Panel) or EPC/ECFC (B, left panel; arrowheads: hemopoietic mononucleated cells). In A, the right panel shows a monolayer of spindle-shaped endothelial-like monocytes. In B, the right panel shows a representative image of CFU-EC after in vitro expansion. Original magnification: 20X and 25X for the inset. In C, frequency of detection of CFU-EC and EPC/ECFC in PBMC of ACS patients, divided on the basis of the time of blood withdrawal after the hospital admission for the acute cardiovascular events. doi:10.1371/journal.pone.0056377.gEndothelial Progenitor Cells in ACS Patientstert-Butylhydroquinone chemical information Figure 2. 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. Alprenolol biological activity 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 boxplots). 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. doi:10.1371/journal.pone.0056377.gEndothelial Progenitor Cells in ACS PatientsFigure 3. Identification of optimal culture conditions for the ex-vivo expansion of ACS PB-derived EPC/ECFC. Primary EPC/ECFC colonies were generated by plating patient PBMC in M5100 medium, as detailed in the Method section. In A, after the colony identification (at day 5 after plating), medium was change (arrow) and replaced either with fresh M5100, or MEGM or M199 and the development of the colonies was monitored over the time. The growth kinetics of a representative experiment out of five independent experiments is shown. At each indicated time point, the mean cell number/ECFC was determined by two independent operators; standard deviations were below 10 and are not shown. Asterisk, p,0.05. In B, immunocytochemical analysis of in vitro expanded EPC/ECFC documenting positivity for CD105 antigen (original magnification: 20X) and for the specific endothelial marker Factor VIII (original magnification: 40X). In C, FISH analysis performed on in vitro expanded EPC/ECFC by using the centromeric enumeration probe CEP9 (white arrows) documenting a normal diploid chromosomal pattern (original magnification: 40X). doi:10.1371/journal.pone.0056377.g1B, left panel). Differently from CFU-EC, EPC/ECFC could be derived from the PBMC of a limited subset of the ACS patients (9/ 70) all harvested at late time points (between 7?4 days) after acute cardiovascular events (Figure 1C). Of interest, EPC/ECFC when kept in culture became a larger monolayer (Figure 1B, right panel), thus exhibiting 15900046 in vitro expansion capacity, differently from CFU-EC.Analysis of pro-angiogenic cytokines release by PBMCWe 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) CFUECpos vs CFU-ECneg; ii) EPC/ECFCpos vs EPC/ECFCneg. The investigated cytokines were expressed by the PBMC at vari.Panel) or EPC/ECFC (B, left panel; arrowheads: hemopoietic mononucleated cells). In A, the right panel shows a monolayer of spindle-shaped endothelial-like monocytes. In B, the right panel shows a representative image of CFU-EC after in vitro expansion. Original magnification: 20X and 25X for the inset. In C, frequency of detection of CFU-EC and EPC/ECFC in PBMC of ACS patients, divided on the basis of the time of blood withdrawal after the hospital admission for the acute cardiovascular events. doi:10.1371/journal.pone.0056377.gEndothelial Progenitor Cells in ACS PatientsFigure 2. 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 boxplots). 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. doi:10.1371/journal.pone.0056377.gEndothelial Progenitor Cells in ACS PatientsFigure 3. Identification of optimal culture conditions for the ex-vivo expansion of ACS PB-derived EPC/ECFC. Primary EPC/ECFC colonies were generated by plating patient PBMC in M5100 medium, as detailed in the Method section. In A, after the colony identification (at day 5 after plating), medium was change (arrow) and replaced either with fresh M5100, or MEGM or M199 and the development of the colonies was monitored over the time. The growth kinetics of a representative experiment out of five independent experiments is shown. At each indicated time point, the mean cell number/ECFC was determined by two independent operators; standard deviations were below 10 and are not shown. Asterisk, p,0.05. In B, immunocytochemical analysis of in vitro expanded EPC/ECFC documenting positivity for CD105 antigen (original magnification: 20X) and for the specific endothelial marker Factor VIII (original magnification: 40X). In C, FISH analysis performed on in vitro expanded EPC/ECFC by using the centromeric enumeration probe CEP9 (white arrows) documenting a normal diploid chromosomal pattern (original magnification: 40X). doi:10.1371/journal.pone.0056377.g1B, left panel). Differently from CFU-EC, EPC/ECFC could be derived from the PBMC of a limited subset of the ACS patients (9/ 70) all harvested at late time points (between 7?4 days) after acute cardiovascular events (Figure 1C). Of interest, EPC/ECFC when kept in culture became a larger monolayer (Figure 1B, right panel), thus exhibiting 15900046 in vitro expansion capacity, differently from CFU-EC.Analysis of pro-angiogenic cytokines release by PBMCWe 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) CFUECpos vs CFU-ECneg; ii) EPC/ECFCpos vs EPC/ECFCneg. The investigated cytokines were expressed by the PBMC at vari.
