EBF1 is expressed in pericytes and contributes to pericyte cell commitment

Abstract

Early B-cell factor-1 (EBF1) is a transcription factor with an important role in cell lineage specification and commitment during the early stage of cell maturation. Originally described during B-cell maturation, EBF1 was subsequently identified as a crucial molecule for proper cell fate commitment of mesenchymal stem cells into adipocytes, osteoblasts and muscle cells. In vessels, EBF1 expression and function have never been documented. Our data indicate that EBF1 is highly expressed in peri-endothelial cells in both tumor vessels and in physiological conditions. Immunohistochemistry, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and fluorescence-activated cell sorting (FACS) analysis suggest that EBF1-expressing peri-endothelial cells represent bona fide pericytes and selectively express well-recognized markers employed in the identification of the pericyte phenotype (SMA, PDGFRβ, CD146, NG2). This observation was also confirmed in vitro in human placenta-derived pericytes and in human brain vascular pericytes (HBVP). Of note, in accord with the key role of EBF1 in the cell lineage commitment of mesenchymal stem cells, EBF1-silenced HBVP cells showed a significant reduction in PDGFRβ and CD146, but not CD90, a marker mostly associated with a prominent mesenchymal phenotype. Moreover, the expression levels of VEGF, angiopoietin-1, NG2 and TGF-β, cytokines produced by pericytes during angiogenesis and linked to their differentiation and activation, were also significantly reduced. Overall, the data suggest a functional role of EBF1 in the cell fate commitment toward the pericyte phenotype.

Keywords: Angiogenesis; Cell fate commitment; Mesenchymal stem cells; Pericytes.

Fig. 1

EBF1 is expressed in a cell population within the vessel wall in both neoplastic and non-neoplastic conditions. a Using an anti-pan-EBF antibody, glomeruloid vascular proliferation in glioblastoma (upper image, H&E staining, ×20 original magnification) shows immunoreactivity for EBFs (lower image, anti-pan-EBF immunostain, ×40 original magnification). b When specific antibodies for all the different EBF family members (EBF1–4) were employed, only EBF1 showed positive immunostaining (all images are ×40 original magnification). c Representative sections from different tumor samples show that newly formed tumor vessels selectively express EBF1 independently of their histotype, suggesting that EBF1 expression is a common feature of tumor vessels (all images are ×10 original magnification). d EBF1 is also expressed in the walls of small vessels in physiological and pathological non-neoplastic conditions, i.e. surgical scars (left image; ×40 original magnification), granulation tissue (upper right image; ×20 original magnification) and inflammatory lesions such as encephalitis (lower right image; ×40 original magnification). e EBF1 is strongly expressed in the vessel wall in embryonic (upper image, ×40 original magnification) and fetal tissue (lower image, ×20 original magnification). From panel (c–e) all immunostaining was performed with a specific antibody for EBF1. Scale bars: ×10, ×20 and ×40 original magnification, corresponding respectively to 200 μm, 100 μm and 50 μm

Fig. 2

EBF1-expressing cells are pericytes. Double immunostaining combining the specific antibody for EBF1 and lineage-specific markers was applied for glioblastoma samples with prominent glomeruloid vascular proliferation in order to identify the phenotype of EBF1-expressing cells. Double immunostaining using endothelial markers CD31, CD34, FVIII showed no double-positive cells (a), and the same was found for monocyte/macrophage markers (CD68, CD163, Tie-2) (b) and for the histological hallmarks of tumor-derived glioma cells (GFAP, EGFRvIII, IDH1-R132H) (c). In contrast, double immunostaining using the most widely recognized mesenchymal/pericyte markers (PDGFRβ, SMA, CD146, NG2 and CD90) (d) revealed the pericyte phenotype of EBF1-expressing cells. Chromogen used to identify immunoreactivity is either brown or blue according to the corresponding label, as indicated. All images are ×40 original magnification; scale bar corresponds to 50 μm

Fig. 3

EBF1-expressing cells are pericytes. Double immunostaining for EBF1 combined with either pericyte (SMA) or endothelial (CD31) lineage-specific markers on representative tissue sections from other neoplastic lesions (squamous cell carcinoma; left upper and lower images), physiological conditions (skin scar; middle upper and lower images) and inflammatory conditions (encephalitis; right upper and lower images) confirmed the pericyte phenotype of EBF1-expressing cells. Remarkably, the double staining with the endothelial markers clearly highlights the peri-endothelial distribution of the EBF1-expressing cells. In double immunostaining, EBF1 shows nuclear staining, while all the other markers showed cytoplasmic or membrane staining. Chromogen used to identify immunoreactivity is either brown or blue according to the corresponding label, as indicated. Higher magnification of the co-localized areas. All images are ×40 original magnification; scale bar corresponds to 50 μm. Insets are photographic enlargement of a representative field, as indicated

Fig. 4

EBF1 is expressed in pericytes isolated from both human placenta and brain. a Representative section from human placenta (left image, H&E staining; ×20 original magnification) shows that EBF1-positive cells within the vessel wall have a pericyte phenotype, as previously described. Double immunostaining for EBF1 (brown nuclear) and SMA, PDGFRβ (blue cytoplasmic); ×20 original magnification. b Placental-derived pericytes (PL-PC; left images, ×40 original magnification) were phenotypically characterized by immunocytochemistry on cell blocks (upper middle panels; ×40 original magnification) and immunofluorescence on cultured cells (lower middle panels; ×100 original magnification) using pericyte markers (PDGFRβ, SMA, CD146, calponin), confirming their pericyte phenotype. PL-PCs at passage II (P-II) were also analyzed by flow cytometry using specific antibodies for CD90-APC, CD146-PE, CD45-FITC and CD31-Pe-Cy7 (red). PL-PCs were positive for the pericyte/mesenchymal markers CD90 and CD146 and negative for CD45 (hematopoietic marker) and CD31 (endothelial marker). The negative control is also shown (no antibodies, light blue) (upper right images). RT-qPCR analysis (lower right image) shows that the pericyte phenotype is maintained in culture at least until P-IV, as indicated by the expression of PDGFRβ, CD90 and CD146 and the negativity for CD31. The endothelial counterpart (HUVECs used as control) shows a higher level of CD31 expression, as expected. Of note, CD146 was also detected in HUVECs, in accord with reported data (Du et al. 2015). c Commercially available HBVPs were used as a second pericyte cell line of different embryological origin (neuroectoderm) as compared to PL-PCs (mesoderm) (left image, ×40 original magnification). Their pericyte phenotype was confirmed by RT-qPCR (right image). As expected, HBVPs expressed PDGFRβ, CD90 and CD146, whereas HCMECs, the endothelial counterpart, expressed CD31. HBVPs maintained the pericyte phenotype in culture at least until P-VI even with a progressive decrease in PDGFRβ expression. d The expression of the EBF family members was assayed by RT-qPCR in both PL-PCs and HBVPs and their endothelial counterparts. As expected, both PL-PCs (left histogram) and HBVPs (right histogram) expressed high levels of EBF1, while the expression of the other EBF family members was very low or barely detected. Of note, the absolute expression level of EBF1 in HBVPs was more than 20-fold higher than that of PL-PCs, probably related to their different embryological origin. Scale bars: ×20, ×40 and ×100 original magnifications, corresponding respectively to 100 μm, 50 μm and 20 μm

Fig. 5

EBF expression allows pericytes to be distinguished from MSCs. a MSCs isolated from human adipose tissue (MSC-AT) showed, as expected, significantly lower levels of PDGFRβ as compared to HBVPs (p < 0.01), with high levels of CD90 that conversely was barely detected in HBVPs (p < 0.01). Of note, CD146 was expressed, albeit at low levels, only in HBVPs (p < 0.01). b The expression levels of the EBF family members in MSC-AT and HBVPs showed that EBF1 was expressed also in MSC-AT, albeit at a significant lower level as compared to HBVPs (p < 0.05). While EBF2 was expressed at a comparable level in both MSC-AT and HBVPs, EBF3 was detected only in MSC-AT, albeit at a very low level (p < 0.01). Of note, EBF4 was detected only in HBVPs, although at a very low level (p < 0.05). These data suggest that the EBF expression profile allows MSC-AT to be distinguished from pericytes. *p < 0.05, **p < 0.01

Fig. 6

EBF1 plays a key role in the pericyte phenotype cell commitment. a Significant downregulation of EBF1 mRNA levels (about 75% decrease; p < 0.01) was obtained in HBVPs by means of siRNA technology, measured by RT-qPCR 24 h after transfection (upper histogram). Immunoblotting performed with protein extracts confirmed the reduction in the synthesis of EBF1 protein (about 50% decrease; p < 0.05). The representative image on the right shows the corresponding EBF1 band at 66 kDa. b Histogram shows that after EBF1 silencing, the expression of EBF3, a marker linked to the MSC phenotype, is significantly increased (p < 0.05). c Silencing of EBF1 does not affect cell proliferation as assessed by the evaluation of the nuclear antigen Ki-67 expression by RT-qPCR, 24 h after seeding cells with or without the addition of different proliferative stimuli, such as the medium from U87 glioblastoma cells cultured under hypoxic conditions or the complete culture medium containing PGS and 10% FBS. d We then investigated whether EBF1 silencing affects the phenotype of the pericytes. Indeed, the histograms show a significant reduction in the main pericyte markers PDGFRβ (p < 0.05) at both the transcriptional (p < 0.05) and protein (p < 0.01) levels. e Additionally, we found a significant reduction in the pericyte marker CD146 (p < 0.01). The expression levels of CD90, a marker linked to a mesenchymal phenotype, did not show a significant reduction. f Of note, the expression levels of VEGF, Ang-1, NG2 and TGF-β, cytokines produced by pericytes during different phases of angiogenesis are significantly reduced in EBF1-silenced HBVPs (p < 0.05). Overall, data confirmed a functional role of EBF1 in pericyte cell fate commitment and functionality. *p < 0.05, **p < 0.01. CTRL (control); siRNA (EBF1-silenced); SCR (scramble, control siRNA); Basal (basal conditions); Hypoxic (hypoxic conditions)