Ectopic calcification in ß-thalassemia patients is associated with increased oxidative stress and lower MGP carboxylation.

A number of beta-thalassemia (ß-thal) patients in the course of the disease exhibit ectopic calcification affecting skin, eyes and the cardiovascular system. Clinical and histopathological features have been described similar to those in pseudoxanthoma elasticum (PXE), although different genes are affected in the two diseases. Cultured dermal fibroblasts from ß-thal patients with and without PXE-like clinical manifestations have been compared for parameters of redox balance and for the expression of proteins, which have been already associated with the pathologic mineralisation of soft connective tissues. Even though oxidative stress is a well-known condition of ß-thal patients, our results indicate that the occurrence of mineralized elastin is associated with a more pronounced redox disequilibrium, as demonstrated by the intracellular increase of anion superoxide and of oxidized proteins and lipids. Moreover, fibroblasts from ß-thal PXE-like patients are characterized by decreased availability of carboxylated matrix Gla protein (MGP), as well as by altered expression of proteins involved in the vitamin K-dependent carboxylation process. Results demonstrate that elastic fibre calcification is promoted when redox balance threshold levels are exceeded and the vitamin K-dependent carboxylation process is affected decreasing the activity of MGP, a well-known inhibitor of ectopic calcification. Furthermore, independently from the primary gene defect, these pathways are similarly involved in fibroblasts from PXE and from ß-thal PXE-like patients as well as in other diseases leading to ectopic calcification, thus suggesting that can be used as markers of pathologic mineralisation.

Fibroblast protein profile analysis highlights the role of oxidative stress and vitamin K recycling in the pathogenesis of pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is a genetic disorder associated to mutations in the ABCC6 gene; however, the pathogenetic mechanisms leading to elastic fibre calcifications and to clinical manifestations are still unknown. Dermal fibroblasts, directly involved in the production of the extracellular milieu, have been isolated from healthy subjects and from patients affected by PXE, cultured in vitro and characterized for their ability to produce reactive oxygen species, for structural and functional properties of their cell membranes, for changes in their protein profile. Data demonstrate that oxidative stress has profound and endurable consequences on PXE fibroblast phenotype being responsible for: reduced levels of global DNA methylation, increased amount of carbonylated proteins and of lipid peroxidation products, altered structural properties of cell membranes, modified protein expression. Data shed new light on the pathogenetic pathways in PXE, by identifying a network of proteins affecting elastic fibre calcification through inefficient vitamin K recycling, and highlight the role of differentially expressed proteins as targets for validating the efficacy of future therapeutic strategies aiming to delay and/or revert the pathologic phenotype of PXE fibroblasts. Moreover, data open new perspectives for investigating PXE-like phenotypes in the absence of ABCC6 mutations.

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.

A new generation of sterile and radiopaque impression materials: an in vitro cytotoxicity study.

Impression materials are largely used to record the geometry of dental tissue. Hence, the assessment of their possible cytotoxicity is a necessary step in the evaluation of their biocompatibility. The present study is carried out to evaluate the cytotoxicity of a new elastomeric sterile and radiopaque impression material. Human gingival fibroblasts, cultured in vitro are exposed directly to Elite Implant in three different viscosities, heavy, medium, and light. At 3, 9, 24, 48, and 72 h, the cellular proliferation is evaluated. In parallel, human gingival fibroblasts are exposed indirectly by means of fluid extracts of Elite Implant. The cellular viability is evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, (MTT) assay (Sigma, St Louis, Mo). The gingival fibroblasts proliferation and viability are unaffected by the presence of Elite Implant. This new impression material may represent a safe medical device for clinical and surgical applications. In addition, this material is radiopaque and, thus, can be identified radiographically.