CCN2/connective tissue growth factor is essential for pericyte adhesion and endothelial basement membrane formation during angiogenesis.

Abstract

CCN2/Connective Tissue Growth Factor (CTGF) is a matricellular protein that regulates cell adhesion, migration, and survival. CCN2 is best known for its ability to promote fibrosis by mediating the ability of transforming growth factor β (TGFβ) to induce excess extracellular matrix production. In addition to its role in pathological processes, CCN2 is required for chondrogenesis. CCN2 is also highly expressed during development in endothelial cells, suggesting a role in angiogenesis. The potential role of CCN2 in angiogenesis is unclear, however, as both pro- and anti-angiogenic effects have been reported. Here, through analysis of Ccn2-deficient mice, we show that CCN2 is required for stable association and retention of pericytes by endothelial cells. PDGF signaling and the establishment of the endothelial basement membrane are required for pericytes recruitment and retention. CCN2 induced PDGF-B expression in endothelial cells, and potentiated PDGF-B-mediated Akt signaling in mural (vascular smooth muscle/pericyte) cells. In addition, CCN2 induced the production of endothelial basement membrane components in vitro, and was required for their expression in vivo. Overall, these results highlight CCN2 as an essential mediator of vascular remodeling by regulating endothelial-pericyte interactions. Although most studies of CCN2 function have focused on effects of CCN2 overexpression on the interstitial extracellular matrix, the results presented here show that CCN2 is required for the normal production of vascular basement membranes.

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CCN2/Connective Tissue Growth Factor Is Essential for
Pericyte Adhesion and Endothelial Basement Membrane
Formation during Angiogenesis
Faith Hall-Glenn1., R. Andrea De Young2.¤a, Bau-Lin Huang3¤b, Ben van Handel1,
Jennifer J. Hofmann1,4¤c, Tom T. Chen1¤d, Aaron Choi2, Jessica R. Ong1, Paul D. Benya2,
Hanna Mikkola1, M. Luisa Iruela-Arispe1,4,5, Karen M. Lyons1,2,4,5*
1Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America, 2Department of
Orthopaedic Surgery, University of California Los Angeles, Los Angeles, California, United States of America, 3Department of Oral Biology, University of California Los
Angeles, Los Angeles, California, United States of America, 4Molecular Biology Institute, University of California Los Angeles, Los Angeles, California, United States of
America, 5Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, California, United States of America
Abstract
CCN2/Connective Tissue Growth Factor (CTGF) is a matricellular protein that regulates cell adhesion, migration, and survival.
CCN2 is best known for its ability to promote fibrosis by mediating the ability of transforming growth factor b (TGFb) to
induce excess extracellular matrix production. In addition to its role in pathological processes, CCN2 is required for
chondrogenesis. CCN2 is also highly expressed during development in endothelial cells, suggesting a role in angiogenesis.
The potential role of CCN2 in angiogenesis is unclear, however, as both pro- and anti-angiogenic effects have been
reported. Here, through analysis of Ccn2-deficient mice, we show that CCN2 is required for stable association and retention
of pericytes by endothelial cells. PDGF signaling and the establishment of the endothelial basement membrane are required
for pericytes recruitment and retention. CCN2 induced PDGF-B expression in endothelial cells, and potentiated PDGF-B-
mediated Akt signaling in mural (vascular smooth muscle/pericyte) cells. In addition, CCN2 induced the production of
endothelial basement membrane components in vitro, and was required for their expression in vivo. Overall, these results
highlight CCN2 as an essential mediator of vascular remodeling by regulating endothelial-pericyte interactions. Although
most studies of CCN2 function have focused on effects of CCN2 overexpression on the interstitial extracellular matrix, the
results presented here show that CCN2 is required for the normal production of vascular basement membranes.
Citation: Hall-Glenn F, De Young RA, Huang B-L, van Handel B, Hofmann JJ, et al. (2012) CCN2/Connective Tissue Growth Factor Is Essential for Pericyte Adhesion
and Endothelial Basement Membrane Formation during Angiogenesis. PLoS ONE 7(2): e30562. doi:10.1371/journal.pone.0030562
Editor: Costanza Emanueli, University of Bristol, United Kingdom
Received October 3, 2011; Accepted December 19, 2011; Published February 20, 2012
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported by NIH AR052686, Scleroderma Foundation, and the Jonsson Comprehensive Cancer Center, UCLA (KML), UCLA Vascular
Biology Training Grant HL069766 and Ruth L. Kirschstein National Research Service Award T32HL69766 (FHG), and NIH HL097766 (HM). The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: klyons@mednet.ucla.edu
¤a Current address: Amgen, Thousand Oaks, California, United States of America
¤b Current address: Center for Cancer Research, National Cancer Institute, Frederick, Maryland, United States of America
¤c Current address: Karolinska Institutet, Stockholm, Sweden
¤d Current address: South San Francisco, California, United States of America
. These authors contributed equally to this work.
Introduction
CCN2, also known as connective tissue growth factor, is a
member of the CCN (CCN1-6) family of matricellular proteins.
CCN family members are cysteine-rich and contain an N-terminal
secretory peptide, followed by four multi-functional domains that
interact with a diverse array of binding partners [1,2]. Proteins
that interact with CCN2 through recognition of these domains
include integrins, low-density lipoprotein receptor-related proteins
(LRPs), growth factors, and extracellular matrix (ECM) compo-
nents. The first domain shares homology to insulin-like growth
factor binding proteins (IGFBPs), but has very low affinity for IGF
[3]. The second domain encodes a von Willebrand type C (VWC)
repeat. This motif mediates CCN2 interactions with growth
factors such as bone morphogenetic proteins (BMPs) and
transforming growth factor b (TGFb) [4]. The third domain is a
type-1 thrombospondin (TSP) repeat, known to mediate the ability
of CCN2 to bind to ECM proteins, matrix metalloproteinases
(MMPs) and integrin a6b1 [5,6] The final C-terminal (CT) motif
contains a cysteine knot similar to those present in many growth
factors, including members of the TGFb superfamily, platelet
derived growth factor (PDGF), and nerve growth factor (NGF).
This motif mediates interactions with integrins avb3, a5b1, and
a6b1 [7–13].
CCN2 was originally isolated from human umbilical vein
endothelial cells (HUVECs) [14]. In situ hybridization and
immunohistochemical studies demonstrated that CCN2 is ex-
pressed predominantly in endothelial cells in embryonic and adult
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vasculature [15–18]. The physiological role of CCN2 in
angiogenesis is unclear, however, as it appears to have both pro-
and anti-angiogenic activities in vitro. For example, CCN2 induces
corneal angiogenesis, and anti-CCN2 antibodies block angiogen-
esis in the chick chorioallantoic membrane assay [19,20]. On the
other hand, anti-angiogenic activities have been reported;
although Ccn2 expression is induced by VEGF [21], CCN2 binds
to and sequesters VEGF in an inactive form [5], and combined
administration of CCN2 and VEGF inhibits VEGF-induced
angiogenesis [22]. The role of CCN2 in angiogenesis in vivo is
unknown.
The majority of studies have focused on the role of CCN2 as a
stimulator of excess ECM production in the context of
pathological fibrosis [23]. CCN2 is overexpressed in all fibrotic
conditions described to date, and depending on the tissue involved,
induces collagen type I deposition and increased susceptibility to
injury [24]. Conversely, the loss of CCN2 in fibroblasts results in
decreased collagen deposition and resistance to chemically
induced skin fibrosis [25,26]. In addition to its role as a mediator
of fibrosis, CCN2 is required for ECM production in cartilage
[27]. Ccn2 knockout mice survive in Mendelian ratios throughout
gestation, but die within minutes of birth. They exhibit severe
chondrodysplasia as a result of decreased collagen type II and
aggrecan expression by chondrocytes in vivo and in vitro [27,28].
CCN2 regulates cell survival, adhesion, migration, and ECM
production in multiple cell types by regulating integrin expression
and activation [13]. In Ccn2 mutant chondrocytes, integrin a5b1
expression and downstream focal adhesion kinase (FAK) and
extracellular signal-related kinase (ERK1/2) signaling are de-
creased, indicating that CCN2 regulates ECM production through
integrins [28].
In endothelial cells, CCN2 mediates adhesion, migration and
survival through binding to integrin avb3 [7]. CCN2 is also a
ligand for a5b1 and a6b1 [13], and these integrins are required
for endothelial basement membrane formation and vessel
stabilization in vitro [29]. Taken together, these studies implicate
CCN2 as an important regulator of cellular adhesion and ECM
production during angiogenesis, but do not address its role in vivo.
As CCN2 is the major mediator of excess ECM production during
fibrosis, and has also been implicated in tumor angiogenesis [30],
it is important to understand its function in normal tissues.
Therefore, the function of CCN2 in angiogenesis was investigated
through analysis of Ccn2 mutant mice.
Results
CCN2 is expressed in the developing vasculature
Using transgenic mice in which lacZ expression is driven by the
4 kb proximal Ccn2 promoter [31], CCN2 expression was seen
throughout the vasculature and microvasculature at E16.5
(Figure 1A). Expression was observed in large vessels, arterioles
and capillaries at all stages examined (E13.5-P0). CCN2 was
detected as early as E13.5 in developing dermal microvasculature
(Figure 1B), where lacZ is present in large and small caliber vessels
(Figure 1A,B). Similar results were seen using bacterial artificial
chromosome (BAC) transgenic mice expressing enhanced green
fluorescent protein (EGFP) under the control of the Ccn2 locus
(CCN2-EGFP) [32]. This analysis revealed Ccn2 expression in
endothelium of arterial and venous elements, and in capillaries. In
large arteries, CCN2-EGFP was expressed in both endothelial and
vascular smooth muscle cells (vSMCs) (Figure 1C,E). CCN2 was
also expressed in developing capillary networks (Figure 1D).
Endothelial-specific expression in microvasculature was also
shown by immunostaining for CCN2 (Figure 1F–H). Specificity
of the antibody was confirmed by the absence of staining sections
from Ccn2 mutants (Figure 1H). Punctate intracellular staining was
observed, most likely within the Golgi and in secretory vesicles, as
reported previously [33]. Cell-associated expression was also seen
on the abluminal surface of the endothelium (Figure 1G). Co-
immunostaining with the endothelial-specific marker PECAM
(CD31) revealed CCN2 expression in endothelial cells and in
mural cells (Figure S1A). Thus, Ccn2 is expressed in both
endothelial and mural cells in blood vessels and capillaries during
development.
Ccn2 mutant mice exhibit vascular defects
Ccn2 mutant mice exhibit perinatal lethality due to a severe
chondrodysplasia [27]. CCN2 expression in developing blood
vessels raised the possibility of an additional role in vascular
development. Ccn22/2 embryos were examined to investigate
this possibility. No overt differences between Ccn2 mutants and
WT littermates were apparent during the initial formation of the
vasculature from E9.5–E13.5 (data not shown). Moreover,
placentas were normal in appearance, weight, and vascularity
throughout development (Figure S1B,C, and data not shown).
However, beginning at E14.5, minor enlargement of vessels was
observed in mutants (Figure S1D,E), which became more
pronounced at later stages (Figure 2A,B). Local edema was seen
in E18.5 mutant dermis (Figure 2C,D). Immunofluorescence
analysis of the vSMC marker smooth muscle actin (SMA) and
PECAM (CD-31) did not reveal obvious evidence that SMC
coverage of large vessels was affected in mutants (Figure S1F–I).
However, comparison of hematoxylin and eosin-stained sections of
the aorta at thoracic and lumbar levels from E16.5 embryos
showed defects in the organization of the tunica media (Fig. 2E–
H). In WT embryos, SMCs had a spindle-like morphology and
were circumferentially oriented around the vessel lumen in distinct
layers (Figure 2E,G). In mutants, SMCs failed to adopt this
spindle-like morphology, were more heterogeneous in size, and
were not organized into distinct layers (Figure 2F,H). The large
vessel phenotype will be reported in more detail elsewhere. Here
we focus on the microvascular phenotype.
Morphological examination (Figure S1J,K) revealed that
arterial-venous identity appeared to be maintained in mutants
(see also Figure S1H,I). Ephrin B2 (expressed on arterial elements)
and EphB4 (preferentially expressed on veins) staining demon-
strated no defects in arterial-venous identity (Figure S1L,M, and
data not shown). However, inspection of E18.5 dermal microvas-
culature revealed evidence of defective remodeling in Ccn2
mutants. Consistent with a defect in remodeling, vessel density
was increased in Ccn2 mutants (Figure 2I–L and Figure S2A–C).
Moreover, mutant capillaries had multiple protrusions along their
surfaces (Figure 2M,N). Electron microscopy revealed numerous
luminal and abluminal protrusions in mutant capillaries, consistent
with the confocal analysis (Figure 2O,P).
CCN2 mutants exhibit defects in vascular remodeling
PCNA labeling and TUNEL analyses were performed to assess
whether defects in proliferation and/or survival might contribute
to the microvascular abnormalities in Ccn2 mutants. No differences
were detected in mutants in comparison to WT littermates (Figure
S2D–G). During vascular remodeling, immature vascular beds
become less dense, arterioles become smaller in diameter than
venules, and pericytes form stable associations with endothelial
tubes [34]. Angiopoetin 1 (Ang1) is required for stabilizing
endothelial-pericyte interactions and is expressed primarily by
mural cells [35]. Ang1 mRNA levels were diminished in Ccn22/2
skin (Figure S2H). No differences were detected in levels of
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expression of Tie2, the endothelial-specific receptor for Ang1 (data
not shown). However, levels of the mRNA encoding the bio-active
VEGF isoform 164 were elevated in mutants (Figure S2I).
Versican is the principal chondroitin sulfate proteoglycan in blood
vessels and exists in at least four isoforms, V0, V1, V2, and V3
[36]. Embryonic endothelial cells express more V0 than other
isoforms, and V0 expression declines during vascular maturation
[37]. No differences were seen in levels of versican V1 in Ccn2
mutants and WT littermates (Figure S2J); however, Ccn2 mutants
exhibited increased levels of V0 (Figure S2K). Therefore, the loss
of Ccn2 leads to diminished expression of vessel maturation marker
Ang1 and elevated expression of markers of immature vasculature,
indicative of a potential defect in vascular remodeling.
The vascular phenotype in Ccn2 mutants bears some resem-
blance to mice lacking platelet-derived growth factor-B (PDGF-B)
or its receptor, PDGFRb [38,39]. In particular, defective pericyte
recruitment is seen in these mice. Therefore, we examined pericyte
recruitment in Ccn2 mutants. Pericytes, which express NG2 and
desmin, become associated with small diameter vessels during
vessel maturation [40]. Consistent with the gene expression
analysis described above, confocal analysis of desmin expression
revealed incomplete coverage of microvessels by pericytes in the
dermis of Ccn2 mutants at E16.5 and E18.5 (Figure 3A–C; data
not shown). Similar results were seen for NG2 expression in the
lung liver, and brain microvasculature (Figure 3D–F, and data not
shown). Thus, the loss of CCN2 affects the microvasculature in
multiple tissues. Flow cytometric analysis of lung, liver, and brain
samples from E16.5 embryos for cells negative for the endothelial
cell marker PECAM, but expressing the pericyte markers NG2
and PDGFRb [41] revealed normal numbers of endothelial cells
and pericytes in Ccn2 mutants (Figure S3, and data not shown).
This suggests that the reduced pericyte coverage in Ccn2 mutants is
not caused by a decrease in pericyte number or migration, but
possibly by defects in the ability of pericytes to make stable
associations and elongate along endothelial cells in Ccn2 mutant
mice.
Confocal analysis of E16.5 dermal and lung microvasculature
co-stained with NG2, desmin, and PECAM supports this
possibility. NG2 staining demonstrated that pericytes associated
with WT vessels were in close contact with the capillary
endothelium and appeared elongated along the endothelial surface
(Figure 3G). In contrast, pericytes associated with capillaries in
mutants were more rounded and exhibited less elongation
(Figure 3H). Immunostaining with desmin also suggested a defect
in pericyte association with endothelial cells in mutants. In WT
capillaries, pericytes were elongated and covered the surface of
endothelial tubes (Figure 3I,J and Figure S4A,B). In contrast,
pericytes on mutant capillaries were rarely elongated, and vessel
coverage was incomplete (Figure 3K,L and Figure S4C,D). Taken
together, these findings indicate that the ability of pericytes to form
stable associations with microvascular endothelium is defective in
Ccn2 mutants.
CCN2 potentiates PDGF signaling in vascular cells
PDGF-B, produced by endothelial cells, and its receptor,
PDGFRb expressed in pericytes, are required for pericyte
recruitment to nascent vessels [38]. CCN2 was originally identified
as a protein that competes with PDGF-B for binding to NIH 3T3
cells, leading to the suggestion that CCN2 binds to PDGF
receptors [42]. However, subsequent studies using a C-terminal
isoform of CCN2 showed no interaction between CCN2 and
PDGF receptors [43]. We tested whether full-length CCN2
interacts with PDGF-B or its receptor through co-immunoprecip-
itation and found no evidence for a direct physical interaction
Figure 1. Expression of Ccn2 in developing vasculature. (A) b-galactosidase activity in Ccn2-lacZ transgenic mice reveals Ccn2 promoter
activity throughout the vasculature in E16.5 embryos. (B) Ccn2-lacZ expression in dermal microvessels at E13.5. (C–E) EGFP fluorescence in CCN2-EGFP
BAC transgenic mice demonstrates CCN2 expression in the endothelium of arterial elements (C and E), venous elements (C), and developing capillary
networks (D). Arrowheads in (C) and (E) demarcate arterial element. Arrow in (C) identifies endothelial cells of a venous element. Arrowhead in (E)
highlights EGFP expression in mural cells in the arterial element. Arrow in (E) highlights expression in endothelial cells in the arterial element. (F)
Immunofluorescence and (G,H) immunohistochemical staining with an aCCN2 antibody on paraffin sections through dermis, demonstrating CCN2
expression in endothelial cells. Arrows in (F) highlight endothelial cells in E18.5 microvasculature. Specificity of the aCCN2 antibody is demonstrated
by the absence of reactivity in the Ccn22/2 section (H). Arrows in (G) and (H) demarcate abluminal surface of the endothelium. Asterisks in (G) and
(H) identify blood cells within the vessels. aCCN2 staining in (G) shows punctate intracellular expression, presumably with the Golgi, in addition to the
surface expression marked by the arrow.
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(Figure S5, Methods S1). These findings suggest that CCN2 does
not influence PDGF signaling by interacting directly with PDGF-B
or PDGFRb.
Next, we investigated whether CCN2 could induce PDGF-B
expression in endothelial cells. Recombinant CCN2 (rCCN2)
induced PDGF-B protein expression in human umbilical vein
endothelial cells (HUVECs) at 1 and 4 hours of stimulation
(Figure 4A). This was confirmed using HUVECs transfected with
a CCN2-GFP adenovirus (adCCN2GFP). AdCCN2GFP-trans-
fected cells induced PDGF-B protein expression at all time points
tested, and the level of PDGF-B induction correlated with levels of
CCN2 expression (Figure 4B). Given that CCN2 induces PDGF-B
expression in endothelial cells, the potential effects of CCN2 on
PDGF signaling pathways in mural cells, which express PDGFRb,
were investigated. CCN2 on its own did not activate Stat3,
ERK1/2, or AKT, whereas PDGF activated all of these pathways.
Furthermore, CCN2 had no effect on PDGF-B-induced ERK1/2
or Stat3 activation, but Akt activation was elevated and prolonged
upon treatment with PDGF and CCN2 (Figure 4C). Thus CCN2
can potentiate PDGF signaling between endothelial cells and
mural cells.
Components of the endothelial basement membrane are
compromised in Ccn2 mutants
Decreased expression of PDGF-B and reduced PDGF signaling
are unlikely to be the entire basis for the Ccn2 mutant phenotype
because endothelial-specific loss of PDGF-B is compatible with
survival, and mice having as much as a 90% decrease in pericyte
number survive as adults [44]. The basement membrane is
essential for coordinating key signaling events that stabilize the
vasculature during angiogenesis [45]. The expression of fibronec-
tin (FN) by endothelial cells is an early event in vascular basement
membrane formation [46]. The provisional fibronectin matrix
provides organizational signals to endothelial cells, and establishes
a framework for the incorporation of permanent basement
membrane components such as collagen type IV [29,46,47].
Defects in basement membrane formation lead to severe defects in
angiogenesis [48–51]. Because overexpression of CCN2 leads to
Figure 2. Vascular abnormalities in Ccn2 mutant embryos. (A) E18.5 WT and (B) Ccn22/2 littermate, showing vessel dilation throughout the
mutant embryo. (C, D) H&E-stained paraffin sections through the lumbar dorsal dermis of (C) E18.5 WT and (D) Ccn22/2 littermate. Arrowheads point
to vessels. Bars highlight the enlarged distance between the hypodermal and epidermal layers in the mutant, indicative of local edema. (E,F)
Hematoxylin and eosin-stained sections through E16.5 WT (E) and Ccn22/2 (F) descending aorta at thoracic level. Smooth muscle cells in the tunica
media are spindle-shaped and arranged in layers in the WT embryo, but are more cuboidal and disorganized in the Ccn22/2 littermate. (G,H) Higher
magnification images through aorta at lumbar level in E16.5 (G) WT and (H) Ccn22/2 littermate showing spindle-shaped smooth muscle cells
(arrowheads) in WT that have a cuboidal shape in the mutant. (I,J) Confocal images of PECAM-stained dorsal dermal vasculature in (I) WT and (J)
Ccn22/2 littermates. Arrows demarcate arterial elements; arrowheads demarcate venous elements; asterisks identify capillary beds. (K,L) Higher
magnification confocal images of (K) WT and (L) Ccn22/2 dorsal dermal capillary beds, showing increased capillary density in the mutant. (M,N) High
magnification confocal image of (M) WT and (N) Ccn22/2 dorsal dermal capillaries, showing numerous abluminal protrusions (arrows in (N)) on the
mutant capillary. (O,P) Electron micrographs of newborn (P0) (O) WT and (P) Ccn22/2 dermal capillaries, showing abluminal and luminal (arrows in
(P)) protrusions.
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thickening of glomerular and retinal capillary basement mem-
branes in diabetic mice [52,53], we investigated whether CCN2 is
required for the formation of endothelial basement membranes
during development.
Electron microscopy provided evidence for defects in micro-
vascular endothelial basement membrane assembly in Ccn2
mutants. In WT microvessels, the interstitial matrix was compact
and localized near the surface of the plasma membrane
(Figure 5A). It was more diffuse in mutants (Figure 5B). Therefore,
expression of FN and Col4a2 was investigated through confocal
analysis. FN expression and association with vessels is significantly
decreased in E16.5 Ccn2 mutant skin and lung vasculature
(Figure 5C–F, and data not shown). Collagen type IV expression
was also diminished and discontinuous in vascular basement
Figure 3. Defective endothelial-pericyte interactions in Ccn2 mutants. (A, B) Co-immunofluorescence staining for desmin and PECAM in
E18.5 dermis from (A) WT and (B) Ccn22/2 mice analyzed by confocal microscopy. (C) Quantification of vessel coverage by pericytes in E18.5 dermis;
asterisk, p,0.05. (D, E) Co-immunofluorescence staining for NG2 and PECAM in E16.5 lung from (D) WT and (E) Ccn22/2 mice analyzed by confocal
microscopy. (F) Quantification of vessel coverage by pericytes in E16.5 lung; asterisk, p,0.05. (G,H) Confocal analysis of NG2 and PECAM
immunostaining in (G) WT and (H) Ccn22/2 E16.5 dermis. Pericytes are elongated around the microvessel in (G), whereas in mutants (H), pericytes
(arrows) are associated with the endothelium, but are rounder, and fewer of them have elongated along the endothelial surface. (I–L) Confocal
sections through E16.5 dermis analyzed for desmin (green) and PECAM (red) immunofluorescence. (I,J) WT desmin positive pericytes appear
elongated and cover most of the surface of the microvessels. (K.L) Ccn22/2 desmin-positive pericytes have a rounder appearance and show less
extensive coverage of the surface of the endothelium.
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membranes in mutants (Figure 5G–J). Western blot analysis of Ad-
CCN2GFP-transfected cells demonstrated that CCN2 induced
expression of FN in HUVECs compared to empty vector-
transfected controls (Figures 5K and S6). CCN2 had no apparent
effect on Col4a2 expression (Figures 5K and S6).
Discussion
Endothelial cells proliferate and migrate toward the sources of
angiogenic signals during development. Upon removal of the
angiogenic trigger, a switch to a maturation phase occurs,
involving cessation of cell proliferation and migration, followed
by the recruitment of mural cells to the vessels, and deposition of
the basement membrane. Although the importance of the
basement membrane in vascular maturation is widely accepted,
the roles of specific ECM components have been difficult to
ascertain, especially in vivo [45]. Here we show that the
matricellular protein CCN2 is a crucial regulator of vascular
remodeling.
The results reported here suggest that CCN2 is required for
pericyte recruitment in part by potentiating PDGF signaling. We
have shown that CCN2 induces expression of PDGF-B in
endothelial cells. In turn, CCN2 is induced in pericytes in
response to serum or TGFb [54]. Thus, PDGF and CCN2 appear
to be components of a positive feedback loop that operates
between endothelial cells and pericytes.
In addition to regulating levels of PDGF-B expression, CCN2
potentiates Akt activation by PDGF-B in vSMCs. Our findings
extend previous studies [42] that indicate CCN2 does not interact
directly with PDGF-B or PDGFRb in vascular cells. Thus, CCN2
most likely potentiates the ability of PDGF-B to activate PDGFRb
in mural cells through indirect mechanisms. One of the most
plausible of these involves interactions between CCN2 and
integrin avb3. This integrin is expressed in endothelial cells and
pericytes [55,56]. CCN2 binds to integrin avb3 to promote
endothelial cell migration and proliferation [9]. Moreover, avb3
associates with and potentiates signaling through PDGFRb [55].
Although our in vivo studies cannot address the physiological
consequences of altered Akt signaling to the Ccn22/2 vascular
phenotype, the Ccn22/2 phenotype is consistent with the
possibility that reduced activation of Akt makes a contribution;
Akt12/2 vasculature is characterized by an incomplete basement
membrane [57].
As discussed above, reduced PDGF signaling alone cannot
explain the severity of the Ccn22/2 endothelial phenotype.
Rather, the data indicate an essential role for CCN2 in formation
of the vascular provisional ECM and basement membrane. The
relationship between CCN2 and FN expression and function is
likely to be complex. CCN2 binds to FN and FN receptors
(integrins a4, a5 and b1) [12,58,59]. Moreover, loss of CCN2
leads to defective adhesion and spreading of cells on FN,
suggesting that these physical interactions are essential for certain
cell types, at least in vitro [28,59]. Other studies have shown that
CCN2 is required for FN protein and mRNA expression in
pathological processes in vivo [60,61]. Studies employing siRNA
knockdown approaches demonstrate that CCN2 induces FN
expression in various cell types [25,62]. The studies reported here
show that CCN2 induces FN expression in endothelial cells, and
that CCN2 is required for normal levels of FN expression during
development in vivo. While we have focused here on the role of
CCN2 as a mediator of FN production by vascular cells, decreased
FN synthesis was also seen in fibroblasts in Ccn22/2 dermis
(Figure 5E,F). These data are consistent with previous studies
showing that CCN2 is required for FN synthesis in fibroblasts in
vitro [61]. Additional studies employing tissue-specific CCN2
knockouts will be required to determine whether the defect in
FN synthesis in dermal fibroblasts has physiological consequences.
The reduced deposition of collagen IV in Ccn2 mutants reveals
that CCN2 is an essential regulator of vascular basement
membrane formation. The underlying mechanisms by which
Figure 4. CCN2 potentiates PDGF-B signaling. (A) rCCN2 induces
PDGF-B expression in HUVEC cells. Right panel, representative Western
blot. Left panel, Quantification of relative expression levels of PDGF-B in
cells treated with or without rCCN2 from three separate experiments. *,
p,0.02. (B) Adenovirally expressed CCN2 induces PDGF-B expression in
HUVECs compared to transfection with an empty adenoviral control.
The extent of PDGF-B induction correlated with levels of CCN2
expression. As reported previously, a higher molecular weight isoform
of CCN2, presumably a result of post-translational modification [1], is
detected 4 and 8 hours post-infection. Relative level of PDGF-B
expression was assessed using ImageJ software. The experiment was
repeated three times, with similar results each time. The induction of
PDGF-B in the presence of CCN2 was statistically significant for each
time point; p,0.05. A representative Western blot is shown. (C) Effects
of rPDGF-B, and/or pcDNA3-CCN2-HA expression on activation of PDGF
pathways in MOVAS cells. PDGF-B stimulated activation of Stat3, ERK,
and Akt, whereas CCN2-HA on its own had no effect. However,
combined treatment with PDGF-B and CCN2-HA led to prolonged Akt
activation (arrows). Relative levels of pAKT expression were assessed
using ImageJ software. All experiments were performed in triplicate and
repeated three times, with similar results each time. The increase in
pAKT levels in the presence of CCN2 was statistically significant at each
time point; p,0.05. A representative Western blot is shown.
doi:10.1371/journal.pone.0030562.g004
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CCN2 mediates basement membrane formation are unknown.
Our studies indicate that CCN2 does not directly regulate levels of
expression of Col4a2. Therefore, the loss of collagen IV expression
in vascular basement membranes may be a secondary conse-
quence of altered FN synthesis and folding. As discussed above,
CCN2 directly interacts with FN and its receptors. Increased
expression of matrix metalloproteinases (MMPs) that target type
IV collagen might also contribute to reduced type IV collagen
deposition in endothelial basement membranes. Additional in vivo
studies will be required to evaluate these possibilities. A growing
body of literature implicates CCN2 in abnormal basement
membrane thickening in pathological processes. Glomerular
basement membrane thickening is prevented in diabetic Ccn2+/
2 mice compared to WT littermates [52]. Moreover, one of the
most prominent features in transgenic mice overexpressing CCN2
from the type I collagen promoter is a thickening of endothelial
basement membranes [63]. Taken together with the data reported
here, CCN2 appears to be a critical mediator of basement
membrane formation. CCN2 is required for normal elaboration of
the basement membrane during developmental angiogenesis, but
CCN2 overexpression leads to basement membrane thickening in
multiple fibrotic processes.
The formation of mature endothelial basement membranes
involves both pericytes and endothelial cells. While we have
focused here on effects of CCN2 in endothelial cells in vivo, it is
very conceivable that primary defects in both endothelial cells and
pericytes in Ccn22/2 mice contribute to the basement membrane
defects seen in these mutants. It is likely that CCN2 has direct
effects on ECM production in pericytes, as CCN2 promotes ECM
production and fibroblast activation in vitro [64]. Moreover, our
preliminary analysis reveals that in addition to the microvascula-
ture, large vessels are impacted by loss of CCN2. This finding
Figure 5. Endothelial basement membrane defects in Ccn2 mutants. Electron microcopic images of endothelial basement membranes in
dermal capillaries of E16.5 (A) WT and (B) Ccn22/2 littermates. Arrows demarcate the plasma membrane (bottom arrow) and top of the interstitial
matrix (top arrow). (C,D) Confocal images of dermis of E16.5 WT (C) and Ccn22/2 (D) mice analyzed by immunofluorescence for fibronectin (FN) and
PECAM. Arrows identify an arteriole. The arteriole in (C) is surrounded by several layers of FN. The arteriole in (D) is incompletely invested with FN.
(E,F), Lower magnification confocal images through (E) WT and (F) Ccn22/2 E16.5 dermis, illustrating less fibronectin throughout the dermis in
mutants. (G,H) Confocal images of dermis of E16.5 (G) WT and (H) Ccn22/2 mice analyzed by immunofluorescence for ColIV (Col4a2) and PECAM.
Arrows identify an arteriole. ColIV coverage of the mutant vasculature is incomplete. (I,J) Confocal images of lungs of E16.5 (I) WT and (J) Ccn22/2
mice analyzed by immunofluorescence for ColIV and PECAM. Most of the vascular elements in the WT lung are surrounded by ColIV. Coverage is
incomplete in the Ccn2 mutant lung. Arrows in (J) identify vessels lacking coverage by ColIV. (K) CCN2 induces expression of FN and ColIV in HUVECS.
HUVECs were infected with Ad-CCN2-GFP or Ad-control. Lysates were collected at the indicated time points post-infection. Levels of FN are elevated
8 hours after infection, concomitant with accumulation of CCN2. There appeared to be an increase in FN levels at 12 hours in the presence of CCN2 in
the blot shown, but this was not seen in every experiment and the result did not reach statistical significance at this time point. Similarly, there was a
trend towards increased expression of Col IV at 12 hr, but this increase did not reach statistical significance (p=0.065). The experiment was repeated
three times. A representative blot is shown. Quantification of levels of FN and Col IV are shown in Figure S6.
doi:10.1371/journal.pone.0030562.g005
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raises the possibility that CCN2 plays a direct role in SMCs in
addition to pericytes. It is noteworthy that the related matricellular
protein CCN1 (Cyr61) is expressed in major vessels, and Ccn12/
2 mice die early in embryogenesis as a result of defects in large
vessel integrity [65]. Although vascular basement membranes have
not been investigated in Ccn12/2 mice, the defects in vessel
integrity raise the possibility that CCN1 and CCN2 will exhibit
functional redundancy in vascular elements. It will thus be of
interest in future studies to investigate vascular cell recruitment
and basement membrane assembly in Ccn1 and Ccn1/Ccn2
mutants.
Finally, the use of tissue-specific Ccn2 knockouts and co-culture
experiments will be required to understand the physiological
relevance of CCN2 produced by endothelial and mural cells in
large vessels.
Methods
Ethics Statement
All the experiments related to mice were performed in
accordance with National Institutes of Health guidelines for care
and use of animals, and also approved by the UCLA Institutional
Animal Care and Use Committee (IACUC), protocol #95-018.
Transgenic Mice
Ccn22/2 mice.
described previously [27]. As previously described, Ccn2+/2 mice
appear indistinguishable from WT littermates, and are viable and
fertile [27]. Ccn22/2 embryos and neonates were obtained by
intercrossing Ccn2+/2 mice. The 4 kb proximal promoter LacZ
mice were generated and genotyped as previously described [31].
CCN2-eGFP mice were ordered from the Mutant Mouse
Resource Center (MMRC, UC Davis) [32]. All mice were
treated andeuthanizedin
Institutional Animal Care and Use Committee (ARC # 1995-
018-52A), and the Association of Assessment and Accreditation of
Laboratory Animal Care International (AAALAC) guidelines.
Histochemical and Immunofluorescent Staining.
isolated embryos were fixed and embedded in paraffin wax as
described previously [27]. 5 mm sections were stained with
hematoxylin and eosin using standard protocols. LacZ staining
was performed as described [66]. Immunofluorescence was
performed as described previously [27]. Briefly, paraffin, sections
were boiled for 15 min in citrate buffer. Sections were blocked
with 5% goat or donkey serum for 1 hour and incubated with
primary antibody overnight at 4uC, followed by incubation with
secondary antibody for 1 hour at room temperature, then with
fluorophore for 30 minutes at room temperature. The following
antibodieswereused:PECAM
Biosciences), CCN2 (1:500; L-20 Santa Cruz Biotechnology),
NG2 (1:100; Abcam), Collagen IV (1:500; Abcam and Santa Cruz
Biotech), Desmin (1:1000; Abcam), anti-Smooth Muscle Actin-
FITC (1:500; Sigma), Col4a2 (1:1,000; Abcam) and Fibronectin
(1:1,000; Santa Cruz Biotech). Secondary antibodies were
conjugated with Alexa-Fluor-555
(Invitrogen).Sectionswere
(Vectashield). Immunofluoresence was visualized on a Leica
TCS-SP Confocal Microscope. For TUNEL staining, the
fluorescein In Situ Cell Death Detection Kit (Roche) was used
according to manufacturer’s protocol. PCNA staining was
performed on paraffin sections as described previously [27] using
an anti-PCNA antibody (Zymed) and, vessels were identified by
PECAM immunofluorescence. The percentage of TUNEL- or
PCNA-positive endothelial cells (PECAM-positive) was quantified
The generation of Ccn22/2 mice was
accordance withtheUCLA
Freshly
(1:500;MEC13.3, BD
andAlexa-Fluor-488
with counterstainedDAPI
on digital photomicrographs processed with Photoshop software
(Adobe), usingImage-Pro software.
microvasculature was quantified as described [67]. Capillary
density was quantified as the area of PECAM1-positive cells on
anti-PECAM1 immunostained images as described [68]. Ten
images each for WT and Ccn22/2 mice, obtained from 5
independent pairs of littermates, were analyzed. Statistical analysis
was performed using Student’s t test. A p value of less than 0.05
was considered statistically significant.
Confocal Microscopy.
Confocal laser scanning microscopy
was performed at the CNSI Advanced Light Microscopy/
Spectroscopy Shared Resource Facility at UCLA, supported
with funding from NIH-NCRR grant (CJX1-443835-WS-29646)
and NSF grant (CHE-0722519). Representative images are shown.
Real-time quantitative polymerase chain reaction.
was isolated using TRIZOL (Invitrogen) according to the
manufacturer’s protocol. Synthesis of cDNA was performed with
Superscript III(Invitrogen).
performed with 20 ng reverse-transcribed RNA. Amplifications
were performed for 30 cycles, followed by a 5 min extension at
72uC. Reaction products were gel electrophoreses and quantified
using Image Quant software (Molecular Dynamics). Primers for
the genes investigated by semi-quantitative RT-PCR were: VegfA
and C: VEGFACF 59-GAA GTC CCA TGA AGT GAT CAA G-
39, VEGF164 59-CAA GGC TCA CAG TGA TTT TCT GGC-
39; ANG1: ANG1F 59-CAT TCT TCG CTG CCA TTC TG,
ANGR 59-GCA CAT TGC CCA TGT TGA ATC-39; PECAM:
PECAMF 59- GAG CCC AAT CAC GTT TCA GTT T-39,
PECAMR 59-TCC TTC CTG CTT CTT GCT AGC T-39;
Versican0: V0F 59-TTC ACA GAA CGC CAC CCT TGA GTC
C-39, V0R 59-CTA GCT TCT GCA GCT GGC CGG GTC C-
39; Versican1-3: V1F 59- GCA GCT TGG AGA AAT GGC TTT
GAC C-39, V1R 59- CGA GTA GTT GTG GGT GAT TCC
GTG G-39; PDGFBF 59-GATCCGCTCCTTTGATGATC-39,
PDGF-BR 59-GTCTCACACTTGCATGCCAG-39; PDGFRbetaF
59-AATGTCTCCAGCACCTTCGT-39, PDGFRbetaR 59-AGC-
GGATGTGGTAAGGCATA-39 [69]; GAPDH, GapdhF 59-GCA
GTG GCA AAG TGG AGA TT-39; GapdhR 59-AGT GGA TGC
AGG GAT GAT GT. cDNA was amplified using Sybr Green I PCR
Master Mix (Applied Biosystems). Amplicons were generated and
analyzed with the ABI 7000 Real-time PCR system (Applied
Biosystems). Data were normalized to the levels of Gapdh. Triplicate
assays were run and analyses were repeated three times. Specificity
was tested by measurement of Tm-values and by gel electrophoresis
of the amplicons. Data are represented as the means of relative levels
of expression+the S.E. of the mean, and statistical analysis was
performed with Student’s t test. A p value of less than 0.05 was
considered statistically significant.
Flow Cytometry.
FACS
previously described [70]. Brain, liver and lung samples were
harvested from E16.5 CCN2 wild type and mutant embryos.
Single cell suspensions were created by serial syringe digestion in
0.2% Collagenase (Sigma Clostridium histolyticum C2674-6), 0.05%
Dispase (Invitrogen 17105-041), 0.0075% DnaseI (Sigma D4513),
0.02% Penicillin Streptomycin (GIBCO-Invitrogen 15140148) in
16 PBS/10%Fetal Bovine Serum (GIBCO-Invitrogen 10437-
028). Cell suspensions were incubated with the following primary
antibodies: CD45-APC Cy7 (1:200;Abcam); NG2 (1:200; Abcam);
CD31-PE (1:200; Abcam); PDGFRb-APC (1:50; Invitrogen). A
secondary goat anti-rabbit conjugated antibody 488 (Invitrogen)
was used for the unconjugated NG2 antibody. FITC, APC, APC-
Cy7, PE control beads (Invitrogen) and 488 secondary alone were
used as controls to correct for background florescence and gate
parameters. FACS sorting was performed using the LSRII FACS
Pericytecoverage of
RNA
Semi-quantitativePCRwas
analysis wasperformed as
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analyzer and cell counts were plotting by FlowJo analysis
(TreeStar).
Transmission Electron
analysis was performed on dermal microvasculature by the
University of California, Los Angeles, Electron Microscopy Core
Facility. 10 images were taken from each E18.5 embryo. Four
Ccn22/2
andfour WT
Representative images are shown.
Celllinesandtreatments.
endothelial cells (HUVECs a gift from Dr. Jau-Nian Chen) were
maintained in HUVEC culture media (Sigma) as described
previously [71]. HUVECs were maintained in 0.5% serum for
12 hr prior to treatment with recombinant protein. Cells were
treated with 150 ng/ml recombinant (r) CCN2 (Peprotech) and/
or 150 ng/ml rPDGF-B (Peprotech), using serum free treated cells
as control. Mouse vascular smooth muscle (MOVAS) (ATTC) cells
were cultured in DMEM, 10% FBS. MOVAS cells were washed
with Hepes buffered saline (HBS) containing 5 mM MgCl2
(HBS+Mg), and treated with or without 150 ng/ml rPDGF-B in
DMEM, 0.5% FBS for the indicated times. In other experiments,
MOVAS cells were transiently transfected with pcDNA3-CCN2-
HA [72] using Lipofectamine (Invitrogen), and treated with
150 ng/ml rPDGF-B 24 hrs later for the indicated time periods.
Each experiment was performed in triplicate and repeated at least
twice. HUVECswere also
adenovirus and adenoviral control vectors at a multiplicity of
infection (MOI) of 200 (a kind gift of Dr. Fayez Safadi).
Western blot analysis.
Cells were lysed with RIPA buffer
with 16 protease (Complete Mini Roche) and 16 phosphatase
inhibitors (Cocktail 2, Sigma). Lysates were separated by 6–12%
SDS-PAGE and transferred to nitrocellulose membrane (0.45 um;
BioRad). Membranes were incubated with antibodies against
CCN2 (L-20; 1:2,000, Santa Cruz Biotechnology), PDGF-B
(1:2000, Cell Signaling), PDGFR b (1:2,000 Cell Signaling),
STAT3 (1:1,000,Cell Signaling),
Signaling), total AKT (1:2,000, Cell Signaling), phospho-AKT
(1:2000, CellSignaling),phospho-ERK1/2
Signaling), Collagen type IV (1:2,000; Abcam), Fibronectin
(1:2,000; Santa Cruz Biotech) and actin (1:5,000, Sigma).
Antibody-antigen complexes were detected with HRP-conjugated
secondary goat and rabbit antibodies (Bio-Rad). Western blots were
performed in triplicate and normalized to actin. Quantification was
performed using ImageJ. Statistical analysis was performed using
the Student’s t-Test, and a p-value less than 0.05 was considered
significant. Representative western blots are shown.
Microscopy.
Ultrastructural
littermates wereexamined.
Human umbilicalvein
transfected withCCN2-GFP
pSTAT3(1:2,000,Cell
(1:2,000,Cell
Supporting Information
Methods S1
blot analysis (Figure S5).
(DOCX)
Methods for co-immunoprecipitation and western
Figure S1
vascular defects in Ccn2 mutants. (A) Confocal image of
dermal microvasculature immunostained for CCN2 (green) and
PECAM (red). Yellow indicates co-expression in endothelial cells.
The staining is punctate, as reported previously [30]. Associated
mural cells expressing CCN2 (green) are indicated by arrows.
Endothelium demonstrating CCN2 expression is indicated by
arrowheads. (B,C) Confocal images of fetal placenta from E16.5
WT (B) and Ccn22/2 (C) littermates immunostained for NG2
(green) and PECAM (red) and counterstained with DAPI showing
no obvious changes in vascular organization. (D) E14.5 WT and
(E) Ccn22/2 littermate. Arrows highlight dilation of cerebral
vessels in the mutant. Dilated vessels are apparent in the mutant.
Expression of CCN2 in vasculature and
(F–I) Confocal images of immunofluorescence staining for aSMA
(green) and PECAM (red) in dorsal dermis of newborn (P0) WT
(F,H,) and Ccn22/2 (G,I,) littermates. Arrows in (F–I) indicate
arteries; arrowheads demarcate veins. (J,K) Confocal images of
immunofluorescence staining for aSMA (green) and PECAM (red)
in dorsal dermis of newborn (P0) WT (J) and Ccn22/2 (K)
littermates showing paired arterioles (arrows) and venules
(arrowheads). (L,M) Confocal images of immunofluorescence
staining for EphB4 (green) and PECAM (red) of E16.5 WT (L)
and Ccn22/2 littermate (M) dorsal dermal microvasculature.
(TIF)
Figure S2
Quantification of microvessel density. (B,C) Additional representative
confocal images of PECAM-immunostained dorsal dermal microvas-
culature from WT (B) and Ccn22/2 (C) E18.5 littermates showing
increased vessel density in mutants. (D) Representative image of
paraffin section through E16.5 dorsal dermis analyzed by aPECAM
and aPCNA co-immunofluorescence and counterstained with DAPI,
used to assess endothelial cell proliferation. Image from WT dermis is
shown. Arrows point to PCNA-positive endothelial cells. (E)
Quantification of PCNA-positive cells revealed no differences in
proliferation in WT versus mutant vessels. (F) Representative images
of paraffin section through E16.5 dorsal dermis analyzed by
immunostaining for PECAM and TUNEL-positive endothelial cells
and counterstained with DAPI. Image from WT dermis is shown. (G)
Quantification of TUNEL-positive endothelial cells revealed no
evidence for altered levels of cell death in Ccn2 mutant vasculature.
(H–K) Quantitative RT-PCR analysis of relative levels of expression
of (H) Ang1, (I) Vegf164, (J) Versican1, and (K) Versican0 mRNA in WT
and Ccn22/2 E16.5 vasculature. *, p,0.05.
(TIF)
Altered gene expression in Ccn2 mutants. (A)
Figure S3
number in Ccn2 mutants. (A, C) FACS analysis of (A) WT and
(C) Ccn22/2 skin samples analyzed for expression of PDGFRb.
(B, D) FACS analysis of (B) WT and (D) Ccn22/2 skin samples
analyzed for expression of NG2. (E) Quantification of percentages
of PDGFRb, NG2, and PECAM-expressing cells revealed no
differences.
(TIF)
FACS analysis of pericyte or endothelial cell
Figure S4
lium in Ccn2 mutants. Paraffin sections through E16.5 dermis
immunostained with desmin (red) and counterstained with DAPI.
(A,B) WT desmin positive pericytes appear elongated and cover
most of the surface of the microvessels. (C,D) Ccn22/2 desmin-
positive pericytes have a rounder appearance and desmin staining
has a less uniform appearance.
(TIF)
Defective pericyte association with endothe-
Figure S5
PDGF-B or PDGFRb. (A) No physical interactions between
CCN2 and PDGF-B. MOVAS cells were infected with a lentiviral
vector encoding CCN-HA (M-CCN2 cells). Non-crosslinked or
DSP-crosslinked lystaes (see Supplementary Materials and Meth-
ods) were immunoprecipitated with aHA antibody. Western blots
of the immunoprecipitates were probed with aCCN2 and
aPDGFB antibodies. First lane in each panel shows rCCN2 and
rPDGFB standards. TXsol and TX insol, triton X-soluble and –
insoluble pellets, respectively. (B) No direct interactions between
CCN2 and PDGFRb. M-CCN2 cells were treated with or without
PDGF-B, followed by immunoprecipitation with aHA antibody.
Western blots of the immunoprecipitates were probed with
aPDGFRb (PDGFR) or aphospho (Y751) PDGFRb antibody.
(TIF)
No physical interaction between CCN2 and
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Figure S6
endothelial cells. Quantification of relative levels of expression
of fibronectin (FN) and Col IV in endothelial cells in the presence
or absence of CCN2. See legend to Figure 5 for experimental
details. Induction of FN was seen by 8 hrs. There was a trend
towards increased FN at 12 hrs (p,0.06), but this did not reach
statistical significance. *, p,0.05. There was no significant
increase in Col IV levels at any time point.
(TIF)
CCN2 induces fibronectin expression in
Acknowledgments
We thank Bonnie Lee and Kevin Mouillesseaux for technical assistance.
Author Contributions
Conceived and designed the experiments: KML MLI-A HM. Performed
the experiments: FH-G RAD B-LH BvH JJH TTC JRO AC PDB.
Analyzed the data: FH-G MLI-A HM PDB KML. Contributed reagents/
materials/analysis tools: PDB HM MLI-A. Wrote the paper: FH-G KML.
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