated fatty acids on vascular function, endothelial progenitor cells and microparticles

Vascular dysfunction is recognised as an integrative marker of CVD. While dietary strategies aimed at reducing CVD risk include reductions in the intake of SFA, there are currently no clear guidelines on what should replace SFA. The purpose of this review was to assess the evidence for the effects of total dietary fat and individual fatty acids (SFA, MUFA and n-6 PUFA) on vascular function, cellular microparticles and endothelial progenitor cells. Medline was systematically searched from 1966 until November 2010. A total of fifty-nine peer-reviewed publications (covering fifty-six studies), which included five epidemiological, eighteen dietary intervention and thirty-three test meal studies, were identified. The findings from the epidemiological studies were inconclusive. The limited data available from dietary intervention studies suggested a beneficial effect of low-fat diets on vascular reactivity, which was strongest when the comparator diet was high in SFA, with a modest improvement in measures of vascular reactivity when high-fat, MUFA-rich diets were compared with SFA-rich diets. There was consistent evidence from the test meal studies that high-fat meals have a detrimental effect on postprandial vascular function. However, the evidence for the comparative effects of test meals rich in MUFA or n-6 PUFA with SFA on postprandial vascular function was limited and inconclusive. The lack of studies with comparable within-study dietary fatty acid targets, a variety of different study designs and different methods for determining vascular function all confound any clear conclusions on the impact of dietary fat and individual fatty acids on vascular function.

The Sphingolipid Rheostat: A Potential Target for Improving Pancreatic Islet Survival and Function

Beta cell apoptosis and suboptimal islet function are implicated in the development of Type I (T1D) and Type II (T2D) diabetes, as well as the failure of the only current clinical beta cell replacement therapy for T1D, islet transplantation. Sphingosine kinase (SK) is a ubiquitous lipid kinase that controls the balance between prosurvival and proapoptotic precursors (e.g. sphingosine-1-phosphate (S1P) and ceramide, respectively), the so-called 'sphingolipid rheostat', in many cell types. S1P, a potent lipid mediator, acts intracellularly through second messengers and extracellularly through five G-protein coupled receptors (S1P1-5), to promote calcium mobilization, intracellular signaling events, cytoskeleton rearrangements and mitogenesis. SK is important for revascularization responses, regulating the maturation of vascular endothelial progenitors and controlling cellular recruitment. The aim of this review is to highlight the sphingolipid rheostat in pancreatic biology as a therapeutic target for pharmacological and therapeutic intervention for diabetes and islet transplantation. SK and the sphingolipid rheostat are likely to be important for both islet function and beta cell survival and represent a common therapeutic target to protect the beta cell from diabetogenic insults and ultimately improve pancreatic islet function. A number of SK inhibitors and S1P receptor agonists/antagonists (including FTY720 (fingolimod) and its newer derivatives) have been recently described, with some now being used in the clinic. Recent developments in SK biochemistry and islet biology indicate the potential importance of the sphingolipid rheostat in determining islet survival and function. Pharmacological manipulation of this pathway represents a novel therapeutic strategy to prevent diabetes and improve islet transplantation outcomes.

Progenitor Cells in the Pulmonary Circulation

The mechanisms involved in the regulation of pulmonary vascular endothelial cells to replace aged or injured cells remains poorly understood, although differences in proliferative potential between the microvascular and macrovascular endothelium are well described. The presence of resident pulmonary vascular endothelial progenitor cells in rats and mice has been recently reported. These resident endothelial progenitor cells display clonal proliferative potential, restricted expression of cell surface molecules to those typical of lung endothelium, and in vivo vessel-forming ability upon transplantation into recipient animals. The rat pulmonary microvascular endothelium is enriched in resident progenitor cells, with the highest proliferative potential compared with the pulmonary macrovascular endothelium. Preliminary evidence suggests that resident endothelial progenitor cells are present in the human lung vasculature, but whether differences in enrichment of the progenitors in various pulmonary vascular beds exist remains to be determined.

The heart endocardium is derived from vascular endothelial progenitors

The embryonic heart is composed of two cell layers: the myocardium, which contributes to cardiac muscle tissue, and the endocardium, which covers the inner lumen of the heart. Whereas significant progress has been made toward elucidating the embryonic origins of the myocardium, the origins of the endocardium remain unclear. Here, we have identified an endocardium-forming field medial to the cardiac crescent, in a continuum with the endothelial plexus. In vivo live imaging of quail embryos revealed that endothelial progenitors, like second/anterior heart field progenitors, migrate to, and enter, the heart from the arterial pole. Furthermore, embryonic endothelial cells implanted into the cardiac crescent contribute to the endocardium, but not to the myocardium. In mouse, lineage analysis focusing on endocardial cells revealed an unexpected heterogeneity in the origins of the endocardium. To gain deeper insight into this heterogeneity, we conditionally ablated Flk1 in distinct cardiovascular progenitor populations; FLK1 is required in vivo for formation of the endocardium in the Mesp1 and Tie2 lineages, but not in the Isl1 lineage. Ablation of Flk1 coupled with lineage analysis in the Isl1 lineage revealed that endothelium-derived Isl1(-) endocardial cells were significantly increased, whereas Isl1(+) endocardial cells were reduced, suggesting that the endocardium is capable of undergoing regulative compensatory growth. Collectively, our findings demonstrate that the second heart field contains distinct myocardial and endocardial progenitor populations. We suggest that the endocardium derives, at least in part, from vascular endothelial cells.