Association of Endothelial Microparticles (EMP) and Endothelial Progenitors with Serum Cholesterol Levels.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1819-1819
Author(s):  
Joaquin J. Jimenez ◽  
Alexander Ferreira ◽  
Hannah J. Dodson ◽  
Katherine M. Lens ◽  
Lucia M. Mauro ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Colony Formation ◽  
Vascular Endothelial Cell ◽  
Endothelial Progenitor ◽  
Endothelial Microparticles ◽  
Cholesterol Levels ◽  
Circulating Endothelial Progenitor Cells

Abstract INTRODUCTION: High cholesterol (HC) is known to adversely affect endothelial cells (EC) and has been shown to correlate with decreased levels of circulating endothelial progenitor cells (CEPC). We assayed endothelial microparticles (EMP), a sensitive indicator of EC perturbation, to investigate relations among HC, CEPC, and injury of coronary artery endothelial cells (CAEC), both in vivo and in vitro. METHODS: Twelve subjects with normal cholesterol (150 ±30 mg/dL, control) and 12 with HC (250 ±25) were studied. EMP were assayed by flow cytometry using fluorescent antibodies and CAEC were cultured as previously described [Jimenez et al, Thromb Res 109:175, 2003]. CEPC were isolated, cultured, and assayed for endothelial colony formation (CFU) as described [Hill et al, NEJM 348:593, 2003]. RESULTS: Comparing the two groups, EMP measured by CD31+/CD42b− were nearly 2.5-fold elevated in HC as compared to controls (1.7 ±0.5 ×106/mL vs.0.35 ±0.02 ×106/mL; p<0.01). Cholesterol levels correlated well with this measure of EMP (R=0.60, p=0.002). However, no significant correlation was found between CD62E+ EMP and cholesterol levels. Assay of CEPC revealed a nearly 2.5-fold decrease in CFU in HC vs. controls (10 ±2 vs. 25 ±4; p<0.01). In studies in vitro, CEPC from controls were cultured in presence of 20% 0.1μm filtered plasma from members of both groups. The HC group plasma inhibited CEPC colony formation by almost 50% (23 ±3.5 CFU for control plasma vs. 13 ±4 colonies for HC plasma). We next assessed the longer-term effect of HC plasma on CAEC cultures. Six-day culture of CAEC in the presence of 20% plasma resulted in a significant increase of CD31+/CD42b− EMP from CAEC treated with HC plasma vs. normal plasma (6.5 ±0.7 ×106/mL vs. 0.23 ±0.03 ×106/mL; p=0.02). CONCLUSION: These results suggest that EMP are useful markers to monitor cholesterol mediated-EC changes. High EMP levels inversely reflect the vascular endothelial cell regeneration potential due to decreased circulating endothelial progenitor cells.

Circulating Endothelial Progenitor Cells Are Increased in Patients with Myelofibrosis and Do Not Harbor V617F JAK-2 or W515L MPL Mutations

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1535-1535 ◽  
Author(s):  
Elisa Bonetti ◽  
Vittorio Rosti ◽  
Laura Villani ◽  
Rita Campanelli ◽  
Gaetano Bergamaschi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Healthy Subjects ◽  
Mononuclear Cells ◽  
Median Frequency ◽  
Endothelial Progenitor ◽  
Circulating Endothelial Progenitor Cells ◽  
Mutational Status

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.

Unresolved questions, changing definitions, and novel paradigms for defining endothelial progenitor cells

Blood ◽  
2005 ◽  
Vol 106 (5) ◽  
pp. 1525-1531 ◽  
Author(s):  
David A. Ingram ◽  
Noel M. Caplice ◽  
Mervin C. Yoder
Keyword(s):  
Endothelial Cells ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Circulating Endothelial Cells ◽  
Proliferative Potential ◽  
Endothelial Progenitor ◽  
Detailed Understanding ◽  
Circulating Endothelial Progenitor Cells

Abstract The field of vascular biology has been stimulated by the concept that circulating endothelial progenitor cells (EPCs) may play a role in neoangiogenesis (postnatal vasculogenesis). One problem for the field has been the difficulty in accurately defining an EPC. Likewise, circulating endothelial cells (CECs) are not well defined. The lack of a detailed understanding of the proliferative potential of EPCs and CECs has contributed to the controversy in identifying these cells and understanding their biology in vitro or in vivo. A novel paradigm using proliferative potential as one defining aspect of EPC biology suggests that a hierarchy of EPCs exists in human blood and blood vessels. The potential implications of this view in relation to current EPC definitions are discussed.

Shear stress regulates inflammatory and thrombogenic gene transcripts in cultured human endothelial progenitor cells

2010 ◽  
Vol 104 (09) ◽  
pp. 582-591 ◽  
Author(s):  
Trine Lund ◽  
Stig Hermansen ◽  
Thomas Andreasen ◽  
Jan Olsen ◽  
Bjarne Østerud ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Mrna Expression ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Mrna Levels ◽  
Endothelial Progenitor ◽  
Gene Transcripts ◽  
Nos Inhibitor

SummaryShear stress has an established effect on mature endothelial cells, but less is known about how shear stress regulates endothelial progenitor cells (EPCs). In vitro expanded EPCs isolated from adult human blood represent a novel tool in regenerative vessel therapy. However, in vitro culturing may generate cells with unfavourable properties. The aim of the present study was therefore to assess whether shear stress may influence the inflammatory and thrombotic phenotype of in vitro expanded EPCs. In late outgrowth EPCs, 6 hours of shear stress (6.0 dynes/ cm2) significantly reduced the mRNA levels of IL-8, COX2, and tissue factor (TF) compared to static controls. This was associated with a reduced TF activity. In contrast, mRNA expression of NOS3 was significantly increased following 6 and 24 hours of shear stress. In accordance with this, NOS3 protein expression was increased following 24 hours of shear stress. Overall stimulation with the proinflammatory mediator, TNFα, for the final 2 hours increased the mRNA expression of IL-6, IL-8, MCP-1, ICAM1, and TF. However exposure to 6 hours of shear stress significantly suppressed the inductory potential of TNFα to increase the mRNA levels of IL-6, IL-8, COX2, and TF. Additionally, TNFα increased TF activity approximately 10 times, an effect that was also significantly reduced by exposure to 6 and 24 hours of shear stress. The effect of shear on the gene levels of TF and NOS3 were not blocked by the NOS inhibitor L-NAME. These observations suggest that EPCs are capable of functionally responding to shear stress.

Abstract 894: Leptin Potentiates the Angiogenic Properties of Endothelial Progenitor Cells In Vitro and In Vivo

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Nana-Maria Heida ◽  
Marco R Schroeter ◽  
I-Fen Cheng ◽  
Elena I Deryugina ◽  
Thomas Korff ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Tube Length ◽  
Endothelial Progenitor ◽  
Signal Transduction Inhibitors ◽  
Stimulation Of

Endothelial progenitor cells (EPC) have been reported to contribute to neovascularization. We have previously shown that the adipocytokine leptin may enhance the adhesive properties of EPC by upregulating specific integrins. To investigate whether the angiogenic effects of leptin may be mediated by modulation of EPC function, mononuclear cells were isolated from healthy human volunteers and cultivated under endothelial cell conditions for 7 days. In the matrigel assay, pretreatment of EPC with recombinant leptin for 24 hours dose-dependently enhanced their incorporation into tubular structures provided by mature endothelial cells. For example, 138.3 ± 7.6% (P = 0.001) and 145.3 ± 5.5% (P = 0.0001) CM-DiI-labeled EPC were detected after stimulation with 10 and 100 ng/mL leptin, respectively (control-treated EPC defined as 100%). Furthermore, in the spheroid angiogenesis assay, stimulation of EPC with 10 ng/mL leptin increased the number of sprouts (P < 0.0001) and tube length (P < 0.0001) of coincubated mature endothelial cells, and the outgrowth of EPC (P < 0.0001). Addition of 100-fold excess of leptin-neutralizing or leptin-receptor-binding antibodies completely reversed these effects. Moreover, EPC adhesion onto endothelial cell tubules could be reduced by addition of RGD peptides (from 159 ± 13.7% to 101.8 ± 14.6%; P = 0.02), or of neutralizing antibodies against αvβ3 (from 165.3 ± 11.8% to 103.8 ± 13.3%; P = 0.006) or αvβ5 (to 93.5 ± 15.8%; P = 0.005). Further experiments using specific signal transduction inhibitors (10 μM of LY294002, PD98059, or SB203580), as well as Western blot analysis, revealed that leptin signaling in EPC involves phosphoinositide-3 kinase and p42/44, but not by p38 MAP kinase. The effects of leptin could also be confirmed under in vivo conditions. Stimulation of EPC with 100 ng/mL leptin potentiated the insprout of newly formed avian vessels into collagen onplants placed on the chorion allantoic membrane of chicken embryos (angiogenic index, 0.58 ± 0.24) compared to control-treated EPC (0.44 ± 0.27; P = 0.07) and endothelial basal medium alone (0.31 ± 0.26; P = 0.0007). Thus, our in vitro and in vivo results suggest that the angiogenic effects of leptin may partly depend on its specific interaction with endothelial progenitor cells.

Bovine Vessel-Derived Endothelial Cells Are Comprised of a Complete Hierarchy of Endothelial Progenitor Cells, with the Highest Proliferative Progenitors Residing in the Aorta.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3912-3912
Author(s):  
Matthew M. Harkenrider ◽  
Scott A. Johnson ◽  
Laura E. Mead ◽  
David A. Ingram ◽  
Mervin C. Yoder
Keyword(s):  
Endothelial Cells ◽  
Single Cell ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Ex Vivo ◽  
Proliferative Potential ◽  
Endothelial Progenitor ◽  
Apparent Paradox ◽  
Endothelial Colony Forming Cells

Abstract Endothelial cell replication in large and small vessels is generally thought to occur at a rate of 0.1–0.6% daily. Despite this low level of cell turnover, endothelial cells derived from a variety of bovine vessels display vigorous patterns of proliferation in vitro. This apparent paradox has not been resolved to date. We have recently determined that human endothelial cells are derived through a process of endopoiesis via a hierarchy of endothelial progenitor cells (EPCs) (Blood, 2004). We have developed a single cell proliferation assay that has resolved endopoiesis into distinct stages of progenitor cell development: 1) high proliferative potential-endothelial colony forming cells (HPP-ECFC; 2001-> 10,000 cells/colony) that replate into secondary and tertiary HPP-ECFC, 2) low proliferative potential-endothelial colony forming cells (LPP-ECFC; 51–2,000 cells/colony) that form colonies greater than 50 cells but fail to replate into LPP-ECFC, 3) endothelial clusters (EC-clusters; 2–50 cells/colony) that contain fewer than 50 cells, and 4) mature differentiated endothelial cells that are non-proliferative. We hypothesized that the proliferative behavior of the bovine vessel-derived endothelial cells was due to the presence of EPCs. We purchased bovine aortic endothelial cells (BAEC), bovine pulmonary artery endothelial cells (BPAEC), and bovine coronary artery endothelial cells (BCAEC) from a commercial vendor and cultured the cells as recommended. As predicted, the endothelial cells displayed a cobblestone morphology and ingested acetylated low density lipoprotein consistent with an endothelial phenotype. We initially plated the monolayer of cells of each type at 10, 25, or 100 cells per collagen I coated 6-well tissue culture wells and determined that cells from each artery gave rise to heterogenous colony sizes with different growth potentials during a 7 day culture. We then utilized flow cytometry to single cell sort the endothelial cells of each arterial type and determined the number of cells that divided in a 14 day culture. As depicted in the TABLE, the entire hierarchy of EPCs (similar to that determined for human adult peripheral blood and umbilical cord blood) is present in the endothelial cells isolated from the bovine vessels. Of interest, our preliminary data indicate that the frequency of the most proliferative progenitors (HPP-ECFC) is higher in the BAEC than the BPAEC or BCAEC samples. These data provide a new conceptual framework for understanding the mechanisms of endothelial replacement and/or repair of aged or damaged endothelial cells. While EPCs clearly circulate, they also engraft and reside in the vessel wall. We speculate that it is the presence of these EPCs that accounts for the ability of isolated BAEC, BPAEC, and BCAEC cells to proliferate ex vivo. Single Cell Sort Colony Distributions Cell Line BAEC-1 % BAEC-2 % BCAEC % BPAEC % Mature EC 31.33 39.33 56.67 53.67 EC-clusters 2.00 2.33 10.00 5.00 LPP-ECFC 5.00 9.00 12.00 11.00 HPP-ECFC 61.67 49.33 21.33 30.33 Total colonies 68.67 60.67 43.33 46.33

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