Angiogenesis

, Volume 16, Issue 2, pp 309–327

Multivalent proteoglycan modulation of FGF mitogenic responses in perivascular cells

  • Sabrina Cattaruzza
  • Ugur Ozerdem
  • Martin Denzel
  • Barbara Ranscht
  • Pietro Bulian
  • Ugo Cavallaro
  • Daniela Zanocco
  • Alfonso Colombatti
  • William B. Stallcup
  • Roberto Perris
Original Paper

DOI: 10.1007/s10456-012-9316-7

Cite this article as:
Cattaruzza, S., Ozerdem, U., Denzel, M. et al. Angiogenesis (2013) 16: 309. doi:10.1007/s10456-012-9316-7

Abstract

Sprouting of angiogenic perivascular cells is thought to be highly dependent upon autocrine and paracrine growth factor stimulation. Accordingly, we report that corneal angiogenesis induced by ectopic FGF implantation is strongly impaired in NG2/CSPG4 proteoglycan (PG) null mice known to harbour a putative deficit in pericyte proliferation/mobilization. Conversely, no significant differences were seen between wild type and knockout corneas when VEGF was used as an angiocrine factor. Perturbed responsiveness of NG2-deficient pericytes to paracrine and autocrine stimulation by several FGFs could be confirmed in cells isolated from NG2 null mice, while proliferation induced by other growth factors was equivalent in wild type and knockout cells. Identical results were obtained after siRNA-mediated knock-down of NG2 in human smooth muscle-like cell lines, as also demonstrated by the decreased levels of FGF receptor phosphorylation detected in these NG2 deprived cells. Binding assays with recombinant proteins and molecular interactions examined on live cells asserted that FGF-2 bound to NG2 in a glycosaminoglycan-independent, core protein-mediated manner and that the PG was alone capable of retaining FGF-2 on the cell membrane for subsequent receptor presentation. The use of dominant-negative mutant cells, engineered by combined transduction of NG2 deletion constructs and siRNA knock-down of the endogenous PG, allowed us to establish that the FGF co-receptor activity of NG2 is entirely mediated by its extracellular portion. In fact, forced overexpression of the NG2 ectodomain in human smooth muscle-like cells increased their FGF-2-induced mitosis and compensated for low levels of FGF receptor surface expression, in a manner equivalent to that produced by overexpression of the full-length NG2. Upon FGF binding, the cytoplasmic domain of NG2 is phosphorylated, but there is no evidence that this event elicits signal transductions that could bypass the FGFR-mediated ones. Pull-down experiments, protein–protein binding assays and flow cytometry FRET coherently revealed an elective ligand-independent association of NG2 with FGFR1 and FGFR3. The NG2 cooperation with these receptors was also corroborated functionally by the outcome of FGF-2 treatments of cells engineered to express diverse NG2/FGFR combinations. Comprehensively, the findings suggest that perivascular NG2 may serve as a dual modulator of the availability/accessibility of FGF at the cell membrane, as well as the resulting FGFR transducing activity.

Keywords

Proteoglycan Angiogenesis FGF signalling NG2/CSPG4 Pericytes 

Supplementary material

10456_2012_9316_MOESM1_ESM.eps (38 mb)
Supplemental Figure 1. Relative knock-down efficacy and surface recovery of NG2 following transient siRNA-mediated abrogation in SK-LMS-1 cells (analogous results were obtained in SK-UT-1 cells). (a) Determination of the relative knock-down efficacy of siRNA probes directed against different regions of the NG2 transcript (a scrambled sequence probe was used as a control) based upon flow cytometry using mAb 7.1. (b, c) Kinetics of NG2 surface knock-down and subsequent cell surface recovery, as determined by flow cytometry using the same anti-NG2 mAb at the indicated days following siRNA transfection, or on day one to six starting from the fourth day after transfection (i.e. Day 1 of recovery = day 4 after transfection). Immunofluorescence (anti-NG2 mAb B5/M28) images show representative cases of maximal knock-down and following surface recovery (i.e. at Day 3 of knock-down and Day 6 of recovery; nuclei were counterstained with TO-PRO-3). When surveying the panel of mesenchymal cell lines used in this study we could estimate by flow cytometry (and corroborate by parallel semi-quantitative Western blotting) that an average of 75–82 % of the cells expressed detectable surface levels of NG2. Following transient siRNA-mediated knock-down, the abrogation efficiency that was achieved was in the range of 53–67 % reduction of cell surface expression of the PG when compared to untreated cells. Supplementary material 1 (EPS 38874 kb)
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Supplemental Figure 2. Patterns of constitutive syndecan and glypican expression in aortic smooth muscle cells from wild type NG2+/+ and knockout NG2−/− mice, and the two primary human smooth muscle cell-like lines used in the study. Expression levels were determined semi-quantitatively at the (a) mRNA (qPCR) and (b) protein level (flow cytometry; representative analyses of SK-LMS-1 cells are shown in the lower panel). Primers and antibodies were as detailed in Materials and Methods. Flow cytometric scoring of relative surface expression of the detected antigens was as follows: “−”, 1–15 %; “+”, 16–40 %; “++”, 41–65 %; and “+++”, 66–95 %. Scoring for relative levels of transcript expression were based upon direct comparisons with calibrating human bone marrow-derived mesenchymal progenitor cells, HUVEC, primary human dermal fibroblasts, A375 human melanoma cells, HT1080 fibrosarcoma, MCF-7 breast carcinoma, HepG2 hepatocellular carcinoma, NIH3T3 cells, B16 murine melanoma cells and mouse embryonic fibroblasts (MEF). The cumulative expression scoring was as follows: “−”, weak mRNA expression and no detectable surface levels of the protein; “+”, moderate transcript levels and weak surface levels of the protein; “++”, high transcript levels and moderate surface levels of the protein; and “+++”, high transcript levels and high surface expression of the protein. Supplementary material 2 (EPS 3515 kb)
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Supplemental Figure S3. (a) FGF-2 mitogenic responses of FGFR1-expressing murine lymphocytic BaF3 cells after transient transduction with rodent NG2 (BaF3NG2). Inset to the right shows the relative levels of ectopic NG2 expression as determined by flow cytometry using the D3 anti-NG2 antiserum. Left inset shows detection of ectopically expressed rodent NG2 in whole cell lysates subjected (BaF3NG2/Chase) or not (BaF3NG2) chondroitinase ABC digestion. (b) Representative immunostaining of untreated and anti-NG2 siRNA-treated SK-LMS-1 cells exposed for 72 h to the indicated FGFs (10 ng/ml) in the absence of serum. (c) One out of four cell-cycle analyses performed by flow cytometry by propidium iodide incorporation on untreated and anti-NG2 siRNA-treated SK-UT-1 cells exposed to FGF-2. Supplementary material 3 (EPS 14437 kb)
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Supplemental Figure S4.(a) Recovery of FGF-responsiveness in SK-UT-1 cells upon surface recovery of transiently knocked down NG2 (Supplemental Fig. S2). Cells were either treated with an effective anti-NG2 siRNA probe (siRNA3289), or with an ineffective one (siRNA2938), and stimulated with 10 ng/ml of FGF-2. Assessment of cell proliferation was as described for other growth factor stimulation experiments. (b) Verification of the preserved ability of NG2-deficient cells to respond to GAG-activated exogenous FGF-2. SiRNA-treated cells were exposed to 10 ng/ml FGF-2 in the presence of soluble heparin (high molecular weight; 10 μg/ml), HS oligosaccharides of different sizes and compositions (HS6-HS12; 1 μg/ml), or the unrelated GAGs chondroitin sulfate (CS; 10-50 μg/ml) and keratan sulfates (KS; 10-50 μg/ml). Adding heparin or HS oligosaccharides to cells not treated with siRNA probes did not augment their FGF response level. Supplementary material 4 (EPS 463 kb)
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Supplemental Figure S5. Patterns of FGFR expression and endogenously produced FGFs in the cell types used in the study, including smooth muscle cells from wild type (NG2+/+) and NG2 null (NG2−/−) mice. Determination of the patterns of FGFR expression was based upon combined qPCR (a) and flow cytometry. (b) shows representative flow cytometry analyses of FGFR expression in wild type (red line) and NG2 null (green line) smooth muscle cells. Endogenous FGFs production was determined by qPCR for the corresponding transcripts (c). Arbitrary scoring of the levels of expression of FGFRs was similar to that detailed for PGs in Supplemental Fig. S2. Analyses of FGFs were restricted to those currently known to be preferred ligands of FGFR1 and FGFR3. Supplementary material 5 (EPS 17012 kb)
10456_2012_9316_MOESM6_ESM.eps (41.9 mb)
Supplemental Figure S6.(a) Schematic overview of the strategy adopted for generating NG2 mutant cells by combined siRNA-mediated mRNA knock-down and gene transduction. SK-LMS-1 cells were stably transduced with a human NG2 construct encoding the entire extracellular domain plus the membrane-spanning portion (NG2extra), or with a human NG2 construct encoding the transmembrane and cytoplasmic domains and a minor portion of the extracellular domain (NG2cyto). Transduced cells were then treated with either a C-terminus-directed siRNA (in the case of NG2extra), or an N-terminus-directed siRNA (in the case of NG2cyto). This approach resulted in an effective abrogation of the expression of the endogenous NG2 (see also Supplemental Fig. S2), while the transduced deletion constructs were not targeted. (b) qPCR-based assessment of the expression levels of the NG2extra and NG2cyto constructs when compared to the endogenous NG2 mRNA. (c, d) Flow cytometry and immunoblotting documenting the overall NG2 surface expression in NG2extra and NG2cyto cells as detected by mAb 7.1 (flow cytometry) and mAb B5/M28 (immunoblotting). The somewhat reduced surface levels of NG2 in NG2extra mutant cells, when compared to wild type cells, are presumably due to a previously observed ineffective membrane localization of NG2 molecules deprived of their cytoplasmic tail. (e) Summarizes the evidence for a putative involvement of NG2 in autocrine FGF and PDGF signallings through activity of its ectodomain. NG2extra, NG2cyto mutant SK-LMS-1 cells or SK-LMS-1 cells in which NG2 was knocked down were grow in low serum content (0.5 %) for the indicated time in the presence or absence of the FGFR antagonist PD173074, or the PDGF receptor antagonist Imatinib, and their proliferation rates were scored. Untreated cells and cells treated with a non-effective siRNA were consistently used in a comparative manner as controls. Supplementary material 6 (EPS 42893 kb)
10456_2012_9316_MOESM7_ESM.doc (87 kb)
Supplementary material 7 (DOC 87 kb)
10456_2012_9316_MOESM8_ESM.doc (60 kb)
Supplementary material 8 (DOC 60 kb)

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Sabrina Cattaruzza
    • 1
  • Ugur Ozerdem
    • 2
  • Martin Denzel
    • 3
  • Barbara Ranscht
    • 3
  • Pietro Bulian
    • 4
  • Ugo Cavallaro
    • 5
  • Daniela Zanocco
    • 1
  • Alfonso Colombatti
    • 1
  • William B. Stallcup
    • 3
  • Roberto Perris
    • 1
    • 6
  1. 1.S.O.C. for Experimental Oncology 2The National Cancer Institute Aviano, CRO-IRCCSAvianoItaly
  2. 2.La Jolla Bioengineering InstituteLa JollaUSA
  3. 3.The Sanford-Burnham Institute for Medical ResearchLa JollaUSA
  4. 4.S.O.C. for Experimental and Clinical Onco-HematologyThe National Cancer Institute Aviano, CRO-IRCCSAvianoItaly
  5. 5.IFOM-IEO CampusThe FIRC Institute of Molecular OncologyMilanItaly
  6. 6.COMT, Centre for Molecular and Translational OncologyUniversity of ParmaParmaItaly

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