REVIEW ARTICLE published: 05 March 2013 doi: 10.3389/fgene.2013.00022 Fibroblast involvement in soft connective tissue calcification Ivonne Ronchetti1, Federica Boraldi1,2, Giulia Annovi1,2, Paolo Cianciulli 3 and Daniela Quaglino1,2* 1 PXELab, University of Modena and Reggio Emilia, Modena, Italy 2 Department of Life Science, University of Modena and Reggio Emilia, Modena, Italy 3 U.O.D.S Unit, S. Eugenio Hospital, Rome, Italy Edited by: Soft connective tissue calcification is not a passive process, but the consequence of Olivier M. Vanakker, Ghent University metabolic changes of local mesenchymal cells that, depending on both genetic and Hospital, Belgium environmental factors, alter the balance between pro- and anti-calcifying pathways. Reviewed by: While the role of smooth muscle cells and pericytes in ectopic calcifications has been Fan Zhang, Mount Sinai School of Medicine, USA widely investigated, the involvement of fibroblasts is still elusive. Fibroblasts isolated Noriko Hiroi, Keio University, Japan from the dermis of pseudoxanthoma elasticum (PXE) patients and of patients exhibiting *Correspondence: PXE-like clinical and histopathological findings offer an attractive model to investigate the Daniela Quaglino, Department of Life mechanisms leading to the precipitation of mineral deposits within elastic fibers and to Science, University of Modena and explore the influence of the genetic background and of the extracellular environment Reggio Emilia, Via Campi 287, Modena 41125, Italy. on fibroblast-associated calcifications, thus improving the knowledge on the role of e-mail: [email protected] mesenchymal cells on pathologic mineralization. Keywords: fibroblasts, PXE, PXE-like disorders, elastin, extracellular matrix, ectopic calcification, mesenchymal stromal cells CALCIFICATIONS IN SOFT CONNECTIVE TISSUES of genetic diseases, in metabolic disorders, such as uremia, For long time, unwanted calcification, as that occurring in arte- hyper-parathyroidism, and diabetes, or in areas without adja- rial calcification and in nephrolithiasis, has been considered as a cent inflammation or atherosclerosis. Due to the heterogeneity passive, physical–chemical phenomenon representing a degener- of factors contributing to the development of calcifications, many ative, irreversible process often associated with aging (Shroff and studies have been carried out in order to find common patho- Shanahan, 2007). Many recent investigations, however, pointed genetic mechanisms and to identify possible druggable targets out that calcium and phosphate precipitation are the result of (i.e., single molecules and/or signaling pathways). Within this complex and highly regulated series of events in which the balance framework, numerous proteins have been identified to be involved between calcification inducers and inhibitory mechanisms may in bone calcification as well as in ectopic mineralization. It become severely deranged locally and/or systemically. has been suggested that an active and dynamic balance of pro- The deposition of calcium and phosphate in soft connective tis- and anti-calcifying mechanisms occurs in both physiological and sues can be classified into three major categories: metastatic calci- pathological calcification (Abedin et al., 2004) and that mesenchy- fication, dystrophic calcification, and calcinosis (Black and Kanat, mal cells are key players, not only because they synthesize most 1985). Metastatic calcification occurs when calcium–phosphorous of the mineral regulatory proteins, but also because they are levels are elevated mainly due to metabolic/hormonal alterations responsible for the qualitative and quantitative characteristics of and/or to tumor-associated complications. Dystrophic calcifica- the extracellular environment, where apatite ectopic deposition tion takes place in the presence of damaged or necrotic tissue as arises. in atherosclerosis. Calcinosis is generally associated to hypovas- cularity or hypoxia, it may involve a localized area or it may be ROLE OF PRO- AND ANTI-CALCIFYING FACTORS IN ECTOPIC widespread, causing secondary muscle atrophy, joint contractures CALCIFICATION and skin ulceration, with recurrent episodes of inflammation or The role of calcitropic hormones, namely catecholamines, infection (Boulman et al., 2005). parathyroid hormone (PTH), and vitamin D or 1,25(OH)2D3 on In most cases, mineral deposition develops in the extracel- calcium metabolism is well-known (Rizzoli and Bonjour, 1998). lular environment without being localized on specific matrix However, in the last decade, a growing number of evidence is components/structures. A typical example is represented by “cal- highlighting the importance of many other molecules as part ciphylaxis,” a rare disease in which a generalized calcification is of a composite network that, on the basis of common struc- associated with thrombotic cutaneous ischemia and necrosis, thus tural components, exhibits peculiar interactions and/or undergoes causing a mortality rate ranging from 60 to 80% due to wound different regulatory mechanisms depending on the tissue [e.g., infection, sepsis, and subsequent organ failure (Arseculeratne osteoprotegerin (OPG) or matrix Gla protein (MGP) in bone and et al., 2006; Hoff and Homey, 2011). vascular tissue; Kornak, 2011] and on the environmental con- As clearly shown by several experimental findings and clin- text. In addition, these molecules can be produced and locally ical observations, calcification may also occur in a number secreted by mesenchymal cells, or can diffuse from circulation to www.frontiersin.org March 2013 | Volume 4 | Article 22 | 1 “fgene-04-00022” — 2013/3/12 — 13:07 — page1—#1 Ronchetti et al. Fibroblasts in ectopic calcifications peripheral tissues, where they may exert different effects on local constituents, for instance, has been never associated with MVs, calcium/phosphate homeostasis (Figure 1). indicating that fibroblasts, differently from smooth muscle cells, The mechanisms of calcification in skeletal and dental tissues can be responsible for mineral deposition, even in the absence of have been under investigation since long time. One common fea- MVs. It could be, therefore, hypothesized that the role of mes- ture to almost all physiological mineralization mechanisms seems enchymal cells in ectopic calcification may differ depending on the involvement of small (20–200 nm) membrane particles, called the ability of the cell type to acquire a bone-oriented phenotype. matrix vesicles (MVs). They bud off from the plasma membrane To further increase the complexity and the heterogeneity of of mineralizing cells and are released into the pre-mineralized mechanisms regulating pathologic calcification there are stud- organic matrix serving as a vehicle for the concentration of ion ies demonstrating that factors promoting or inhibiting ectopic or ion-enriched substrates, which are required for the activity calcifications are under the control of different genes, as in of membrane-bound enzymes triggering mineral deposition at the case of extracellular pyrophosphate (PPi), a small molecule specific sites. made of two phosphate ions, linked by an ester bond, that reg- The observation that MV-like membranes are present in a num- ulates cell differentiation and serves as an essential physiologic ber of ectopic calcification processes supports the concept that the inhibitor of calcification by negatively interfering with crystal mechanisms of vascular calcification are similar to those seen in growth (Terkeltaub, 2001). The amount of extracellular PPi is reg- normal skeletal development (Golub, 2011). ulated by two different gene products, as it originates either from However, soft connective tissue calcifications activate a num- the breakdown of nucleotide triphosphates by the ectonucleotide ber of common pathways, but, at the same time they may exhibit pyrophosphatase/phosphodiesterase (PC-1/ENPP1) or from the local specific variations (e.g., in different tissue/body regions), PPi transport by the transmembrane ankylosis protein homolog possibly depending on the genotypic/phenotypic peculiarities of (ANKH). Consistently, either mutations or knockdown of these each mesenchymal cell type/subtype. Mineralization of dermal genes can induce hyper-mineralization of aorta (i.e., generalized FIGURE 1 | Major factors involved in mineral deposition. AMP, Glu- and Gla-MGP,uncarboxylated- and carboxylated-matrix Gla protein; adesosine monophosphate; ANKH, ankylosis protein homolog; ATP, OPG, osteoprotegerin; OPN, osteopontin; Pi, inorganic phosphate; Pit-1, adenosine triphosphate; BMP2, bone morphogenetic protein-2; BMP2R, phosphate transporter-1; PPi, pyrophosphate; RANKL, receptor activator of bone morphogenetic protein-2 receptor; BSP,bone sialoprotein; Ca, nuclear factor kappa-B ligand; TNAP,tissue non-specific alkaline calcium; ENPP1, ectonucleotide pyrophosphatase/phosphodiesterase; phosphatase. Frontiers in Genetics | Systems Biology March 2013 | Volume 4 | Article 22 | 2 “fgene-04-00022” — 2013/3/12 — 13:07 — page2—#2 Ronchetti et al. Fibroblasts in ectopic calcifications arterial calcification in infants or GACI) and of ligaments and heart valves, and skeletal muscle. It is involved in the early stages articular cartilage (i.e., chondrocalcinosis) in humans and mice, of mineralization and bone desorption, since it is immobilized respectively (Okawa et al., 1998; Ho et al., 2000; Pendleton et al., on collagen fibrils where the poly-glutamic acid sequences of BSP 2002; Rutsch et al., 2003). act as possible nucleation sites for hydroxyapatite