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 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 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 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

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

Clinical characteristics of cKS patients.

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 ^(53,54).</p><p>A indicates slow evolution; B, rapid evolution; rapid denotes an increase in the total number of nodules/plaques or in the total area of plaques in three months following the last examination.</p

B cells from cKS patients show increased resistance to spontaneous apoptosis and unaffected <i>in vivo</i> turnover.

<p>Apoptotic cells were detected by annexin V binding after 24 h culture in unstimulated conditions. <i>A</i>, representative flow cytometric analysis showing annexin V binding gated on total B cells, CD27⁻, MZ-like (CD27⁺IgD^lo) and switched memory (CD27⁺IgD⁻) B cells, as indicated; comparison between one HC (upper row) and one cKS patient (lower row). B, B cells from cKS patients (grey bars) showed significantly lower annexinV binding than B cells from HCs (white bars); this lower annexin V binding was evident on CD27⁻ and MZ-like (CD27⁺IgD^lo) B cells, roughly composing the preimmune/natural effector compartment, but not on antigen-experienced switched memory (CD27⁺IgD⁻) B cells. Data shown as mean ± SE. <i>C, In vivo</i> cell turnover was estimated by Ki67 staining of freshly isolated PBMCs. Representative flow cytofluorimetric analysis showing Ki67 binding gated on total B cells, CD27⁻ and CD27⁺ B cells, as indicated; comparison between one HC (upper row) and one cKS patient (lower row). <i>D</i>, The proportion of Ki67⁺ cells within total B cells, CD27⁻ and CD27⁺ B cells did not differ between cKS patients (grey bars) and HCs (white bars). Data shown as mean ± SE. <i>P</i>-values calculated using the Student <i>t</i> test for independent samples.*<i>P</i><.05; **<i>P</i><.01.</p

B cells from cKS patients show a low state of activation.

<p>B cells from cKS patients (grey bars) expressed lower levels of the costimulatory molecules CD80 and CD86 and higher levels of CD20 than B cells from HCs (white bars). The expression of the indicated markers on total B cells and their CD27⁻ and CD27⁺ subsets is shown. Data presented as mean ± SE of mean fluorescence intensity (MFI) values. <i>P</i>-values calculated using the Student <i>t</i> test for independent samples. *<i>P</i><.05; **<i>P</i><.01; ***<i>P</i><.001.</p

HHV-8 load in isolated PBMCs, B cells and non-B cells from cKS patients and healthy HHV-8-seropositive (HSP) controls according to their peripheral blood B cells count.

a<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.</p>b<p>gEq =  genome Equivalents.</p