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In vitro characterization of circulating endothelial progenitor cells isolated from patients with acute coronary syndrome.

Campioni D, Zauli G, Gambetti S, Campo G, Cuneo A, Ferrari R, Secchiero P - PLoS ONE (2013)

Bottom Line: Indeed, after expansion, EPC/ECFC exhibited a normal diploid chromosomal setting by FISH analysis and an immunophenotype characterized by: i) uniform positivity for the expression of CD105, CD31, CD146 and Factor VIII, i) variable expression of the CD34, CD106 and CD184 markers, and iii) negativity for CD45, CD90, CD117 and CD133.Of interest, in single-cell replanting assays EPC/ECFC exhibited clonogenic expansion capacity, forming secondary colonies characterized by variable proliferation capacities.Our data indicate that a careful characterization of true EPC is needed in order to design future studies in the clinical autologous setting of patients with ACS.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Sciences, Section of Hematology, Azienda Ospedaliero-Universitaria, Arcispedale Sant'Anna, University of Ferrara, Ferrara, Italy.

ABSTRACT

Background: The current understanding of the functional characteristics of circulating endothelial progenitor cells (EPC) is limited, especially in patients affected by cardiovascular diseases. In this study, we have analyzed the in vitro clonogenic capacity of circulating EPC, also known as endothelial colony-forming cells (ECFC), in patients with acute coronary syndrome (ACS), in comparison to the colony forming unit-endothelial-like cells (CFU-EC) of hematopoietic/monocytic origin.

Methodology/principal findings: By culturing peripheral blood mononuclear cells (PBMC) of patients with ACS (n = 70), CFU-EC were frequently isolated (from 77% of ACS patients), while EPC/ECFC were obtained only in a small subset (13%) of PBMC samples, all harvested between 7-14 days after the acute cardiovascular event. Notably, ex-vivo generation of EPC/ECFC was correlated to a higher in vitro release of PDGF-AA by the corresponding ACS patient PBMC. By using specific endothelial culture media, EPC/ECFC displayed in vitro expansion capacity, allowing the phenotypic and functional characterization of the cells. Indeed, after expansion, EPC/ECFC exhibited a normal diploid chromosomal setting by FISH analysis and an immunophenotype characterized by: i) uniform positivity for the expression of CD105, CD31, CD146 and Factor VIII, i) variable expression of the CD34, CD106 and CD184 markers, and iii) negativity for CD45, CD90, CD117 and CD133. Of interest, in single-cell replanting assays EPC/ECFC exhibited clonogenic expansion capacity, forming secondary colonies characterized by variable proliferation capacities.

Conclusion/significance: Our data indicate that a careful characterization of true EPC is needed in order to design future studies in the clinical autologous setting of patients with ACS.

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Related in: MedlinePlus

Analysis of pro-angiogenic cytokines release by PBMC derived from ACS patients.After 48 hour of culture, PBMC conditioned media were collected and analyzed for the release of angiogenic cytokines. Cytokine levels were analyzed in relation to the ability of the PBMC ACS patient samples to generate EPC/ECFC and/or CFU-EC colonies: EPC/ECFCneg vs EPC/ECFCpos gray box-plots) or CFU-ECneg vs CFU-ECpos (white box-plots). Horizontal bars are median, upper and lower edges of box are 75th and 25th percentiles, lines extending from box are 10th and 90th percentiles. Asterisk, p<0.01.
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pone-0056377-g002: Analysis of pro-angiogenic cytokines release by PBMC derived from ACS patients.After 48 hour of culture, PBMC conditioned media were collected and analyzed for the release of angiogenic cytokines. Cytokine levels were analyzed in relation to the ability of the PBMC ACS patient samples to generate EPC/ECFC and/or CFU-EC colonies: EPC/ECFCneg vs EPC/ECFCpos gray box-plots) or CFU-ECneg vs CFU-ECpos (white box-plots). Horizontal bars are median, upper and lower edges of box are 75th and 25th percentiles, lines extending from box are 10th and 90th percentiles. Asterisk, p<0.01.

Mentions: We have next investigated the potential correlation between the occurrence of CFU-EC and/or EPC/ECFC and the release of pro-angiogenic cytokines by the same patient PBMC. For this purpose, patient PBMC conditioned media were collected and analyzed for the release of angiogenic cytokines, such as HB-EGF, KGF, TPO, PDGF-AA, VEGFR-1, VEGFR-2. As shown in Figure 2, cytokine levels were analyzed by subdividing and comparing the ACS patient PBMC samples on the basis of their ability to generate CFU-EC and/or EPC/ECFC colonies: i) CFU-ECpos vs CFU-ECneg; ii) EPC/ECFCpos vs EPC/ECFCneg. The investigated cytokines were expressed by the PBMC at variable levels, with HB-EGF and KGF either under or very close to the detection limit of the assay (3.7 and 1.95 pg/ml, respectively) in most samples and without any significant difference among the PBMC subgroups. On the other hand, TPO, PDGF-AA, VEGFR-1, VEGFR-2 were released at consistent levels by the PBMC samples assessed. Of interest, a significant higher release of PDGF-AA (p<0.01) characterized the EPC/ECFCpos PBMC, with respect to the other subgroups (Figure 2), suggesting a correlation between the release of these cytokines and the circulating EPC/ECFC, which was confirmed by Pearson analysis (R = 0.75, p<0.01). No significant correlations were found between the generation of CFU-EC and the levels of the different cytokines tested.


In vitro characterization of circulating endothelial progenitor cells isolated from patients with acute coronary syndrome.

Campioni D, Zauli G, Gambetti S, Campo G, Cuneo A, Ferrari R, Secchiero P - PLoS ONE (2013)

Analysis of pro-angiogenic cytokines release by PBMC derived from ACS patients.After 48 hour of culture, PBMC conditioned media were collected and analyzed for the release of angiogenic cytokines. Cytokine levels were analyzed in relation to the ability of the PBMC ACS patient samples to generate EPC/ECFC and/or CFU-EC colonies: EPC/ECFCneg vs EPC/ECFCpos gray box-plots) or CFU-ECneg vs CFU-ECpos (white box-plots). Horizontal bars are median, upper and lower edges of box are 75th and 25th percentiles, lines extending from box are 10th and 90th percentiles. Asterisk, p<0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569417&req=5

pone-0056377-g002: Analysis of pro-angiogenic cytokines release by PBMC derived from ACS patients.After 48 hour of culture, PBMC conditioned media were collected and analyzed for the release of angiogenic cytokines. Cytokine levels were analyzed in relation to the ability of the PBMC ACS patient samples to generate EPC/ECFC and/or CFU-EC colonies: EPC/ECFCneg vs EPC/ECFCpos gray box-plots) or CFU-ECneg vs CFU-ECpos (white box-plots). Horizontal bars are median, upper and lower edges of box are 75th and 25th percentiles, lines extending from box are 10th and 90th percentiles. Asterisk, p<0.01.
Mentions: We have next investigated the potential correlation between the occurrence of CFU-EC and/or EPC/ECFC and the release of pro-angiogenic cytokines by the same patient PBMC. For this purpose, patient PBMC conditioned media were collected and analyzed for the release of angiogenic cytokines, such as HB-EGF, KGF, TPO, PDGF-AA, VEGFR-1, VEGFR-2. As shown in Figure 2, cytokine levels were analyzed by subdividing and comparing the ACS patient PBMC samples on the basis of their ability to generate CFU-EC and/or EPC/ECFC colonies: i) CFU-ECpos vs CFU-ECneg; ii) EPC/ECFCpos vs EPC/ECFCneg. The investigated cytokines were expressed by the PBMC at variable levels, with HB-EGF and KGF either under or very close to the detection limit of the assay (3.7 and 1.95 pg/ml, respectively) in most samples and without any significant difference among the PBMC subgroups. On the other hand, TPO, PDGF-AA, VEGFR-1, VEGFR-2 were released at consistent levels by the PBMC samples assessed. Of interest, a significant higher release of PDGF-AA (p<0.01) characterized the EPC/ECFCpos PBMC, with respect to the other subgroups (Figure 2), suggesting a correlation between the release of these cytokines and the circulating EPC/ECFC, which was confirmed by Pearson analysis (R = 0.75, p<0.01). No significant correlations were found between the generation of CFU-EC and the levels of the different cytokines tested.

Bottom Line: Indeed, after expansion, EPC/ECFC exhibited a normal diploid chromosomal setting by FISH analysis and an immunophenotype characterized by: i) uniform positivity for the expression of CD105, CD31, CD146 and Factor VIII, i) variable expression of the CD34, CD106 and CD184 markers, and iii) negativity for CD45, CD90, CD117 and CD133.Of interest, in single-cell replanting assays EPC/ECFC exhibited clonogenic expansion capacity, forming secondary colonies characterized by variable proliferation capacities.Our data indicate that a careful characterization of true EPC is needed in order to design future studies in the clinical autologous setting of patients with ACS.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Sciences, Section of Hematology, Azienda Ospedaliero-Universitaria, Arcispedale Sant'Anna, University of Ferrara, Ferrara, Italy.

ABSTRACT

Background: The current understanding of the functional characteristics of circulating endothelial progenitor cells (EPC) is limited, especially in patients affected by cardiovascular diseases. In this study, we have analyzed the in vitro clonogenic capacity of circulating EPC, also known as endothelial colony-forming cells (ECFC), in patients with acute coronary syndrome (ACS), in comparison to the colony forming unit-endothelial-like cells (CFU-EC) of hematopoietic/monocytic origin.

Methodology/principal findings: By culturing peripheral blood mononuclear cells (PBMC) of patients with ACS (n = 70), CFU-EC were frequently isolated (from 77% of ACS patients), while EPC/ECFC were obtained only in a small subset (13%) of PBMC samples, all harvested between 7-14 days after the acute cardiovascular event. Notably, ex-vivo generation of EPC/ECFC was correlated to a higher in vitro release of PDGF-AA by the corresponding ACS patient PBMC. By using specific endothelial culture media, EPC/ECFC displayed in vitro expansion capacity, allowing the phenotypic and functional characterization of the cells. Indeed, after expansion, EPC/ECFC exhibited a normal diploid chromosomal setting by FISH analysis and an immunophenotype characterized by: i) uniform positivity for the expression of CD105, CD31, CD146 and Factor VIII, i) variable expression of the CD34, CD106 and CD184 markers, and iii) negativity for CD45, CD90, CD117 and CD133. Of interest, in single-cell replanting assays EPC/ECFC exhibited clonogenic expansion capacity, forming secondary colonies characterized by variable proliferation capacities.

Conclusion/significance: Our data indicate that a careful characterization of true EPC is needed in order to design future studies in the clinical autologous setting of patients with ACS.

Show MeSH
Related in: MedlinePlus