The Multifaceted Complexity of Genetic Diseases: A Lesson from Pseudoxanthoma Elasticum

Pseudoxanthoma elasticum (PXE) is a rare genetic disorder characterized by mineralization of elastic fibers within all connective tissue, although the most important clinical manifestation affect skin, eyes and the cardiovascular system. Despite the dramatic involvement of the extracellular matrix, the first attempts made by researchers to find out the gene defect among those coding for matrix molecules failed and in 2000 three groups, independently, demonstrated that PXE is due to mutation in the ABCC6 gene belonging to the ABC family of membrane transporters. Today the physiological substrate of this transporter is not know and still elusive are the pathogenetic mechanisms linking a defective cellular transporter mainly expressed in liver and kidney to ectopic calcification of connective tissues. This disease may therefore represent a very interesting example of the complexity that regulate molecular pathways, on the influence of metabolism on several other organs/systems. Moreover, there are also evidence that similar endpoints (i.e. clinical and histological alterations) can be observed in some patients starting from different gene defects (Pseudoxanthoma, Beta-thalassemia, vitamin-k dependent coagulation deficiency). These data support the importance of using wide-spread technologies as transcriptomic or proteomic analysis to have a broader view of the cellular pathways that may be involved. Moreover recent findings in the literature highlights the role of polymorphisms in other genes that could be responsible for phenotypic changes and for a different severity of clinical manifestation in this monogenic disorder

Comparison of ex vivo and in vitro human fibroblast ageing models

Several studies have analyzed modulation of gene expression during physiological ageing with interesting, but often contradictory results, depending on the model used. In the present report we compare age-related metabolic and synthetic parameters in human dermal fibroblasts (HDF) isolated from young and old subjects (ex vivo ageing model) and cultured from early up to late cumulative population doublings (CPD) (in vitro ageing model) in order to distinguish changes induced in vivo by the aged environment and maintained in vitro, from those associated with cell senescence and progressive CPD. Results demonstrate that fibroblasts from aged donors, already at early CPD, exhibit an impaired redox balance, highlighting the importance of this parameter during ageing, even in the presence of standard environmental conditions, which are considered optimal for cell growth. By contrast, several proteins, as those related to heat shock response, or involved in endoplasmic reticulum and membrane trafficking, appeared differentially expressed only during in vitro ageing, suggesting that, at high CPD, the whole cell machinery becomes permanently altered. Finally, given the importance of the elastic component for a long-lasting connective tissue structural and functional compliance, this study focuses also on elastin and fibulin-5 synthesis and deposition, demonstrating a close relationship between fibulin-5 and ageing

Cytotoxic Evaluation of Elastomeric Dental Impression Materials on a Permanent Mouse Cell Line and on a Primary Human Gingival Fibroblast Culture

The need for clinically relevant in vitro tests of dental materials is widely recognized. Nearly all dental impression materials are introduced into the mouth just after mixing and allowed to set in contact with the oral tissues. Under these conditions, the materials may be toxic to cells or may sensitize the tissues. The aim of the present study is to evaluate the potential cytotoxicity of new preparations of elastomeric dental impression materials: A) four vinylpolysiloxanes: Elite H-D Putty and Elite H-D Light Body (Zhermack, Badia Polesine, Rovigo, Italy); Express Putty and Express Light Body (3M ESPE AG Seefeld, Germany) and B) two polyethers: Impregum Penta and Permadyne Penta L (3M ESPE AG Seefeld, Germany). The cytotoxicity of these impression materials were examined using two different cell lines: Balb/c 3T3 (permanent cell line) and human gingival fibroblasts (primary cell line) and their effects were studied by indirect and direct tests. The direct tests are performed by placing one sample of the impression materials in the centre of the Petri dishes at the time of the seeding of cells. The cell growth was evaluated at the 12th and 24th hours by cell number. The indirect tests were performed by incubating a square of 1 cm diameter impression material in 5 mL of medium at 37 °C for 24 hours (“eluates”). Subconfluent cultures are incubated with “eluates” for 24 hours. The MTT-formazan production is the method used for measuring the cell viability. The results indicate that: a) polyether materials are cytotoxic under both experimental conditions; b) among vinylpolysiloxanes, only Express Light Body (3M ESPE AG Seefeld, Germany) induces clear inhibition of cellular viability of Balb/c 3T3 evaluated by direct and indirect tests and c) the primary cell line is less sensitive to the toxic effect than the permanent cell line

Fibroblast involvement in soft connective tissue calcification

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 mineralizatio

Changes in dermal fibroblasts from Abcc6-/- mice are present before and after the onset of ectopic tissue mineralization

Pseudoxanthoma elasticum (PXE), a rare genetic disease caused by mutations in the ABCC6 gene, is characterized by progressive calcification of elastic fibers in the skin, eyes, and the cardiovascular system. The pathomechanism of the mineralization is still obscure. Several hypotheses have been proposed, one of them suggesting a role for fibroblasts in controlling the amount and the quality of the calcified extracellular matrix. This hypothesis raises the question whether changes in mesenchymal cells are the cause and/or the consequences of the calcification process. In this study, fibroblasts were isolated and cultured from Abcc6+/+ and Abcc6-/- mice of different ages to investigate parameters known to be associated with the phenotype of fibroblasts from PXE patients. Results demonstrate that a few changes (Ank and Opn downregulation) are already present before the occurrence of calcification.By contrast, a modification of other parameters (intracellularO2- content, Tnap activity, and Bmp2 upregulation) can be observed inAbcc6-/- mice after the onset of tissue mineralization.These data suggest that in the Abcc6 -/- genotype, dermal fibroblasts actively contribute to changes that promote matrix calcification and that these cells can be further modulated with time by the calcified environment, thus contributing to the age-dependent progression of the disease

Histology-directed and imaging mass spectrometry: An emerging technology in ectopic calcification

The present study was designed to demonstrate the potential of an optimized histology directed protein identification combined with imaging mass spectrometry technology to reveal and identify molecules associated to ectopic calcification in human tissue. As a proof of concept, mineralized and non-mineralized areas were compared within the same dermal tissue obtained from a patient affected by Pseudoxanthoma elasticum, a genetic disorder characterized by calcification only at specific sites of soft connective tissues. Data have been technically validated on a contralateral dermal tissue from the same subject and compared with those from control healthy skin. Results demonstrate that this approach 1) significantly reduces the effects generated by techniques that, disrupting tissue organization, blend data from affected and unaffected areas; 2) demonstrates that, abolishing differences due to inter-individual variability, mineralized and non-mineralized areas within the same sample have a specific protein profile and have a different distribution of molecules; and 3) avoiding the bias of focusing on already known molecules, reveals a number of proteins that have been never related to the disease nor to the calcification process, thus paving the way for the selection of new molecules to be validated as pathogenic or as potential pharmacological targets

Mineralization by mesenchymal stromal cells is variously modulated depending on commercial platelet lysate preparations

Background aims: Numerous cellular models have been developed to investigate calcification for regenerative medicine applications and for the identification of therapeutic targets in various complications associated with age-related diseases. However, results have often been contradictory due to specific culture conditions, cell type ontogeny and aging status. Human platelet lysate (hPL) has been recently investigated as valuable alternative to fetal bovine serum (FBS) in cell culture and bone regeneration. A parallel comparison of how all these multiple factors may converge to influence mineralization has yet to be reported. Methods: To compare mineralization of human mesenchymal cell types known to differ in extracellular matrix calcification potency, bone marrowâderived mesenchymal stromal cells and dermal fibroblasts from neonatal and adult donors, at both low and high passages, were investigated in an ex vivo experimental model by supplementing the osteogenic induction medium with FBS or with hPL. Four commercial hPL preparations were profiled by liquid chromatography/electrospray ionization quadrupole time-of-flight spectrometry, and mineralization was visualized by von Kossa staining and quantified by morphometric evaluations after 9, 14 and 21 days of culture. Results: Data demonstrate that (i) commercial hPL preparations differ according to mass spectra profiles, (ii) hPL variously influences mineral deposition depending on cell line and possibly on platelet product preparation methods, (iii) donor age modifies mineral deposition in the presence of the same hPL and (iv) reduced in vitro proliferative capacity affects osteogenic induction and response to hPL. Conclusion: Despite the standardized procedures applied to obtain commercial hPL, this study highlights the divergent effects of different preparations and emphasizes the importance of cellular ontology, donor age and cell proliferative capacity to optimize the osteogenic induction capabilities of mesenchymal stromal cells and design more effective cell-based therapeutic protocols

Donor's age and replicative senescence favour the in-vitro mineralization potential of human fibroblasts

Aberrant mineralization of soft connective tissues (ectopic calcification) may occur as a frequent age-related complication. Still, it remains unclear the role of mesenchymal cell donor's age and of replicative senescence on ectopic calcification. Therefore, the ability of cells to deposit in-vitro hydroxyapatite crystals and the expression of progressive ankylosis protein homolog (ANKH), ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), tissue non specific alkaline phosphatase (TNAP) and osteopontin (OPN) have been evaluated in human dermal fibroblasts derived from neonatal (nHDF) and adult (aHDF) donors (ex-vivo ageing model) or at low and high cumulative population doublings (CPD) up to replicative senescence (in-vitro ageing model). This study demonstrates that: 1) replicative senescence favours hydroxyapatite formation in cultured fibroblasts; 2) donor's age acts as a major modulator of the mineralizing potential of HDF, since nHDF are less prone than aHDF to induce calcification; 3) donor's age and replicative senescence play in concert synergistically increasing the calcification process; 4) the ANKH+ENPP1/TNAP ratio, being crucial for pyrophosphate/inorganic phosphate balance, is greatly influenced by donor's age, as well as by replicative senescence, and regulates mineral deposition; 5) OPN is only modulated by replicative senescence

The effect of serum withdrawal on the protein profile of quiescent human dermal fibroblasts in primary cell culture

The effect of serum deprivation on proliferating cells is well known, in contrast its role on primary cell cultures, at confluence, has not been deeply investigated. Therefore, in order to explore the response of quiescent cells to serum deprivation, ubiquitous mesenchymal cells, as normal human dermal fibroblasts, were grown, for 48 h after confluence, in the presence or absence of 10% FBS. Fibroblast behaviour (i.e. cell morphology, cell viability, ROS production and elastin synthesis) was evaluated morphologically and biochemically. Moreover, the protein profile was investigated by 2-DE and differentially expressed proteins were identified by MS. Serum withdrawal caused cell shrinkage but did not significantly modify the total cell number. ROS production, as evaluated by the dihydroethidium (DH2) probe, was increased after serum deprivation, whereas elastin synthesis, measured by a colorimetric method, was markedly reduced in the absence of serum. By proteome analysis, 41 proteins appeared to significantly change their expression, the great majority of protein changes were related to the cytoskeleton, the stress response and the glycolytic pathway. Data indicate that human dermal fibroblasts in primary cell culture can adapt themselves to environmental changes, without significantly altering cell viability, at least after a few days of treatment, even though serum withdrawal represents a stress condition capable to increase ROS production, to influence cell metabolism and to interfere with cell behaviour, favouring the expression of several age-related features