Effects of fibronectin and collagen on the immunophenotype of ECFCs.

<p>ECFCs expanded on fibronectin or collagen showed a similar immunophenotype. On the left are reported flow cytometric histograms showing that, independently from the substrate used for cell culture, ECFCs did not express CD45 and CD14, while they expressed at similar levels CD31, VEGFR2, CD144, CD146, CD54 and CXCR4. On the right are reported immunofluorescence photographs showing that ECFCs expanded on both substrates incorporated Dil-ac-LDL and bound lectin UEA-1. Photographs were obtained using an an Olympus Fluoview FV1000 confocal microscope, x40 magnification. One of three representative experiments is reported.</p

Effects of fibronectin and collagen on ECFC cell expansion.

<p>Collagen sustained cell expansion of ECFCs more efficiently than fibronectin. 14 ECFC colonies were divided into portions, with half of them seeded on fibronectin-coated plates and the rest seeded on collagen-coated plates. (A) Representative phase-contrast photographs showing that ECFCs expanded on fibronectin and collagen formed similar cobblestone-like monolayers with endothelial-like morphology. Photographs were obtained using an Olympus inverted microscope IX51, x10 magnification. ECFCs cultured on collagen (gray bar) compared with fibronectin (white bar) achieved (B) higher number of passages, (C) longer lifespan and (D) higher cell yields. p values calculated by the Wilcoxon signed-rank test.</p

Effects of fibronectin and collagen on the ability of ECFCs for in vitro tubulogenesis.

<p>ECFCs expanded on fibronectin or collagen showed a similar ability to form capillary-like structures in vitro. Independently from the substrate used for cell culture, ECFCs cultured in Matrigel gave rise within 12 hours of incubation to vascular structures. Representative phase-contrast photographs showing that the capillary-like structures formed by ECFCs cultured on fibronectin and collagen were similar. Photographs were obtained using an Olympus inverted microscope IX51, x4 magnification.</p

Effects of fibronectin and collagen on cytokine production by ECFCs.

<p>ECFCs expanded on collagen produced higher levels of IL-6 and IL-8 that ECFCs expanded on fibronectin. (A) Representative curves showing that the concentration of IL-6 and IL-8 in the supernatants of ECFCs expanded on fibronectin (white circles) and collagen (gray circles) tended to increase with increasing passages. (B) ECFCs expanded on collagen (gray bars) reached higher levels of IL-6 and IL-8, but not bFGF, than ECFCs cultured on fibronectin (white bars). The mean ± SEM of the highest cytokine concentration reached during culture of the same 14 ECFC colonies reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066734#pone-0066734-g002" target="_blank">Figure 2</a> is shown. p values calculated by the Wilcoxon signed-rank test.</p

Effects of IL-6 and IL-8 on ECFC proliferation.

<p>IL-6 and IL-8 may account, at least in part, for the higher cell proliferation of ECFCs cultured on collagen as compared to fibronectin. Cell proliferation was assessed by the colorimetric CV assay, and expressed as OD. (A) Addition of IL-6 or IL-8 to ECFCs cultured on fibronectin increased cell proliferation in a dose-dependent manner. The mean ± SEM of 7 ECFC cultures is shown. *p = 0.046 and **p = 0.009 compared with untreated ECFCs cultured on fibronectin, as calculated by the Wilcoxon signed-rank test. (B) Addition of IL-6 and IL-8 together in the same culture did not further increase cell proliferation. One representative of three experiments is shown. (C) Addition of neutralizing anti-IL-6 or anti-IL-8 mAb to ECFCs cultured on collagen induced a dose-dependent reduction of cell proliferation. One representative of three experiments is shown.</p

Effects of fibronectin and collagen on isolation of ECFC colonies.

<p>Fibronectin promoted the appearance of ECFC colonies earlier than collagen. (A) PBMCs from 9 donors were seeded on fibronectin-coated plates and PBMCs from other 9 donors were seeded on collagen-coated plates; ECFC colonies from PBMCs seeded on fibronectin (white bar) appeared earlier than those seeded on collagen (gray bar). p value calculated by the Mann-Whitney U-test. (B) PBMCs from 8 donors were divided into portions, with half of them seeded on fibronectin-coated plates and the rest seeded on collagen-coated plates; also in these paired experiments ECFC colonies from PBMCs seeded on fibronectin (white circles) appeared earlier than those seeded on collagen (gray circles). p value calculated by the Wilcoxon signed-rank test.</p

Clinical characteristics of patients with cKS at the time of late-EPC culture.

a<p>Mean±standard error.</p>b<p>cKS patients were classified according to our classification that takes into account the prevalent type of lesions, localization, clinical behaviour, evolutive pattern and presence of complications <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001520#pone.0001520-Brambilla1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001520#pone.0001520-DellaBella1" target="_blank">[8]</a>.</p><p>A = slow evolution; B = rapid evolution; rapid denotes an increase in the total number of nodules/plaques or in the total area of plaques in the three months following the last examination.</p

Flow cytometry analysis showing CD146 expression on different late-EPC populations.

<p>To confirm that late-EPCs with proven endothelial phenotype could support KSHV infection, late-EPCs from one cKS patient underwent cell sorting of CD146+ cells and were cultured for further 2 weeks. Analysis of unsorted late-EPCs stained with isotype control (A) or anti-human CD146 mAb (B); analysis of highly purified CD146+ late-EPCs stained with anti-human CD146 mAb immediately after sorting (C) or after 2 weeks of culture (D).</p

KSHV-DNA in peripheral blood mononuclear cells and in late-EPC cultures from patients with cKS according to their clinical stage and KSHV serology.

a<p>Antibody titers were calculated as the reciprocal of the highest plasma dilution giving positive results.</p>b<p>cKS patients were classified according to our classification that takes into account the prevalent type of lesions, localization, clinical behaviour, evolutive pattern and presence of complications <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001520#pone.0001520-Brambilla1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001520#pone.0001520-DellaBella1" target="_blank">[8]</a>.</p>c<p>In two patients the presence of KSHV-DNA was determined in multiple passages of unstimulated late-EPC cultures.</p>d<p>KSHV-DNA determined in highly pure CD146+ late-EPCs, maintained in culture for further 2 weeks after CD146 sorting.</p><p>KSHV = Kaposi's sarcoma-associated herpesvirus; PBMCs = peripheral blood mononuclear cells; late-EPC = late-endothelial progenitor cell;</p><p>cKS = classic Kaposi's sarcoma; LANA = latency-associated nuclear antigen; GE = genome equivalents.</p

Late-EPCs obtained from patients with cKS support KSHV productive replication.

<p>A) To induce KSHV lytic replication, late-EPCs from cKS patients underwent treatment with <i>n</i>-butyrate (5 patients, solid lines) or TPA (2 patients, hatched lines) for 48 hours. Multiple colonies from each patient were pooled before treatment. B) To confirm that late-EPCs with proven endothelial phenotype could support KSHV lytic replication, late-EPCs from one cKS patient underwent cell sorting of CD146+ cells. Unsorted late-EPCs (solid line) or highly purified CD146+ late-EPCs from the same cKS patient were cultured for further 2 weeks after sorting (hatched line) and underwent treatment with <i>n</i>-butyrate for 48 hours. In any case, KSHV genomes were analyzed by real-time PCR in DNA extracted from late-EPC supernatants. <i>P</i> value was determined using the Wilcoxon signed-rank test.KSHV genomes were analyzed by real-time PCR in DNA extracted from culture supernatants. KSHV = Kaposi's sarcoma-associated herpesvirus; TPA = phorbol 12-myristate 13-acetate.</p