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Soft connective tissue calcification is not a passive process, but the consequence of metabolic changes of local mesenchymal cells that, depending on both genetic and environmental factors, alter the balance between pro- and anti-calcifying pathways. While the role of smooth muscle cells and pericytes in ectopic calcifications has been widely investigated, the involvement of fibroblasts is still elusive. Fibroblasts isolated from the dermis of pseudoxanthoma elasticum (PXE) patients and of patients exhibiting PXE-like clinical and histopathological findings offer an attractive model to investigate the mechanisms leading to the precipitation of mineral deposits within elastic fibers and to explore the influence of the genetic background and of the extracellular environment on fibroblast-associated calcifications, thus improving the knowledge on the role of mesenchymal cells on pathologic mineralization.
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“fgene-04-00022” 2013/3/12 13:07 page1—#1
published: 05 March 2013
doi: 10.3389/fgene.2013.00022
Fibroblast involvement in soft connective tissue
Ivonne Ronchetti
, Federica Boraldi
, Giulia Annovi
, Paolo Cianciulli
and Daniela Quaglino
PXELab, University of Modena and Reggio Emilia, Modena, Italy
Department of Life Science, University of Modena and Reggio Emilia, Modena, Italy
U.O.D.S Unit, S. Eugenio Hospital, Rome, Italy
Edited by:
Olivier M. Vanakker, Ghent University
Hospital, Belgium
Reviewed by:
Fan Zhang, Mount Sinai School of
Medicine, USA
Noriko Hiroi, Keio University, Japan
Daniela Quaglino, Department of Life
Science, University of Modena and
Reggio Emilia, Via Campi 287,
Modena 41125, Italy.
Soft connective tissue calcification is not a passive process, but the consequence of
metabolic changes of local mesenchymal cells that, depending on both genetic and
environmental factors, alter the balance between pro- and anti-calcifying pathways.
While the role of smooth muscle cells and pericytes in ectopic calcifications has been
widely investigated, the involvement of fibroblasts is still elusive. Fibroblasts isolated
from the dermis of pseudoxanthoma elasticum (PXE) patients and of patients exhibiting
PXE-like clinical and histopathological findings offer an attractive model to investigate the
mechanisms leading to the precipitation of mineral deposits within elastic fibers and to
explore the influence of the genetic background and of the extracellular environment
on fibroblast-associated calcifications, thus improving the knowledge on the role of
mesenchymal cells on pathologic mineralization.
Keywords: fibroblasts, PXE, PXE-like disorders, elastin, extracellular matrix, ectopic calcification, mesenchymal
stromal cells
For long time, unwanted calcification, as that occurring in arte-
rial calcification and in nephrolithiasis, has been considered as a
passive, physical–chemical phenomenon representing a degener-
ative, irreversible process often associated with aging (Shroff and
Shanahan, 2007). Many recent investigations, however, pointed
out that calcium and phosphate precipitation are the result of
complex and highly regulated series of events in which the balance
between calcification inducers and inhibitor y mechanisms may
become severely deranged locally and/or systemically.
The deposition of calcium and phosphate in soft connective tis-
sues can be classified into three major categories: metastatic calci-
fication, dystrophic calcification, and calcinosis (Black and Kanat,
1985). Metastatic calcification occurs when calcium–phosphorous
levels are elevated mainly due to metabolic/hormonal alterations
and/or to tumor-associated complications. Dystrophic calcifica-
tion takes place in the presence of damaged or necrotic tissue as
in atherosclerosis. Calcinosis is gener ally associated to hypovas-
cularity or hypoxia, it may involve a localized area or it may be
widespread, causing secondary muscle atrophy, joint contractures
and skin ulceration, with recurrent episodes of inflammation or
infection (Boulman et al., 2005).
In most cases, mineral deposition develops in the extracel-
lular environment without being localized on specific matrix
components/structures. A typical example is represented by cal-
ciphylaxis, a rare disease in which a generalized calcification is
associated with thrombotic cutaneous ischemia and necrosis, thus
causing a mortality rate ranging from 60 to 80% due to wound
infection, sepsis, and subsequent organ failure (Arseculeratne
et al., 2006; Hoff and Homey, 2011).
As clearly shown by several experimental findings and clin-
ical observations, calcification may also occur in a number
of genetic diseases, in metabolic disorders, such as uremia,
hyper-parathyroidism, and diabetes, or in areas without adja-
cent inflammation or atherosclerosis. Due to the heterogeneity
of factors contributing to the development of calcifications, many
studies have been carried out in order to find common patho-
genetic mechanisms and to identify possible druggable targets
(i.e., single molecules and/or signaling pathways). Within this
framework, numerous proteins have been identified to be involved
in bone calcification as well as in ectopic mineralization. It
has been suggested that an active and dynamic balance of pro-
and anti-calcifying mechanisms occurs in both physiological and
pathological calcification (Abedin et al., 2004) and that mesenchy-
mal cells are key players, not only because they synthesize most
of the mineral regulatory proteins, but also because they are
responsible for the qualitative and quantitative characteristics of
the extracellular environment, where apatite ectopic deposition
The role of calcitropic hormones, namely catecholamines,
parathyroid hormone (PTH), and vitamin D or 1,25(OH)
calcium metabolism is well-known (Rizzoli and Bonjour, 1998).
However, in the last decade, a growing number of evidence is
highlighting the importance of many other molecules as part
of a composite network that, on the basis of common struc-
tural components, exhibits peculiar interactions and/or undergoes
different regulatory mechanisms depending on the tissue [e.g.,
osteoprotegerin (OPG) or matr ix Gla protein (MGP) in bone and
vascular tissue; Kornak, 2011] and on the environmental con-
text. In addition, these molecules can be produced and locally
secreted by mesenchymal cells, or can diffuse from circulation to March 2013
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Ronchetti et al. Fibroblasts in ectopic calcifications
peripheral tissues, where they may exert different effects on local
calcium/phosphate homeostasis (Figure 1).
The mechanisms of calcification in skeletal and dental tissues
have been under investigation since long time. One common fea-
ture to almost all physiological mineralization mechanisms seems
the involvement of small (20–200 nm) membrane particles, called
matrix vesicles (MVs). They bud off from the plasma membrane
of mineralizing cells and are released into the pre-mineralized
organic matrix serving as a vehicle for the concentration of ion
or ion-enriched substrates, which are required for the activity
of membrane-bound enzymes triggering mineral deposition at
specific sites.
The observation that MV-like membranes are present in a num-
ber of ectopic calcification processes supports the concept that the
mechanisms of vascular calcification are similar to those seen in
normal skeletal development (Golub, 2011).
However, soft connective tissue calcifications activate a num-
ber of common pathways, but, at the same time they may exhibit
local specific variations (e.g., in different tissue/body regions),
possibly depending on the genotypic/phenotypic peculiarities of
each mesenchymal cell type/subtype. Mineralization of dermal
constituents, for instance, has been never associated with MVs,
indicating that fibroblasts, differently from smooth muscle cells,
can be responsible for mineral deposition, even in the absence of
MVs. It could be, therefore, hypothesized that the role of mes-
enchymal cells in ectopic calcification may differ depending on
the ability of the cell type to acquire a bone-oriented phenotype.
To further increase the complexity and the heterogeneity of
mechanisms regulating pathologic calcification there are stud-
ies demonstrating that factors promoting or inhibiting ectopic
calcifications are under the control of different genes, as in
the case of extracellular pyrophosphate (PPi), a small molecule
made of two phosphate ions, linked by an ester bond, that reg-
ulates cell differentiation and serves as an essential physiologic
inhibitor of calcification by negatively interfering with crystal
growth (Terkeltaub, 2001). The amount of extracellular PPi is reg-
ulated by two different gene products, as it originates either from
the breakdown of nucleotide triphosphates by the ectonucleotide
pyrophosphatase/phosphodiesterase (PC-1/ENPP1) or from the
PPi t ransport by the transmembrane ankylosis protein homolog
(ANKH). Consistently, either mutations or knockdown of these
genes can induce hyper-mineralization of aorta (i.e., generalized
Major factors involved in mineral deposition. AMP,
adesosine monophosphate; ANKH, ankylosis protein homolog; ATP,
adenosine triphosphate; BMP2, bone morphogenetic protein-2; BMP2R,
bone morphogenetic protein-2 receptor; BSP, bone sialoprotein; Ca,
calcium; ENPP1, ectonucleotide pyrophosphatase/phosphodiesterase;
Glu- and Gla-MGP, uncarboxylated- and carboxylated-matrix Gla protein;
OPG, osteoprotegerin; OPN, osteopontin; Pi, inorganic phosphate; Pit-1,
phosphate transporter-1; PPi, pyrophosphate; RANKL, receptor activator of
nuclear factor kappa-B ligand; TNAP, tissue non-specific alkaline
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Ronchetti et al. Fibroblasts in ectopic calcifications
arterial calcification in infants or GACI) and of ligaments and
articular cartilage (i.e., chondrocalcinosis) in humans and mice,
respectively (Okawa et al., 1998; Ho et al., 2000; Pendleton et al.,
2002; Rutsch et al., 2003).
In the extracellular space, phosphate levels are directly con-
trolled by tissue non-specific alkaline phosphatase (TNAP), a cell
membrane-bound ecto-enzyme that increases inorganic phos-
phate availability by releasing it from a variety of phosphate-
enriched substrates and, at the same time, reduces the levels of
calcification inhibitors, promoting the hydrolysis of PPi and the
dephosphorylation of osteopontin (OPN; El-Abbadi et al., 2009;
Orimo, 2010). As a consequence, expression and activity of TNAP
are associated either with physiological and pathological calcifi-
cations, although changes in enzyme activity may not be directly
proportional to the level of mineralization, which is actually the
result of the activity of many genes/proteins (Mendes et al., 2004).
Consistently, increased TNAP expression and activity have been
observed in CD73 deficiency, a disorder that, due to mutations
in NT5E (a gene encoding for the membrane-bound ecto-enzyme
that cleaves adesosine monophosphate (AMP) to adenosine and
inorganic phosphate), is characterized by tor tuosity and calcifica-
tion of lower limb arteries and by mineralization of hand and foot
joint capsules (StHilaire et al., 2011).
Alkaline phosphatase, similarly to other osteogenic genes, as
type I collagen, osteocalcin (OC), and bone sialoprotein (BSP),
can be transcriptionally regulated by bone morphogenetic protein
2 (BMP2; Kim et al., 2004), a powerful cytokine that, by activating
Smad signaling pathways, promotes differentiation of mesenchy-
mal cells into osteoblasts in vitro and induces bone formation in
vivo (Rosen, 2009). Consistently, in fibrodysplasia ossificans pro-
gressiva endochondral ossification is triggered by BMP signaling
in muscle cells (Shen et al., 2009). It has been demonstrated that
treatment of smooth muscle and bone cell cultures with BMP2 (i)
promotes osteogenic phenotype transition of smooth muscle cell
(SMC; i.e., up-regulation of Runx2/Cbfa1 and down-regulation
of SM22 expression), (ii) enhances elevated phosphate-induced
calcification, but does not induce calcification under normal phos-
phate conditions. These results clearly indicate that phosphate
transport via Pit-1 is crucial in BMP2-mediated calcification and
in cell phenotype modulation (Suzuki et al., 2006; Li et al., 2008).
Pit-1 is a type III sodium-dependent phosphate co-transporter
that, through the activation of the Erk 1/2 signaling pathways, pro-
motes calcification and favors changes of vascular smooth muscle
cell (VSMC) toward an osteochondrogenic phenotype. Moreover,
it has been shown that Pit-1 may exert effects also at the endo-
plasmic reticulum level. Studies on VSMC revealed that, when
these cells are treated w ith platelet-derived growth factor (PDGF),
they exhibit increased Pit-1 expression and it has been hypothe-
sized that Pit-1 may regulate anti-calcification proteins (such as
MGP), as well as kinases able to phosphorylate secreted matrix
proteins (such as OPN; Villa-Bellosta et al., 2007). In addition,
recent evidence has been provided that Pit-1 have other unex-
pected functions in cell proliferation and embryonic development
(Lau et al., 2010), thus emphasizing the regulatory importance of
phosphate in cell behavior.
Another protein favoring calcification is BSP, originally identi-
fied in bone and at sites of ectopic calcification in blood vessels,
heart valves, and skeletal muscle. It is involved in the early stages
of mineralization and bone desorption, since it is immobilized
on collagen fibrils where the poly-glutamic acid sequences of BSP
act as possible nucleation sites for hydroxyapatite crystals. BSP,
together with another bone phosphoprotein named OPN, can
modulate crystal shape by adsor ption on a specific face of the
crystals (Ganss et al., 1999).
Osteopontin is in fact a highly phosphorylated and glycosy-
lated secreted protein or iginally discovered in bone, but identified
also in calcified vascular lesions (Giachelli et al., 1993), where it
may counteract apatite deposition by physically inhibiting crys-
tal growth (Boskey et al., 1993) and/or by up-regulating the
expression of genes, as carbonic anhydrase II, favoring mineral
absorption, mainly through the activation of macrophage activi-
ties (Rajachar et al., 2009). These properties depend on the level
of OPN phosphorylation as well as on the targeted tissue (i.e.,
bone or soft connective tissues; Jono et al., 2000). Recent evidence
puts forward that OPN is actually a multi-functional protein able
to interact with several integrin receptors, thus playing a role in
activation, adhesion and migration of many cell types, not only in
tissue mineralization and tumor growth, but also in inflammation
(Jahnen-Dechent et al., 2008). These broad biological activities
underlie the presumed role of OPN in the pathogenesis of cardio-
vascular diseases, including atherosclerosis and abdominal aortic
aneurysm (Giachelli and Steitz, 2000), thus paving the way toward
the clinical use of OPN plasma levels as biomarker of inflamma-
tion and as predictor of the risk for cardiovascular complications
(Cho et al., 2009).
Another interesting protein is OPG that serves as a decoy
receptor for the receptor activator of nuclear factor κB-ligand
(RANKL) and acts as an inhibitor of osteoclastogenesis and osteo-
clast activation by blocking RANK activation (Boyle et al., 2003;
Van Campenhout and Golledge, 2009). As demonstrated in the
KO animals, the absence of OPG is associated with osteoporosis
as well as with calcifications of aorta and renal arteries (Bucay
et al., 1998). Therefore, within the vasculature, OPG may exert a
protective role toward ectopic calcification down-regulating alka-
line phosphatase activity (Van Campenhout and Golledge, 2009
Consequently, serum OPG levels have been significantly associated
with the presence of coronary artery disease (Jonoet al., 2002), sug-
gesting that OPG may represent a strong risk factor for mortality
in dialysis patients (Morena et al., 2006).
Matrix Gla protein belongs to a large family of proteins whose
maturation requires vitamin K-dependent carboxylation of glu-
tamyl residues (Schurgers et al., 2007; O’Young et al., 2011). It is
considered the most active anti-calcifying agent in vessels (Shana-
han et al., 1998; Price et al., 2006), but it is actually produced by
several cell types, among which VSMCs, osteoblasts, and fibrob-
lasts (Davies et al., 2006; Park et al., 2006). The phenotype of
MGP/ mice is characterized by arterial calcification and by
arterial-venous malformations (Yao et al., 2011), suggesting that
MGP has roles in connective tissue development and homeostasis,
as well as in preventing ectopic calcification. The corresponding
human disorder is Keutel syndrome (Munroe et al., 1999), charac-
terized by enhanced mineralization of the growth plate cartilage
leading to reduced longitudinal growth and osteopenia, as well
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chondrocyte transformation of VSMCs. This aberrant cellular dif-
ferentiation could be related to the ability of MGP to act as a
regulator of BMP2 in a dose-dependent manner. Low levels of
MGP relative to BMP2 may result in mild enhancement of BMP2
activity, whereas intermediate levels would inhibit and high lev-
els strongly increase BMP2 activity (Zebboudj et al., 2002). These
findings clearly demonstrate the complexity of the mechanisms
regulating ectopic calcifications, which are dependent not only
on the presence/absence of specific proteins and on their activity
(due, for instance, to post-translational modifications as phospho-
rylation and carboxylation), but also on the ratio among different
A similar vitamin K-dependent carboxylated protein is OC that,
synthesized by osteoblasts, is deposited into bones, where it con-
trols the size and the speed of crystal formation and acts as a
chemo-attractant for osteoclasts (Roach, 1994). Moreover, it is
released into circulation, where it is also used as a biomarker of
bone metabolism and vitamin K status. The increase of under-
carboxylated OC (ucOC) levels in the aging population led to
the hypothesis that vitamin K insufficiency might be related to
the calcification paradox (namely age-dependent bone loss asso-
ciated to vascular calcification), however, clinical trials did not
provide support to the hypothesis that vitamin K supplementa-
tion will reduce bone loss or fracture risk. Very recent results
from in vitro and in vivo experimental models indicate that
ucOC is an active hormone with a positive role on glycemia.
If this hypothesis will be proved also in humans, vitamin K
supplementation, by decreasing ucOC,might exert unknown, pos-
sibly detrimental, effects on glucose metabolism (Gundberg et al.,
2012). This hypothesis sustains the importance to perform broad
and extended investigations when diet regimens, supplemented
with even physiological/endogenous components, are used as ther-
apeutic tools. Interference with a specific molecule may in fact
havedominoconsequences on many other, apparently unrelated,
Finally, a novel γ-carboxyglutamate (Gla)-containing protein,
named Gla-rich protein (GRP) due to its high content in Gla
residues, has been identified in association with chondrocytes
and bone cells. Although its molecular function is yet unknown,
the high content of Gla residues and its accumulation at sites of
pathological calcification in skin, vascular system and breast cancer
tumors suggest that GRP modulates calcium availability, regulates
cartilage matrix organization and influences matrix stability being
associated with fibrillar collagens (Cancela et al., 2012).
Although not synthesized by mesenchymal cells, being secreted
from hepatocytes into the circulation, never the less, fetuin A
exerts its biological role in the periphery, where it inhibits calcifica-
tion by the transient formation of soluble colloidal spheres (Heiss
et al., 2003). It binds calcium phosphate and calcium carbonate
with high affinity and, although with lower efficiency, magnesium
phosphate (Schinke et al., 1996). In rat sera fetuin A is present
in high molecular weight complexes, termed calciprotein parti-
cles, which contain calcium, phosphate and matrix Gla protein.
They act as inhibitors of mineralization in solution and of cell-
mediated miner alization by inhibiting the de novo formation
of calcium phosphate without dissolving preformed minerals
(Schlieper et al., 2007).
The complexity of the mechanisms regulating pathologic cal-
cification is further highlighted by the involvement of apparently
unrelated gene products, as it was noticed for Klotho and multi-
drug resistance protein 6 (MRP6), just to mention few of them.
Klotho is a tr ansmembrane protein with an extracellular (β-
glucosidase domain that can be shed f rom the plasma membrane
by Adamts proteases and, in addition to its enzymatic function,
binds directly to fibroblast growth factor (FGF)23 acting as an
essential FGF-coreceptor. In the kidney, FGF23 signalling leads to a
down-regulation of the sodium/phosphate co-transporter (NaPi)
and of the vitamin D 1α-hydroxylase. Therefore, Klotho defi-
ciency, in spite of high FGF23 levels and of high 1,25-dihydroxy
vitamin D3 and calcium concentrations, leads to osteopenia,
hyper-phosphatemia, and consequently widespread vascular and
soft tissue calcifications (Moe, 2012). By contrast, dysfunction
of the ATP-binding cassette (ABC)-transporter ABCC6 (cod-
ing for the transmembrane protein MRP6 highly expressed in
liver and kidney) causes pseudoxanthoma elasticum (PXE), a
rare disease characterized by mineralization and degeneration
of elastic fibers within soft connective tissues, thus causing skin
laxity, cardiovascular complications, and visual impairment in
a setting of normal le vels of circulating calcium and phosphate
and without bone abnormalities (see ahead for further details;
Quaglino et al., 2011).
Changes in the characteristics of the extracellular matrix and
in the ratio between matrix constituents influence not only the
mechanical properties of connective tissues, but significantly
contribute to modulate cell phenotype by altering integrin expres-
sion, focal adhesions, cytoskeletal organization and consequently
intracellular signaling pathways.
As a consequence, it has been shown that osteogenic differen-
tiation of calcifying VSMCs was promoted by type I collagen and
fibronectin, but it was inhibited by type IV collagen. By contrast,
valvular interstitial cells (a heterogeneous population of fibrob-
lasts, with a small percentage of myofibroblasts and smooth muscle
cells ranging from 5 to 30% in physiological or pathological con-
ditions, respectively) w hen grown on type I collagen or fibronectin
remain in a quiescent fibroblastic state, whereas those cultured on
fibrin surfaces exhibit a myofibroblast phenotype and rapidly form
calcified aggregates (Chen and Simmons, 2011). These data fur-
ther highlight the complex interactions between cells and between
cells and matrix.
Therefore, beside alterations in the balance between pro- and
anti-calcifying factors, changes in the extracellular matrix may
significantly contribute to mineral deposition. It is noteworthy to
mention that in soft connective tissues, if mineralization is not
triggered by necrotic cell debris, elastic fibers seem to represent
the selected target of pathologic mineralization, possibly due to
their low turnover and/or susceptibility to calcium ion-binding
(Pugashetti et al., 2011).
Purified elastin has been demonstrated to have calcium-binding
capabilities (Molinari-Tosatti et al., 1968; Cox et al., 1975; Long
and Ur ry, 1981). Moreover, addition of elastin peptides to cul-
tured SMC enhances Von Kossa positive calcium precipitates in
the phosphate model of in vitro calcification (Hosaka et al., 2009).
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In accordance with these data, elastin degradation due to the
up-regulation of matrix metalloproteinase (MMP)2, MMP9, and
cathepsin S has been shown to increase arterial calcification in the
uremic mice model of ectopic calcification (Pai et al., 2011) and
to favor calcification of native heart valves (Per rotta et al., 2011).
It has been therefore suggested that peptides or frag ments derived
from elastin degradation, due to their high hydrophobicity and
coacervation properties, may enhance abnormal mineralization
(Abatangelo et al., 1975; Long et al., 1975) leading to the formation
of abnormal complexes with high calcium-binding capabili-
ties (Bell et al., 1974; Tamburro et al., 1977; Urry et al., 1982).
These findings sustain the association between inflammation and
ectopic calcification, especially in the vascular compartment (Shao
et al., 2010).
Beside elastin itself, elastic fibers are made of several compo-
nents whose exposure, with age and in pathological conditions,
further contributes to the preferential localization of ectopic cal-
cifications on elastin and on elastic fiber-associated molecules.
For instance, calcium-binding sites have been found on specific
domains in fibrillin I (Handford, 2000), one of the principal
elastin-associated proteins. Therefore, not only elastin per se, but
also elastic fibers, as a whole, could function as nucleation center
for calcium precipitation (Starcher and Urry, 1973). Beside fib-
rillin, proteoglycans (PG), glycosaminog lycans (GAGs), and other
glycoproteins present inside elastic fibers could also represent
additional calcium-binding sites.
We have repeatedly demonstrated that GAGs are present inside
elastic fibers, possibly regulating the mechanical properties and
stability of these fibers (Pasquali-Ronchetti et al., 1984; Contri
et al., 1985). Changes in the type or ratio of GAGs, as those occur-
ring with age, in the course of pathologic conditions or depending
on tissue or on specific physiological requirements (Berenson et al.,
1985; Cherchi et al., 1990; Passi et al., 1997; Qu et al., 2011), may
influence the characteristics of elastic fibers and of the whole extra-
cellular matrix, as demonstrated for instance in the vasculature
where connective tissue molecules follow a gradient depending
on the distance from the heart (Madhavan, 1977). Moreover, we
have also demonstrated that calcified elastic fibers exhibit pecu-
liar type and localization of PG/GAGs such as heparan sulfate,
putting forward the hypothesis that GAGs have a role in elastic
fiber homeostasis as well as in the calcification process (Passi et al.,
1996; Gheduzzi et al., 2005).
Furthermore, it was shown that, in cartilage, PG/GAGs act as
calcium-concentrating agents promoting calcification, but they
also behave as inhibitors of hydroxyapatite formation functioning
as a cation-exchanging calcium reservoir (Hunter, 1991). Consis-
tently, decorin, a small leucine-r ich PG containing one dermatan
sulfate or chondroitin sulfate chain, beside its regulatory role on
transforming growth factor (TGF)-β activ ity and collagen fibril-
logenesis, binds to hydroxyapatite (Boskey et al., 1997; Rees et al.,
2001; Mochida et al., 2009) and colocalizes w ith areas of calcifica-
tion in skeletal tissues, in the adventitia of blood vessels, and in the
skin (Hocking et al., 1998).
A further link between GAGs and the calcification pro-
cess is the ability of BMPs to bind to heparin and to
induce osteoblast differentiation of mesenchymal cells. Moreover,
heparan sulfate and dextran sulfate enhanced BMP2 activity
serving as ligands to their signaling receptors on cell membranes
(Takada et al., 2003).
The presence of perivascular cells closely associated with capillar-
ies was described more than 100 years ago, although their origin
remained elusive for many decades (Díaz-Flores et al.,1991). Some
studies proposed that they may derive from the neural crest,
whereas other studies suggested that pericytes derive from smooth
muscle cells, fibroblasts, endothelial cells, and bone marrow and
that they exhibit a multi-lineage potential being capable of dif-
ferentiating into a variety of cell types including osteoblasts and
chondrocytes, as demonstrated, both in vitro and in vivo experi-
mental models. On the basis of these observations, it was suggested
that pericytes play a role in mediating ectopic calcification (Collett
and Canfield, 2005).
There is now good evidence that angiogenesis regulates ectopic
calcification in several ways: (i) angiogenic factors are mitogenic
for mesenchymal cells and osteoblasts and enhance bone for-
mation in vivo; (ii) cytokines as BMP2 and BMP4, released by
endothelial cells, induce both the differentiation of osteoprogeni-
tor cells and calcification in vitro and in vivo, although this effect
is context-dependent (Shin et al., 2004); (iii) new vessels serve as a
conduit for osteoprogenitor cells that may derive from the circu-
lation or from pericytes themselves. Consistently, the association
between angiogenesis and ectopic calcification has been noted in
several cases, as in ductal carcinoma in situ, in calcifying fibrob-
lastic granuloma, in choroidal osteoma and in the calcifications of
the retina.
When cultured in standard growth medium, pericytes undergo
a process of growth and differentiation characterized by the forma-
tion, w ithin approximately 8 weeks, of large multi-cellular nodules
that, similarly to the matrix found in calcified vessels, contain type
I collagen, OPN, matrix Gla protein and OC and hydroxyapatite
crystals with a calcium to phosphate ratio analogous to that of
bone (Doherty and Canfield, 1999; Abedin et al., 2004).
Studies in human lesions and mouse models of arterial calcifica-
tion as well as in vitro calcification models of human and bovine
VSMC suppor t the concept that mesenchymal-derived vascular
cells participate in mineral deposition by mimicking bone for-
mation, since they exhibit several hallmarks of endochondral
ossification (Liu and Shanahan, 2011).
It has been clearly demonstrated that VSMC (1) undergo
osteoblastic differentiation with loss of smooth muscle-specific
gene expression and gain of osteoblast-like properties, includ-
ing expression of the osteoblast differentiation factor Cbfa-1;
(2) activate the mineralization process in the presence of high
concentrations of extracellular phosphate; (3) may require a
sodium-dependent phosphate co-transporter function to calcify
(Giachelli, 2001; Vattikuti and Towler, 2004). The complexity of
VSMC phenotypic changes associated to ectopic calcifications has
been recently clearly outlined by a whole-genome expression array
approach in uremic rats fed on a high phosphate diet. It was in fact March 2013
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Ronchetti et al. Fibroblasts in ectopic calcifications
demonstrated that the transition from “muscle-related” to bone-
related” gene expression involved the deregulation of at least 53
genes (Román-García et al., 2010) and the activation of Erk1/2
and Wnt pathways (Lau et al., 2010).
Interestingly, it has been shown that, in appropriate culture
conditions, approximately 10–30% of VSMC have the capacity to
express osteoblast differentiation markers and to retain this pheno-
type through in vitro passages (Boström et al., 1993). In agreement
with these in vitro data, a variety of bone matrix proteins and
regulatory factors have now been demonstrated in human calci-
fied plaque, including OC, BSP, osteonectin, collagen I, alkaline
phosphatase, Msx-2, and Cbfa-1.
Many studies performed on VSMC demonstrated that mesenchy-
mal cells, whether locally producing pro- and anti-calcifying
factors or being involved in extracellular matrix synthesis and
degradation, are involved in the mineralization of soft connective
tissues. Never the less, a key question is whether all mesenchy-
mal cells behave similarly, or if differences in their tissue-specific
differentiation may be associated to a different susceptibility of
connective t issues to mineralize.
For instance, skin seems to be only rarely affected by ectopic
calcification in contrast to the vascular system. Very few studies
have been done on fibroblasts and especially on dermal fibroblasts,
although in a number of disorders there is a clear evidence for their
close association to ectopic calcifications (Figure 2).
Among the few reports on fibroblast-associated calcifications
are those showing that a human gingival fibroblast cell line may
exhibit both intracellular and extracellular ectopic mineralization
starting within round and irregularly shaped vesicles contained
in large cytoplasmic vacuoles. This may suggest that mineral
Transmission electron microscopy of a dermal biopsy from
a patient affected by pseudoxanthoma elasticum (PXE). Deformed
calcified elastic fibers (E) are present in close proximity to large fibroblasts
with abundant and dilated cisternae of the endoplasmic reticulum. Collagen
fibrils occasionally organized into collagen flowers (arrows) are also visible.
= 1 μm.
accumulation and transformation of amorphous mineral into
cr ystalline structures take place within cellular vesicular structures
likeMV(Yajima et al., 1984).
By contrast, MV have been never observed within or around
dermal fibroblasts in areas of matrix calcification in vivo nor
in the high phosphate-calcification model in vitro (personal
observations). This finding indicates the occurrence of different
phenotypic characteristics between dermal and gingival fibrob-
lasts, but at the same time demonst rates that mineral deposition
can be observed also independently from MV.
In the attempt to understand the interactions between cells
from hard and soft connective tissues and to unveil the complexity
of the mechanisms involved in fibroblast-associated calcifica-
tion, Yu and coworkers performed a cDNA microarray analysis
on fibroblasts from spinal ligaments cultured in the presence
of conditioned media of osteoclast-like cells. In this environ-
ment fibroblasts exhibited high levels of alkaline phosphatase and
mineral deposition, but more interestingly, microRNA expres-
sion profiles revealed a significant down-regulation of a group
of microRNAs known to negatively interfere with genes associ-
ated with osteogenic differentiation (e.g., BMP2, OC, Runx2).
In the light of these data, it has been hypothesized that osteo-
clasts might induce the osteogenic differentiation of fibroblasts in
vitro and that miRNA may play an important role in the regu-
lation of cell–cell interactions between osteoclasts and fibroblasts
(Yu et al., 2011).
An additional demonstration of the ability of fibroblasts to
modulate their phenotype in response to specific environmen-
tal characteristics has been provided by studies on rat dermal
fibroblasts cultured in the presence of elastin degradation products
and of TGF-beta1. Mineralization was preceded by up-regulation
of alpha-smooth muscle actin, type I collagen and MMP2,
which are characteristic features of myofibroblasts. Thereafter,
osteogenic markers as OC, alkaline phosphatase, and osteoprote-
gerin increased their expression and, after 21 days, multi-cellular
calcified nodules were observed. It was proposed that elastin-
associated mineralization might result from defective/unbalanced
dynamic remodeling events similar to those occurring during the
repair process (Simionescu et al., 2007).
Therefore, it is important to note that, irrespective of the cell
type, a specific environment is required for calcification to occur, in
vivo, but especially in vitro. All cultured mesenchymal cells, in fact,
are dependent for their growth on a variety of cytokines and adhe-
sive molecules as those easily provided by addition of fetal/calf
bovine serum. However, the amount of “serum factors signifi-
cantly higher compared to physiological in vivo concentrations,
provide cells of a number of other components that, depending on
the characteristics of the serum, directly influences the mineraliza-
tion process or may regulate cell behavior and, as a consequence,
the expression of specific gene/proteins. Among these factors,
serum fetuin A, that is usually present in cell culture media, repre-
sents a powerful inhibitor of the calcification process making cells
unable to mineralize in standard cell culture conditions. To over-
come this problem, it is possible to utilize serum-free media (i.e.,
media with chemically defined components and supplements)
and/or to add to standard cell cultures [in Dulbeccos modified
Eagle’s medium (DMEM) plus serum] high concentrations of
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Ronchetti et al. Fibroblasts in ectopic calcifications
phosphate (that can easily precipitate as soon as it forms complexes
with calcium) or of phosphate donor substrates (that require an
active involvement of cells for the enzymatic release of inorganic
phosphate from substrates). Human skin-derived fibroblast pre-
cursor cells, for instance, can acquire an osteoblast-like behavior
and star t to mineralize the newly deposited extracellular matrix
only if cultured in a pro-osteogenic medium (supplemented with
ascorbic acid, beta-glycerophosphate, and dexamethasone); as a
result induced expression of alkaline phosphatase, BSP and OC
leads to mineral deposition (Buranasinsup et al., 2006).
A further difference, between mesenchymal cell types is the
time required in vitro to obtain a calcified matrix, which may be
taken as predictive of the pro-osteoblastic potential of the cells.
In osteoblast and VSMC cultures, mineralization can be obtained
after 3–5 or 8–10 days in culture, respectively (Uchimura et al.,
2003; Li et al., 2004). By contrast, mineralized matrix becomes
clearly evident only after 2–3 weeks in dermal fibroblast cultures
(Boraldi et al., in press; Figure 3).
On the basis of these results it could be hypothesized that der-
mal fibroblasts are rather resistant to be converted into osteoblast-
like cells, in agreement with the uncommon occurrence of dermal
Interestingly, ectopic soft tissue calcification is a well-known
symptom in Werner syndrome (WS), an autosomal recessive
progeroid disorder caused by mutations in RecQ DNA heli-
case. Cultured fibroblasts from WS patients undergo spontaneous
mineralization in vitro at normal phosphate concentration, and
overexpress Pit-1 at mRNA and protein levels. Both calcifica-
tion and Pit-1 up-regulation have been also detected in situ in
the skin of patients (Honjo et al., 2008), supporting the concept
that dermal fibroblasts mimic and retain in vitro at least some of
the pathologic characteristics they have in vivo, thus representing
a valuable model to investigate the pathogenetic mechanisms of
Pseudoxanthoma elasticum is a rare genetic disorder characterized
by skin papules on the neck, axillae, and groin, often associated
with skin redundancy and laxity, by retinal alterations as angioid
streaks and neovascularization, and by middle sized artery narrow-
ing up to occlusion. All these alterations depend on the deposition
of calcium minerals inside or associated with elastic fibers (Truter
et al., 1996; Gheduzzi et al., 2003; Figure 2). The phenomenon is
rather peculiar as calcifications affect only elastic fibers, whereas
collagen does not calcify; moreover, this abnormal mineral-
ization occurs in the absence of increased calcium and phos-
phate levels, in the total absence of inflammation, cell necrosis,
Pseudoxanthoma elasticum has been associated to mutations
in the ABCC6 gene, a member of the ABC family of mem-
brane transporters (it encodes for the membrane transporter
MRP6), that is mainly expressed in liver and kidney (Bergen
et al., 2000; Le Saux et al., 2000; Ringpfeil et al., 2000), whereas
its expression is surprisingly low in tissues specifically involved in
Light microscopy of dermal fibroblasts cultured for 3 weeks
in the presence of DMEM (A) or of DMEM supplemented with
ascorbate, beta-glycerophosphate, and dexamethasone (B). In the
presence of the calcified medium, dermal fibroblasts exhibit areas of
mineralization that can be clearly visualized upon Von Kossa staining as dark
deposits (lower panel; see also Boraldi et al., in press, for methodological
the clinical manifestations of PXE. In this context, it has been
suggested that a still unknown circulating metabolite released
(or not released) by the liver in ABCC6 deficiency may directly
affect elastic fiber formation, stability, and calcification (Le Saux
et al., 2006).
Actually, in a setting of normal calcemia and phosphatemia,
several abnormalities have been documented in the circulation of
PXE patients, from PGs and enzymes involved in their synthe-
sis (Götting et al., 2005; Schön et al., 2006; Volpi and Maccari,
2006), to protein and lipid abnormalities indices of oxidative
stress (Garcia-Fernandez et al., 2008), to high levels of MMP2 and
MMP9 (Diekmann et al., 2009) and of elastin-derived peptides
(Annovazzi et al., 2004; Ta b le 1 ).
Moreover, low levels of fetuin A and of vitamin K have been
measured in the circulation of PXE patients and in the PXE animal
model (Jiang et al., 2010; Vanakker et al.,2010). Low levels of fetuin
A could be explained by the augmented capture of this molecule
by peripheral mineral precipitates (Price et al., 2004; Hendig et al.,
2006), although it cannot be excluded that PXE fibroblasts may March 2013
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Table 1
Genes/molecules involved in the regulation of elastic fiber
calcification in PXE.
Regulators of mineral
PXE findings (reference)
Circulating ion levels Normal Ca and P (Boraldi et al., in press)
Normal serum alkaline phosphatase (ALP;
Boraldi et al., in press)
TNAP in fibroblasts (Boraldi et al., in press)
Calcification inhibitors Gla-MGP (Gheduzzi et al., 2007; Li et al., 2007;
Hendig et al., 2008b)
Fetuin-A (Hendig et al., 2006; Jiang et al.,
Polymorphisms of OPN (Hendig et al., 2007)
Mutation of ENPP1 (Nitschke et al., 2012)
Extracellular matrix
MMP2, MMP9 in serum (Diekmann et al.,
MMP2 in fibroblasts (Quaglino et al., 2005)
Polymorphisms of MMP2 (Zarbock et al., 2010)
Elastin-derived peptides (Annovazzi et al.,
Different ratio PG/GAGs (Tiozzo-Costa et al.,
1988; Passi et al., 1996; Maccari andVolpi, 2008)
Circulating selectins (Götting et al., 2008)
Serum intercellular adhesion molecule (ICAM;
Hendig et al., 2008a)
Different expression of integrins (Quaglino et al.,
Serum XT-I activities (Götting et al., 2005)
Polymorphisms of XT-I (Schön et al., 2006)
Redox balance ROS in serum (Garcia-Fernandez et al., 2008)
ROS in fibroblasts (Pasquali-Ronchetti et al.,
2006; Boraldi et al., 2009)
Polymorphisms of antioxidant genes
(Zarbock et al., 2007)
sequester this inhibitor (Boraldi et al., 2007)asaconsequenceof
higher intracellular uptake that may prevent fetuin A from exert-
ing its regulatory role in peripheral tissues. The recent finding that
in the mouse model of PXE ectopic calcification can be signifi-
cantly reduced by overexpressing fetuin A (Jiang et al., 2010)may
suggest that in PXE the role of this inhibitor should be further
Nevertheless, it is unlikely that all these plasma abnormali-
ties in PXE patients would directly depend on the deficiency of
the membrane transporter MRP6. It would seems more reason-
able that inherited ABCC6 deficiency, along the years, would
induce a series of metabolic adjustments in several tissues pos-
sibly epigenetically involving a network of different genes and
leading to the complexity and heterogeneity of PXE alterations.
Moreover, since each patient has a different genetic background,
the consequences of these “metabolic adjustments” would be
different in each individual thus explaining the extreme variability
of clinical manifestations among patients.
Therefore, clinical and experimental data st rongly suggest that
elastic fiber calcification in PXE is not a passive process merely
due to the presence or absence of one or more abnormal plasma
components, but the result of activities mediated by local cells.
At least in skin, fibroblasts should be considered the principal
candidates for several reasons.
First of all, if elastic fiber calcifications in PXE are a passive pro-
cess due to the infiltration of plasma molecule(s), all elastic fibers
should calcify; on the contrary, skin elastic fiber calcification is
present only in peculiar regions of the body. Moreover, also in
areas prone to calcification, not all elastic fibers mineralize. This is
in agreement with the overgrowing evidence of the diversity of skin
fibroblasts at different anatomical sites, as these cells display dis-
tinct and characteristic transcriptional patterns for a large number
of genes depending on the body region they come f rom (Chang
et al., 2002; Lindner et al., 2012). Therefore, skin fibroblasts may be
considered differentiated cell types that, depending on their loca-
tion, maintain their positional identities even when isolated and
cultured in vitro (Rinn et al., 2008) and probably react in different
ways to abnormal exogenous stimula, such as those present in the
circulation of PXE patients.
A second evidence for the involvement of fibroblasts in skin
abnormalities in PXE is, beside elastic fiber calcification, the
documented presence of huge aggregates of PGs and of various
extracellular matrix proteins in the affected areas of the der-
mis (Pasquali-Ronchetti et al., 1986; Tiozzo-Costa et al., 1988;
Baccarani-Contri et al., 1994; Passi et al., 1996), consistent with
a significant increase of the total amount of GAGs in the skin of
patients (Maccari and Volpi, 2008; Table 1 ) and with the observed
decreased susceptibility of GAG-associated elastin to pancreatic
elastase (Schwartz et al., 1991). Such structural and chemical alter-
ations, very likely responsible for skin redundancy and laxity in the
affected areas, must be under the local control of fibroblasts, which
are responsible for the synthesis of the extracellular milieu in soft
connective tissues.
An indirect indication that also in the vessel wall fibroblasts
are probably involved in the early calcification of elastic fibers is
the observation that calcification in PXE vessels is often present
within the elastic fibers close to the adventitia, in the absence of
any osteoblast-like phenotype of the adjacent cells, that in fact
maintain a fibroblast-like appearance (Gheduzzi et al., 2003).
Finally, several studies by our and other groups have shown
that fibroblasts isolated from the dermis of PXE patients have and
maintain in vitro a metabolic behavior different from fibroblasts
isolated from the same body areas of gender and age-matched nor-
mal subjects. Actually, it has been demonstrated that PXE fibrob-
lasts suffer from an oxidative stress condition (Pasquali-Ronchetti
et al., 2006), produce highly sulfated GAGs (Tiozzo-Costa et al.,
1988; Passi et al., 1996), exhibit abnormal proteoglycanase (Gor-
don et al., 1978, 1983) and higher metalloproteinase activities
(Quaglino et al., 2005), are unable to properly carboxylate MGP
(Gheduzzi et al., 2007; Boraldi et al., in press; Table 1 ), and have a
different protein profile (Boraldi et al., 2009) indicating that their
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Ronchetti et al. Fibroblasts in ectopic calcifications
metabolic behavior is genetically or epigenetically different from
control fibroblasts and is maintained when cells are cultured in
vitro (Rinn et al., 2008).
In the light of these observations, dermal fibroblasts from PXE
patients can be considered a valuable and very informative model
to better understand the contribution of these cells to soft connec-
tive tissue calcifications. Therefore, beside the role of fibroblasts
in regulating the characteristics of the extracellular environment
and, as a consequence, the different susceptibility of tissues and of
elastic fibers to calcify, a key question remains whether the same
osteoblast-related pathways, as those described in SMC, are also
involved in the phenotype of fibroblasts prone to calcify (i.e., PXE
Recent evidence from our laboratory indicates that TNAP activ-
ity, althoug h within normal range in the circulation of patients,
is higher in PXE fibroblasts compared to control cells and that
these differences are further amplified when cells are g rown in
a calcifying medium. The process is rather complex, however,
data suggested that the local increase of phosphorus in the extra-
cellular milieu together with the reduced amount of anti-calcific
molecules, such as carboxylated MGP, may favor hydroxyapatite
formation (Boraldi et al., in press).
Interestingly, low le vels of MGP have been found in the circu-
lation of PXE patients (Hendig et al., 2008b) and in the Abcc6/
mice model of PXE (Li et al., 2007). In accordance, low levels
of carboxylated MGP are produced by skin PXE fibroblasts in
vitro, indicating that the local synthesis of the mature protein is
of paramount importance in preventing elastic fiber calcification
(Gheduzzi et al., 2007) and that fibroblasts are likely involved in the
local secretion of this important anti-calcific protein. Since MGP
γ-carboxylation is a vitamin K-dependent process (Theuwissen
et al., 2012), it was suggested that PXE calcification could be due
to vitamin K deficiency (Borst et al., 2008). Although the level of
vitamin K is low in PXE patients (Vanakker et al., 2010), never-
theless the availability and the cellular utilization of vitamin K do
not seem responsible for MGP under-carboxylation. Both in PXE
fibroblasts and in two different Abcc6/ mice models, addition
of vitamin K did not improve MGP carboxylation (Boraldi et al., in
press) nor prevented calcification in spite of the high serum con-
centration of vitamin K upon treatments (Brampton et al., 2011;
Gorgels et al., 2011; Jiang et al., 2011). Therefore, low vitamin K
does not seem to play a pivotal role in MGP carboxylation nor
in elastic fiber calcification in PXE. Moreover, in PXE, carboxy-
lation of proteins involved in coagulation or in bone calcification
seems adequate, as no defects in coagulation or in bone mineral-
ization have been described in patients. Therefore, both vitamin
K availability and the carboxylase system do not seem directly
involved in PXE mineralization. Recent data from our laboratory
seem to suggest that the low carboxylation rate of MGP by PXE
skin fibroblasts, even in a setting of high vitamin K concentration,
might depend on the intrinsic ability of MGP to be carboxylated
(Boraldi et al., in press)
In addition, evidence has been provided through the years that
the PXE phenotype can be obtained through pathways other than
those caused by ABCC6 mutations. An indirect proof of this is that
PXE-like clinical and histo-pathological manifestations have been
described in a number of patients affected by beta-thalassemia
(Aessopos et al., 1992, 1998; Baccarani-Contri e t al., 2001; Cian-
ciulli et al., 2002) and other hemoglobinopathies (Goldberg et al.,
1971; Nagpal et al., 1976; Aessopos et al., 2002; Fabbri et al., 2009),
in subjects treated with penicillamine (Rath et al., 2005), in cases
of γ-carboxylase gene (GGCX) and ENPP1 mutations (Vanakker
et al., 2007; Le Boulanger et al., 2010
; Li et al., 2012; Nitschke et al.,
2012; see further for additional data) and, more recently, in a few
cases of liver transplantation where the impossibility to examine
the DNA from all donors and recipients made not clear if the
transplanted liver harbored or not ABCC6 mutations (Bercovitch
et al., 2011).
As already mentioned, the metabolic complexity at the basis of
elastic fiber calcification could, at least partially, explain the phe-
notypic similarities of the skin lesions in inherited PXE and in a
number of different unrelated disorders, such as in patients
affected by beta-thalassemia (Aessopos et al., 1992; Baccarani-
Contri et al., 2001).
More than 60 years ago, elastinopathies similar to that in PXE
were documented in sickle cell anemia (Paton, 1959; Suerig and
Siefert, 1964) and, later, in a series of hemoglobinopathies (Nag-
pal et al., 1976), among which β-thalassemia (Aessopos et al., 1992,
1997). Subsequent studies better defined the almost identical clin-
ical and histo-pathological alterations in PXE and in a relevant
number of β-thalassemia patients (Aessopos et al., 1998). In both
these unrelated genetic disorders, angioid streaks (Gibson et al.,
1983; Kinsella and Mooney, 1988; Aessopos et al., 1989, 1992;
O’Donnell et al., 1991), arterial elastorrhexis, and calcification
(Aessopos et al., 1998; Ts omi et al., 2001; Cianciulli et al., 2002)
as well as coalescent skin papules on the posterior/lateral aspect
of neck, axillae, and groin with elastic fiber calcification (Aesso-
pos et al., 1992; Baccarani-Contri et al., 2001)havebeenrepeatedly
Skin abnormalities in genetic PXE and in beta-thalassemia
patients with clinical PXE-like manifestations (β-thal/PXE) have
been carefully analyzed. It was observed that both disorders had
identical elastic fiber calcifications, “collagen flowers” abnormali-
ties, as well as cell and matrix alterations, suggesting that similar
metabolic changes could be involved in both disorders as final
consequence of mutations in apparently unrelated genes.
In agreement with this hypothesis are data from experiments
aiming to verify if the similarities of clinical and histo-pathological
features in genetic PXE and in β-thal/PXE patients could be sus-
tained by analogous similarities in the metabolic behavior of
cultured fibroblasts.
Fibroblasts from healthy subjects, from PXE patients as well
as from indiv iduals affected by beta-thalassemia exhibiting (β-
thal/PXE) or not (β-thal) ectopic calcification, were investigated
for their ability to accumulate and to extr ude calcein-AM (ace-
tomethoxy derivate of calcein; Boraldi et al., 2008), a chemical
widely used for determining cell viability. The non-fluorescent
calcein-AM enters living cells where it is hydrolyzed by intracellu-
lar esterases into the strongly fluorescent g reen anion calcein that
can be retained in the cytoplasm or actively extruded. The accu-
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Ronchetti et al. Fibroblasts in ectopic calcifications
visualized by confocal microscopy and quantified by flow cytom-
etry (Boraldi et al., 2003). Within few minutes of incubation with
calcein-AM (0.1 μM) dermal fibroblasts accumulate the fluores-
cent molecule into discrete granules in the cytoplasm. After a
30-minute incubation, calcein accumulation is much higher in
PXE and in β-thal/PXE cells than in controls and in β-thal fibrob-
lasts. Furthermore, also calcein efflux from β-thal/PXE fibroblasts
is significantly different compared to controls (p < 0.05), whereas
it is identical to that of fibroblasts from patients with inherited PXE
(Figure 4). Therefore, in v itro dermal fibroblasts from β-thal/PXE
patients, in the absence of ABCC6 mutations (Hamlin et al., 2003),
exhibit functional alterations similar to those of fibroblasts isolated
from patients with inherited PXE. In this specific case, the calcein
assay is defective in fibroblasts isolated from subjects with identical
elastic fiber calcification (i.e., PXE and β-thal/PXE), whereas it is
normal in fibroblasts from β-thalassemia patients without PXE-
like clinical alterations. Therefore, these findings further support
the hypothesis that PXE-like clinical manifestations described in
some β-thalassemia patients might derive from metabolic alter-
ations occurring in this particular sub-group of patients and that
similar pathways may be at the basis of elastic fiber calcification in
inherited PXE and in β-thal/PXE patients (Boraldi et al., 2008).
In β-thalassemia patients an abnormal oxidative stress induced
by the iron overload derived from repeated transfusions, by
Calcein uptake and extrusion in cultured fibroblasts
from healthy individuals (Control), PXE patients (PXE), beta-
Thalassemic patients with (
Thal/PXE) and without (βThal) PXE-like
manifestations. Intracellular calcein accumulation is shown after 2 and
30 min by confocal microscopy (A). Calcein uptake (B) and extrusion
(C) have been measured by flow cytometry and are shown in panels
on the right (see also Boraldi et al., 2003, for methodological
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Ronchetti et al. Fibroblasts in ectopic calcifications
unpaired alpha-globin chains (Livrea et al., 1996; Hershko et al.,
1998; Cighetti et al., 2002) and by deficient oxygen transport
to peripheral tissues (Chan et al., 1999; Meral et al., 2000) has
been described. How oxidative stress interferes with connec-
tive tissue metabolism is still largely unknown. It has been
shown that iron overload and the consequent oxidative stress
affect the synthesis of elastin by human dermal fibroblasts in
vitro (Bunda et al., 2005) and that genetic factors as well as
environmental oxidative stress may deeply influence the extra-
cellular matrix and the behavior of cells in vivo (Hanley and
Repine, 1993). It could be suggested that clinical and histolog-
ical alterations in β-thal/PXE patients could be due, at least in
part, to chronic oxidative stress that, similarly to inherited PXE
(Garcia-Fernandez et al., 2008), is not adequately compensated
due to a peculiar genetic/epigenetic background. Interestingly,
the introduction of oral iron chelators markedly increased the
survival of β-thalassemia patients (Borgna-Pignatti et al., 2004)
and reduced the level of oxidative stress, in agreement with
the observation that PXE-like clinical manifestations are never
found in properly treated new β-thalassemia patients (personal
Interestingly, Hbb
mice are characterized by a significant
liver-specific decrease of mrp6 production, due to failure of the
NF-E2p45 transcription factor to bind to the Abcc6 proximal
promoter. Even though this animal model of thalassemia is not
characterized by soft connective tissue mineralization, never the
less it demonstrates that Abcc6 gene expression can be modified by
environmentally-induced changes in transcr iption factor activity
(Martin et al., 2011) and that oxidative stress could play a relevant
role. In this context, it is worthwhile to mention that there are
data in the literature in favor of a relationship between NF-E2p45
and Nrf2 t ranscription factors, as independent groups have shown
the role hemin in stimulating the expression of antioxidant heme
oxygenase 1 (Li et al., 2011) as well as in inducing beta-globin
gene expression through the functional intervention of p45NF-E2
transcription factor (Moore et al., 2006).Moreover,infavorof
a negative effect of oxidative stress on the expression of ABCC6
are data showing that vitamin K3 and oxidant agents induce
down-regulation of ABCC6 expression in HepG2 cells (De Boussac
et al., 2010).
Deficit of vitamin K-dependent gamma-carboxylase system
The vitamin K-dependent gamma-carboxylation system is com-
posed of the gamma-carboxylase and the warfarin-sensitive
enzyme vitamin K(1) 2,3-epoxide reductase (VKOR), which are
located in the endoplasmic reticulum where they interact with
other proteins like calumenin and protein disulfide isomerase,
negative and positive regulators of the vitamin K cycle, respec-
tively (Wajih et al., 2004; Wallin et al., 2008). Different expression
of these two regulatory proteins has been demonstrated on in vitro
cultured fibroblasts to be probably involved in the pathogenesis of
PXE and of PXE-like calcifications (Boraldi et al., 2009).
During vitamin K-dependent post-translational gamma-
glutamyl carboxylation, vitamin K hydroquinone is oxidized to
the epoxide form (K>O) that, in turn, is reduced by the enzyme
VKORC1 (vitamin K epoxide reductase complex component 1) to
complete the vitamin K cycle.
The demonstration that the enzyme VKORC1 is the target for
the anti-coagulant drug warfarin and that patients treated with
this drug develop extensive vascular calcifications (Palaniswamy
et al., 2011) sustain the importance of the vitamin K-dependent
regulatory mechanisms of calcification. In particular, VKORC1
appears to be a rate-limiting step in the biosynthesis of functional
vitamin K-dependent proteins.
Interestingly, skin lesions due to elastic fiber calcification almost
identical to those in PXE have been described in cases of muta-
tions of GGCX (Vanakker et al., 2010). As already mentioned,
this enzyme is necessary for the γ-carboxylation of a number
of proteins some of which are involved in ectopic calcification
(Shanahan et al., 1998; Price et al., 2006). Therefore, mutations in
the GGCX gene are at the basis of an autosomal recessive dis-
order characterized by a generalized deficiency of the Vitamin
K-dependent clotting factors as well as mineralization and fr ag-
mentation of elastic fibers leading to thickened, inelastic skin and
limited retinopathy, associated to accumulation of uncarboxylated
Gla proteins (MGP and OC) in plasma, serum and dermis, in
the presence of normal serum levels of vitamin K (McMillan and
Roberts, 1966). Even though, the deficient carboxylation of coag-
ulation proteins can be restored by vitamin K administration that
increases the level of the electron-donor hydroquinone form of
vitamin K available for GGCX, never the less, 1 year treatment with
vitamin K did not ameliorate skin lesions nor elastic fiber calcifi-
cation in one patient affected by GGCX mutations (unpublished
observations). It could be suggested that carboxylase is essential
for maturation of MGP, but that the electron donor level of vita-
min K does not influence the performance of MGP carboxylation.
These data and those from other laboratories showing that vita-
min K supplementation does not increase the level of circulating
carboxylated MGP in a case of Keutel syndrome (MGP mutations;
Cranenburg et al., 2011) seem to indicate that MGP carboxylation
is under a complex control, only partly dependent on vitamin K, in
agreement with recent results obtained on PXE fibroblasts treated
in vitro with vitamin K supplementation (Boraldi et al., in press).
To further enlarge the spectr um of ectopic calcification
disorders which are clinically and/or pathogenetically related to
PXE, there is a recent report describing a patient, bearing two
ABCC6 mutations and a gain of function single-nucleotide poly-
morphism (SNP) in the GGCX gene, who was characterized by
both classic PXE (papules, retinopathy, and calcifications) and by a
PXE-like syndrome (cutis laxa beyond the flexural areas; Vanakker
et al., 2011).
Mutations in the GGCX or VKORC1 genes are associated with
a h ereditary deficiency of the vitamin K-dependent clotting fac-
tors as well as a clinically relevant dependency of anti-coagulants
(Brenner, 2000; Vanakker et al., 2010). Besides these enzymatic
defects, a deficiency of vitamin K has been described in associ-
ation with coagulation, bone (osteoporosis, osteoarthritis) and
vascular (arteriosclerosis) disorders resulting from insufficient
carboxylation of Gla proteins (Neogi e t al., 2006).
Generalized arterial calcification of infancy
Generalized arterial calcification of infancy is associated with
mutations in the ENPP1 gene and is characterized by mineral-
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Ronchetti et al. Fibroblasts in ectopic calcifications
arteries and stenosis due to myointimal proliferation. Although
survival to adulthood has been reported, most patients die within
the first 6 months of life (Rutsch et al., 2003).
Features of PXE have been recently described in patients
with homozygous missense mutation of the ENPP1 gene (Li
et al., 2012). Cutaneous calcification was never been previously
described in ENPP1 deficiency and this finding is a clear demon-
stration of the role of PPi as a critical anti-calcific agent in PXE
and PXE-like disorders.
It is therefore noteworthy the occurrence of a clinical and
genetic overlapping between PXE and GACI as clearly high-
lighted by a recent study on two brothers born from to
unrelated parents, showing that the elder developed a PXE con-
dition bearing ABCC6 mutations, whereas the younger died
at 15 months of age of a condition clinically reminiscent of
GACI, although it appeared independent of ENPP1 mutations (Le
Boulanger et al., 2010).
The finding that MGP and fetuin A are involved in both
conditions further sustain the hypothesis that ABCC6 muta-
tions account for a significant subset of GACI patients, and
ENPP1 mutations can also be associated with PXE lesions in
young children, thus reflecting two ends of a clinical spectrum
of ectopic calcifications, possibly through the involvement of
common physiological pathways (Nitschke et al., 2012).
In spite of the extreme complexity and still incomplete knowledge
of the various actors involved, we have reported evidence sup-
porting the importance of mesenchymal cells, and of fibroblasts
in particular, in the occurrence and development of soft connec-
tive tissue calcifications. Within this context, fibroblasts from PXE
and PXE-like disorders offer a valuable model to better understand
the complex pathways that end up with elastic fiber mineraliza-
tion. It can be argued that not all mesenchymal cells behave in the
same way and that morpho-functional characteristics of tissues
as well as composition of the extracellular mat rix and exogenous
agents should be taken into account for understanding the sus-
ceptibility/resistance to calcification of different body regions in
physiological conditions, in aging and in both genetic and acquired
Data from our laboratory were obtained from studies supported
by FCRM (Ectocal), PXE International, and PXE Italia Onlus.
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Frontiers in Genetics
Systems Biology March 2013
Volume 4
Article 22
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