Endothelial colony-forming cells from patients with chronic myeloproliferative disorders lack the disease-specific molecular clonality marker

Blood ◽  
2009 ◽  
Vol 114 (14) ◽  
pp. 3127-3130 ◽  
Author(s):  
Giovanna Piaggio ◽  
Vittorio Rosti ◽  
Mirko Corselli ◽  
Francesca Bertolotti ◽  
Gaetano Bergamaschi ◽  
...  

Abstract Two putative types of circulating endothelial progenitor cells have been recently identified in vitro: (1) endothelial colony-forming cell (ECFC) and (2) colony-forming unit–endothelial cell (CFU-EC). Only the former is now recognized to belong to endothelial lineage. We have used the ECFC and CFU-EC assays to readdress the issue of the clonal relation between endothelial progenitor cells and hematopoietic stem cells in patients with Philadelphia-positive and Philadelphia-negative chronic myeloproliferative disorders. Both ECFCs and CFU-ECs were cultured from peripheral blood mononuclear cells, and either BCR-ABL rearrangement or JAK2-V617F mutation were assessed in both types of endothelial colonies. We found that ECFCs lack the disease-specific markers, which are otherwise present in CFU-ECs, thus reinforcing the concept that the latter belongs to the hematopoietic lineage, and showing that in chronic myeloproliferative disorders the cell that gives rise to circulating ECFC has a distinct origin from the cell of the hematopoietic malignant clone.

Author(s):  
WAHYU WIDOWATI ◽  
RIMONTA F. GUNANEGARA ◽  
TERESA LILIANA WARGASETIA ◽  
HANNA SARI WIDYA KUSUMA ◽  
SEILA ARUMWARDANA ◽  
...  

Objective: Circulating EPCs (endothelial progenitor cells) play a role in neovascularization and vascular repair. Oxidative stress impairs endothelial progenitor. Flavonoid is a phytochemical compound for antioxidant activity. Flavonoid effects toward oxidative stress, apoptosis, and expression of the cell markers on EPCs are not fully understood. This study was aimed to elucidate the effects of quercetin, kaempferol, and myricetin toward oxidative stress, apoptosis, and cell markers of peripheral blood-derived-EPCs. Methods: EPCs (endothelial progenitor cells) were isolated from peripheral blood mononuclear cells (PBMNCs) using cultivation under EPCs spesific media. Oxidative stress in EPCs was induced by H2O2 and then treated by quercetin, kaempferol, and myricetin. Cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, while intracellular reactive oxygen species (ROS), apoptosis and characterization of cells, which expressed CD133 and KDR, was measured using flow cytometry. Results: Quercetin, kaempferol, and myricetin at concentration 12.50 µmol/l were not toxic on EPCs as the cells viability were 96.11±4.03%, 95.42±7.75%, and 94.22±9.49%, respectively. Flavonoids decreased intracellular ROS level in EPCs (quercetin: 14.38±1.47%, kaempferol: 20.21±6.25%, and myricetin: 13.88±4.02%) compared to EPCs treated with H2O2 (30.70%±1.04). Percetage of EPCs apoptosis was not significantly different among each treatment. Immunophenotyping showed the increasing of CD133 and KDR expression in EPCs treated with flavonoids. Conclusion: Quercetin, kaempferol, and myricetin were safe for EPCs, decreased ROS levels, and increased CD133 and KDR expression. However, the flavonoids did not significantly affect EPCs apoptosis.


2009 ◽  
Vol 29 (5) ◽  
pp. 933-943 ◽  
Author(s):  
Anna Rosell ◽  
Ken Arai ◽  
Josephine Lok ◽  
Tongrong He ◽  
Shuzhen Guo ◽  
...  

Endothelial progenitor cells (EPCs) may provide novel opportunities for therapeutic angiogenesis after ischemic diseases. However, it is unclear how the angiogenic potential of EPCs might be affected by an inflammatory environment. We examine how the potent cytokine interleukin-1β (IL-1β) affects angiovasculogenic responses in EPCs in culture. Mononuclear cells isolated from mouse spleen were plated on fibronectin-coated wells and grown in EGM-2 MV media. Endothelial progenitor cells were phenotyped using multiple markers (UEA-Lectin, ac-LDL, CD133, CD34, vWillebrand Factor, Flk-1) and to identify the IL-1 Receptor-I. We quantified cell and colony counts and performed MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide) and Matrigel assays, in vitro, under control and IL-1β (10 ng/mL) conditions. Endothelial progenitor cells exposed to IL-1β increased in the number of cells and colonies compared with untreated cells, without any effect on cell metabolic integrity. Furthermore, IL-1β treatment augmented EPC angiogenic function, significantly increasing the number of vessel-like structures in the Matrigel assay. An early phosphorylation of ERK1/2 occurred after IL-1β stimulation, and this pathway was inhibited if IL-1 Receptor-I was blocked. Our results suggest that IL-1β is a potent stimulator of in vitro angiogenesis through ERK signaling in mouse EPCs. Further studies are warranted to assess how interactions between proinflammatory environments and EPC responses may be leveraged to enhance therapeutic angiogenesis.


Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1535-1535 ◽  
Author(s):  
Elisa Bonetti ◽  
Vittorio Rosti ◽  
Laura Villani ◽  
Rita Campanelli ◽  
Gaetano Bergamaschi ◽  
...  

Abstract Bone marrow and spleen neoangiogenesis is a relevant feature of patients with myelofibrosis (MF). We have previously reported that patients with MF have an increased percentage of circulating endothelial progenitor cells (EPC) assessed as CD34+CD133+VEGFR2+ cells compared with patients with other Ph-negative myeloproliferative disorders (polycythemia vera, PV, and essential thrombocytemia, ET) and healthy subjects. However, neither the functional activity of these putative EPC nor their belonging to the malignant clone have been yet fully characterized. In order to address these issues we have grown in vitro EPC-derived colonies from the peripheral blood (PB) of 36 patients with MF, 9 patients with PV or ET and 10 healthy subjects. Seventeen MF patients harbored a V617F JAK-2 mutation (8 heterozygous and 9 homozygous) whereas 2 patients showed a W515L MPL mutation (both heterozygous). Eight out of 9 PV/ET patients had a V617F JAK-2 mutation (5 heterozygous and 3 homozygous). Mononuclear cells were cultured in collagen coated 6 well plates in the presence of EBM-2MV medium according to Ingram et al (Blood104:2752; 2004). The endothelial origin of the colonies was ascertained by assessment of the expression of CD105, CD146, CD144, CD31, vWf, VEGFR-2, CD14 and CD45 antigens. V617F JAK-2 and W515L MPL mutations were assessed by PCR, followed by enzymatic digestion, of endothelial cells after tripsinization of the EPC-derived colonies. The median frequency (number of colonies per 107 mononuclear cells plated) of EPC-derived colonies was statistically higher in MF patients (0.25, range 0–8.1) compared to healthy subjects (0.05, 0–0.3; P=0.037), but not different form that of PV/ET patients (0, 0–4.4; P=NS). Immunophenotyping confirmed that the cells expressed the endothelial antigens CD105, CD146, CD144, CD31, vWf, and VEGFR-2 but not the hematopoietic specific antigens CD45 and CD14. The capacity of colony-derived endothelial cells of MF patients to form capillary-like structures in the Matrigel assay was not different from that of healthy subjects. No correlation was found between the number of colonies and the mutational status of either JAK-2 or MPL. In 11 MF patients harboring either a JAK-2 (n=9) or a MPL (n=2) mutation, colony growth was observed and PCR was performed on EPC-derived colonies. In 0/9 and 0/2 cases neither JAK-2 nor MPL mutations were found, respectively. In addition, no V617F JAK-2 mutation was found in the EPC-derived colonies of 8 PV/ET patients who carried the mutation in their granulocytes. Taken together, our data show that patients with MF have an increased frequency of EPC in their PB compared to healthy subjects and that these mobilized EPC are not clonally-related to the JAK-2 or MPL mutated clone. Whether or not circulating EPC derive from an earlier progenitor cell compared to the one in which the JAK-2/MPL mutations arise remains to be determined.


Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5404-5404
Author(s):  
Eun-Sun Yoo ◽  
Jee-Young Ahn ◽  
KiHwan Kwon ◽  
Soo-Ah Oh ◽  
Moon-Young Choi ◽  
...  

Abstract Background: The identification of circulating endothelial progenitor cells (EPCs) has revolutionized approaches to cell-based therapy for injured and ischemic tissues. Recently, we have demonstrated that there are 2 distinct types of EPCs from UCB having different biologic properties for angiogenic capabilities in vitro and in vivo. In present study, the aim is to directly compare umbilical cord blood (UCB)- and BM-derived late EPC surface phenotypes and in vitro functional capacity. Methods: Mononuclear cells from UCB and BM cultured using EGM-2 medium with VEGF, IGF-1 and FGF for 21 days. Late outgrowing endothelail cells(late OECs) which were in peak growth at third weeks of culture were analyzed for expression of various surface markers by flow cytometry/RT-PCR/IF, tube formation in Matrigel plates, proliferation assay, endothelial colony assay and the role of SDF-1/VEGF on migration. Results: The adherent cells after culture of 7 days exhibited a fibroblast like shape in BM and a cobblestone shaped cells in UCB. Although two sources of OECs were comparable in expression of endothelial and various adhesion molecule markers, BM-derived OECs contained higher proportion of cells expressing smooth muscle cell markers(SMMHC), several adhesion molecule(CD49d, CD62L and VCAM-1), whereas the expression of CXCR-4, PECAM-1 and CD62E and expression of mRNA on endothelial marker genes were higher in UCB-derived OECs. UCB-OECS stained positive for uptake of acetylated low-density lipoprotein and had more migratory ability in the presence of SDF-1 and VEGF compared with BM-OECs. Both sources OECs effectively formed capillary tubes in Matrigel plates. Conclusion: We directly compared OECs derived from UCB and BM and two source of OECs differ in aspect of several adhesion molecule and angiogenic potential in vitro. These difference of UCB render it potentially advantageous for human therapeutic OECs applications for potential applications for a “cell therapy” in the situations on vascular injuries when compared with patients-derived BM.


2010 ◽  
Vol 31 (3) ◽  
pp. 855-867 ◽  
Author(s):  
Akihiko Taguchi ◽  
Pengxiang Zhu ◽  
Fang Cao ◽  
Akie Kikuchi-Taura ◽  
Yukiko Kasahara ◽  
...  

Circulating bone marrow-derived immature cells, including endothelial progenitor cells, have been implicated in homeostasis of the microvasculature. Decreased levels of circulating endothelial progenitor cells, associated with aging and/or cardiovascular risk factors, correlate with poor clinical outcomes in a range of cardiovascular diseases. Herein, we transplanted bone marrow cells from young stroke-prone spontaneously hypertensive rats (SHR-SP) into aged SHR-SP, the latter not exposed to radiation or chemotherapy. Analysis of recipient peripheral blood 28 days after transplantation revealed that 5% of circulating blood cells were of donor origin. Cerebral infarction was induced on day 30 posttransplantation. Animals transplanted with bone marrow from young SHR-SP displayed an increase in density of the microvasculature in the periinfarction zone, reduced ischemic brain damage and improved neurologic function. In vitro analysis revealed enhanced activation of endothelial nitric oxide synthase and reduced activation p38 microtubule-associated protein (MAP) kinase, the latter associated with endothelial apoptosis, in cultures exposed to bone marrow-derived mononuclear cells from young animals versus cells from aged counterparts. Our findings indicate that partial rejuvenation of bone marrow from aged rats with cells from young animals enhances the response to ischemic injury, potentially at the level of endothelial/vascular activation, providing insight into a novel approach ameliorate chronic vascular diseases.


2018 ◽  
Vol 27 (7) ◽  
pp. 1068-1079 ◽  
Author(s):  
Makiko Kado ◽  
Rica Tanaka ◽  
Kayo Arita ◽  
Kayoko Okada ◽  
Rie Ito-Hirano ◽  
...  

The transplantation of endothelial progenitor cells (EPCs) is used to promote wound angiogenesis. In patients with chronic wounds and accompanying morbidities, EPCs are often compromised in number and function. To overcome these limitations, we previously developed a quality and quantity controlled (QQ) culture system to enrich peripheral blood mononuclear cells (PBMNCs) in EPCs. To evaluate the wound healing efficacy of mononuclear cells (MNCs) harvested after QQ culture (QQMNCs), preclinical studies were performed on large animals. MNCs harvested from the blood of healthy human subjects were cultured in the presence of angiogenic cytokines and growth factors in a serum-free medium for 7 days. A total of 5 × 106 QQMNCs per full-thickness skin defect or control saline was injected into wounds induced in cyclosporine-immunosuppressed pigs. EPC colony-forming assays revealed a significantly higher number of definitive (partially differentiated) EPC colony-forming units in QQMNCs. Flow cytometry evaluation of QQMNC surface markers showed enrichment of CD34+ and CD133+ stem cell populations, significant reduction in CCR2+ cell percentages, and a greater than 10-fold increase in the percentage of anti-inflammatory M2-type macrophages (CD206+ cells) compared with PBMNCs. Wounds treated with QQMNCs had a significantly higher closure rate. Wounds were harvested, frozen, and sectioned at day 21 postoperatively. Hematoxylin and eosin staining revealed that the epithelization of QQMNC-treated wounds was more advanced than in controls. Treated wounds developed granulation tissue with more mature collagen and larger capillary networks. CD31 and human mitochondrial co-staining confirmed the presence of differentiated human cells within newly formed vessels. Real-time polymerase chain reaction (PCR) showed upregulation of interleukin 6 (IL-6), IL-10, and IL-4 in the wound bed, suggesting paracrine activity of the transplanted QQMNCs. Our data demonstrate for the first time that QQ culture of MNCs obtained from a small amount of peripheral blood yields vasculogenic and therapeutic cells effective in wound healing.


Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Toshikazu D Tanaka ◽  
Masaaki Ii ◽  
Haruki Sekiguchi ◽  
Kentaro Jujo ◽  
Sol Misener ◽  
...  

Background: Endothelial progenitor cells (EPCs) have been shown to have angiogenic potential contributing to neovascularization. However, the definition of EPC, including surface marker expression of EPCs promoting vasculo-/angiogenesis in ischemic tissue, remains uncertain. We hypothesized that stem/progenitor (c-kit vs. sca-1) and endothelial cell (EC) markers (CD31) may identify cells with enhanced EPC potential. Methods and Results: Mononuclear cells (MNCs) were isolated from mouse bones, and Lin+ cells were depleted by magnetic cell sorting. Lin- cells were further sorted with the following markers (% of total MNCs) by FACS: c-kit+ (1.87%), sca-1+ (0.6%), c-kit+ /CD31+ (1.1%) and sca-1+ /CD31+ (0.28%). Non-sorted MNCs were used as a control. To examine EC phenotype in culture, cells were labeled with DiI and co-cultured with mature ECs (human microvascular endothelial cells: HMVECs). The percent incorporation of DiI labeled cells into HMVEC tube structures 12 hours after co-culture and BS1-lectin positivity/acLDL uptake were: sca-1+ /CD31+ cells (87 ± 2%) > c-kit+ /CD31+ (79 ± 8%) > sca-1+ (62 ± 8%) > c-kit+ (59 ± 5%) > MNC (50 ± 3% ) . Next, we examined homing capacity of these cells to ischemic myocardium using a mouse myocardial infarction (MI) model. DiI-labeled cells (5x10 4 , IV) were injected to splenectomized mice 3 days after MI, and the hearts were excised 24 hours after the cell injection for histological analysis. Interestingly, the number of recruited/retained DiI-labeled-cells in the MI hearts exactly replicated the findings of the in vitro tube formation assay (cells/HPF): sca-1+ /CD31+ (108 ± 26) > c-kit+ /CD31+ (77 ± 16) > sca-1+ (71 ± 14) > c-kit+ (67 ± 1) > MNCs (48 ± 6) , suggesting that sca-1+ /CD31+ cells might have great functional activities as endothelial precursors. Conclusions: Both stem/progenitor marker Sca-1 and EC marker CD31 expressing EPCs exhibited high potential angiogenic capacity with EC phenotypic features compared with c-kit expressing cells. Our data suggest that Sca-1+ /CD31+ cells may represent EPC-rich cell population, and Sca-1/CD31 could be useful markers to enrich for cells with EPC potential. Ongoing studies will determine the in vivo characteristics of these cells for ischemic tissue repair.


2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
F. R. Spinelli ◽  
A. Metere ◽  
C. Barbati ◽  
M. Pierdominici ◽  
C. Iannuccelli ◽  
...  

Endothelial dysfunction has been detected in RA patients and seems to be reversed by control of inflammation. Low circulating endothelial progenitor cells (EPCs) have been described in many conditions associated with increased cardiovascular risk, including RA. The aim of this study was to investigate the effect of inhibition of TNF on EPCs in RA patients. Seventeen patients with moderate-severe RA and 12 sex and age-matched controls were evaluated. Endothelial biomarkers were tested at baseline and after 3 months. EPCs were identified from peripheral blood mononuclear cells by cytofluorimetry using anti-CD34 and anti-vascular endothelial growth factor-receptor 2. Asymmetric dimethylarginine (ADMA) was tested by ELISA and flow-mediated dilatation (FMD) by ultrasonography. Circulating EPCs were significantly lower in RA patients than in controls (P=0.001). After 3 months EPCs increased significantly (P=0.0006) while ADMA levels significantly decreased (P=0.001). An inverse correlation between mean increase in EPCs number and mean decrease of DAS28 after treatment was observed (r=−0.56,P=0.04). EPCs inversely correlated with ADMA (r=−0.41,P=0.022). No improvement of FMD was detected. Short-term treatment with anti-TNF was able to increase circulating EPCs concurrently with a proportional decrease of disease activity suggesting that therapeutic intervention aimed at suppressing the inflammatory process might positively affect the endothelial function.


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