Matrix gla protein and alkaline phosphatase are differently modulated in human dermal fibroblasts from PXE patients and controls.

Mineralization of elastic fibers in pseudoxanthoma elasticum (PXE) has been associated with low levels of carboxylated matrix gla protein (MGP), most likely as a consequence of reduced vitamin K (vit K) availability. Unexpectedly, vit K supplementation does not exert beneficial effects on soft connective tissue mineralization in the PXE animal model. To understand the effects of vit K supplementation and in the attempt to interfere with pathways leading to the accumulation of calcium and phosphate within PXE-mineralized soft connective tissues, we have conducted in vitro studies on dermal fibroblasts isolated from control subjects and from PXE patients. Cells were cultured in standard conditions and in calcifying medium (CM) in the presence of vit K1 and K2, or levamisole, an alkaline phosphatase (ALP) inhibitor. Control and PXE fibroblasts were characterized by a similar dose-dependent uptake of both vit K1 and vit K2, thus promoting a significant increase of total protein carboxylation in all cell lines. Nevertheless, MGP carboxylation remained much less in PXE fibroblasts. Interestingly, PXE fibroblasts exhibited a significantly higher ALP activity. Consistently, the mineralization process induced in vitro by a long-term culture in CM appeared unaffected by vit K, whereas it was abolished by levamisole.

Heparan sulfate affects elastin deposition in fibroblasts cultured from donors of different ages.

Heparan sulfate (HS), due to its presence on the cell surface and in the extracellular milieu and its ability to modulate cell signaling, has a fundamental role in both physiological and pathological conditions. For decades we have demonstrated the occurrence of interactions between glycosaminoglycans (GAGs) and elastic fibers. In particular, we have recently shown that HS is present inside elastic fibers and plays a role in the assembly and stability of elastin coacervates. Elastin represents, within the extracellular matrix, the component most severely affected during aging, and changes in the synthesis and posttranslational modifications of HS have been described, possibly influencing cellular behavior and protein interactions. Thus, the present study has investigated, in two different in vitro experimental models, the role of HS on elastin deposition and assembly. Results demonstrate that: (1) Biological effects of HS are partly dependent on the physicochemical characteristics of the GAGs; (2) HS does not affect attachment, viability, and growth of human dermal fibroblasts; (3) HS does not modify elastin gene expression nor elastin synthesis, but favors α-elastin aggregation and, independently from the age of donors, elastin assembly; (4) HS significantly increases the expression of fibulin 5, and these effects are especially evident in fibroblasts isolated from aging donors. These data provide a better understanding of the biological role of HS and offer new perspectives regarding the possibility of restoring and/or preserving the elastic component with aging.

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.

Hypoxia influences the cellular cross-talk of human dermal fibroblasts. A proteomic approach.

The ability of cells to respond to changes in oxygen availability is critical for many physiological and pathological processes (i.e. development, aging, wound healing, hypertension, cancer). Changes in the protein profile of normal human dermal fibroblasts were investigated in vitro after 96 h in 5% CO(2) and 21% O(2) (pO(2) = 140 mm Hg) or 2% O(2) (pO(2) = 14 mm Hg), these parameters representing a mild chronic hypoxic exposure which fibroblasts may undergo in vivo. The proliferation rate and the protein content were not significantly modified by hypoxia, whereas proteome analysis demonstrated changes in the expression of 56 proteins. Protein identification was performed by mass spectrometry. Data demonstrate that human fibroblasts respond to mild hypoxia increasing the expression of hypoxia inducible factor (HIF1a) and of the 150-kDa oxygen-regulated protein. Other differentially expressed proteins appeared to be related to stress response, transcriptional control, metabolism, cytoskeleton, matrix remodelling and angiogenesis. Furthermore, some of them, like galectin 1, 40S ribosomal protein SA, N-myc-downstream regulated gene-1 protein, that have been described in the literature as possible cancer markers, significantly changed their expression also in normal hypoxic fibroblasts. Interestingly, a bovine fetuin was also identified that appeared significantly less internalised by hypoxic fibroblasts. In conclusion, results indicate that human dermal fibroblasts respond to an in vitro mild chronic hypoxic exposure by modifying a number of multifunctional proteins. Furthermore, data highlight the importance of stromal cells in modulating the intercellular cross-talk occurring in physiological and in pathologic conditions.

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.

17 Beta-estradiol prevents cytotoxicity from hydrophobic bile acids in HepG2 and WRL-68 cell cultures.

BACKGROUND: Epidemiological and clinical studies suggest the possibility that estrogens might have a cytoprotective effect on the liver. The aim of the present study was to test the hypothesis that 17beta-estradiol (E2) prevents hepatocellular damage induced by deoxycholic acid (DCA), a hydrophobic bile acid. METHODS: HepG2 cells were exposed for 24 h to DCA (350 micromol/L). Cell viability, aspartate aminotransferase and lactate dehydrogenase activity and apoptosis were measured as indices of cell toxicity. The effect of DCA was compared to that observed using either a hydrophilic bile acid, ursodeoxycholic acid (UDCA; 100 micromol/L), or E2 at different concentrations (1 nmol/L, 10 nmol/L, 50 nmol/L and 50 micromol/L) or mixtures of E2/DCA or UDCA/DCA. The same experiments were performed using WRL-68 cells that, at variance with HepG2, express a higher level of nuclear estrogen receptor. RESULTS: High concentrations of E2 and UDCA prevented DCA-induced decrease in cell viability, increase in enzyme activity and apoptosis evaluated both by 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) and TdT-mediated dUTP nick-end labeling (TUNEL) assays. In addition, DCA-related apoptosis, assessed by caspase activity, was also prevented by E2 (P < 0.01) in physiological (1-10 nmol/L) doses. The cytoprotective effects of E2 and UDCA was also observed in the WRL-68 cell line. CONCLUSIONS: 17Beta-Estradiol prevents DCA-induced cell damage in HepG2 and WRL-68 cell lines to an extent comparable to UDCA. The hypothesis that the protective effect of E2 may be mediated by a mechanism that is nuclear estrogen receptor independent, deserves further verification.

Oxidative stress in fibroblasts from patients with pseudoxanthoma elasticum: possible role in the pathogenesis of clinical manifestations.

Pseudoxanthoma elasticum (PXE) is a genetic disease characterized by calcification and fragmentation of elastic fibres of the skin, cardiovascular system and eye, caused by mutations of the ABCC6 gene, which encodes the membrane transporter MRP6. The pathogenesis of the lesions is unknown. Based on studies of similar clinical and histopathological damage present in haemolytic disorders, our working hypothesis is that PXE lesions may result from chronic oxidative stress occurring in PXE cells as a consequence of MRP6 deficiency. Our results show that PXE fibroblasts suffer from mild chronic oxidative stress due to the imbalance between production and degradation of oxidant species. The findings also show that this imbalance results, at least in part, from the loss of mitochondrial membrane potential (DeltaPsi(m)) with overproduction of H2O2. Whether mitochondrial dysfunction is the main factor responsible for the oxidative stress in PXE cells remains to be elucidated. However, mild chronic generalized oxidative stress could explain the great majority of structural and biochemical alterations already reported in PXE.

Dermal fibroblasts from pseudoxanthoma elasticum patients have raised MMP-2 degradative potential.

Cultured fibroblasts from the dermis of normal subjects and of Pseudoxanthoma elasticum (PXE) patients were analysed for enzyme activity, protein and mRNA expression of metalloproteases (MMP-2, MMP-3, MMP-9, MT1-MMP) and of their specific inhibitors (TIMP-1, TIMP-2 and TIMP-3). MMP-3, MMP-9 and TIMP-3 mRNAs and proteins failed to be detected in both the medium and the cell layer of both controls and PXE patients. MMP-2 mRNA was significantly more expressed in PXE than in control cell lines, whereas MT1-MMP, TIMP-1 and TIMP-2 mRNAs appeared unchanged. MMP-2 was significantly higher in the cell extracts from PXE fibroblasts than in control cells, whereas differences were negligible in the cell medium. Data suggest that PXE fibroblasts have an increased proteolytic potential, and that MMP-2 may actively contribute to connective tissue alterations in this genetic disorder.

Adhesion and proliferation of human dermal fibroblasts on collagen matrix.

The purpose of this study was to evaluate adhesion and growth of human dermal fibroblasts on a 0.150 mm-thick matrix of reconstituted collagen isolated from horse tendon. Collagen was extracted and polymerized according to the standard procedures (Opocrin, Corlo, Modena, Italy). By light microscopy, the bottom surface of the matrix appeared linear and compact, whereas the superficial one was indented and less homogeneous. By scanning electron microscopy, the collagen fibrils had different diameters and the great majority of them was oriented parallel to the surface of the gel. By transmission electron microscopy, collagen fibrils showed the typical banding. Human dermal fibroblasts were seeded on the collagen matrix, previously equilibrated in growth medium. Fibroblast proliferation stopped in the second week and was always significantly lower than that of the same cell strain seeded on plastic and cultured in parallel. By light microscopy, after six days culture, cells formed a confluent multilayer on the surface of the gel. By scanning and transmission electron microscopy, fibroblasts appeared flat and adherent to the matrix. Contacts of cells among themselves and with the collagen fibrils were observed. Fibroblasts never moved into the collagen gel. In conclusion, human dermal fibroblasts can be grown in a three-dimensional matrix made by horse tendon that, on the other hand, seems to condition their proliferation rate.

Proteome analysis of dermal fibroblasts cultured in vitro from human healthy subjects of different ages.

Aging is a complex multifactorial process still far from being completely understood. The aim of the present study was to compare the proteome of in vitro cultured dermal fibroblasts from healthy subjects of different ages (i.e. 15 +/- 2, 41 +/- 4 and 82 +/- 3 years old). Proteins of the cell layer were separated by two-dimensional electrophoresis and protein identification was performed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry; moreover, synthetic gels were qualitatively and quantitatively analyzed by Melanie 3 software. Our study did not reveal any protein typical of any one age group. On the other hand, we observed 38 proteins exhibiting more than three-fold reproducible variations with aging, some (45%) being reduced such as F-actin capping protein alpha1, proteasome subunit alpha type 3, heat shock protein 27, ubiquitin carboxyl-terminal hydrolase isozyme L1, mitochondrial thioredoxin-dependent peroxide reductase, cathepsin B, glutathione S-transferase P, cyclophilin A and calgizzarin. In contrast, T-complex protein 1, probable protein disulfide isomerase ER60, phosphoglycerate kinase 1, Ran-specific GTPase-activating protein, proteasome subunit alpha type 5, triosephosphate isomerase and superoxide dismutase (Mn) increased with age. Furthermore, annexin 1, elongation factor 1beta, proteasome activator complex subunit 1, phosphoglycerate mutase, superoxide dismutase (Cu-Zn) and cofilin, exhibited the highest levels in adult cells; whereas, septin 2 homolog, RNA-binding protein regulatory subunit and ATP synthase D chain revealed the lowest values in adults. The present investigation, underlining the complexity of the aging process, highlights the role of synthetic and degradative pathways in modulating the whole cell machinery and emphasizes that metabolic impairment with age could depend partly on different expression of a number of genes and leading to an imbalance among functional proteins.

Normal human dermal fibroblasts: proteomic analysis of cell layer and culture medium.

Proteins present within the cell layer and those released in the cell medium from in vitro cultured normal human dermal fibroblasts were separated and characterized in terms of their isoelectric point and molecular weight, by two-dimensional (2-D) gel electrophoresis. All spots in the synthetic gel were firstly analyzed by the Melanie 3 software and compared with those of breast cancer cells, colorectal epithelial cells, HL60, lymphoma cells, and platelets, already available on-line. From the identification of 144 spots from both the cell layer and the medium, we were able to recognize 89 different proteins, since a certain number of spots represented different isoforms of the same molecule. Identifications were performed by matching with on-line 2-D databases, and by matrix assisted laser-desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), in order to confirm the identification by matching, or to identify new proteins. The procedure we used allows (i) to design a highly reproducible reference map of the proteome of adult human normal fibroblasts in culture, (ii) to evaluate protein species produced in the cell layer as well as those released in the culture medium, and (iii) to compare data from gel matching with those obtained by MS. This work represents an essential step for a better knowledge of mesenchymal cells, given the widespread use of this cell type in both clinical and experimental investigations.

Cell-matrix interactions of in vitro human skin fibroblasts upon addition of hyaluronan.

Normal human skin fibroblasts were grown in a three-dimensional collagen gel or in monolayer in the presence or absence of high molecular weight hyaluronan (HA) to assess the influence of extracellular HA on cell-matrix interactions. HA incorporated into the collagen gel or added to the culture medium did not modify lattice retraction with time. The effect was independent from HA molecular weight (from 7.5 x 10(5) to 2.7 x 10(6) Da) and concentration (from 0.1 up to 1 mg/ml). HA did not affect shape and distribution of fibroblasts within the gel, whereas it induced the actin filaments to organise into thicker cables running underneath the plasma membrane. The same phenomenon was observed in fibroblasts grown in monolayer. By contrast, vimentin cytoskeleton and cell-substrate focal adhesions were not modified by exogenous HA. The number of fibroblasts attached to HA-coated dishes was always significantly lower compared to plastic and to collagen type I-coated plates. By contrast, adhesion was not affected by soluble HA added to the medium nor by anti-CD44 and anti-RHAMM-IHABP polyclonals. After 24-h seeding on collagen type I or on plastic, cells were large and spread. Conversely, cells adherent to HA-coated surfaces were long, thin and aligned into rows; alcian blue showed that cells were attached to the plastic in between HA bundles. Therefore, normal human skin fibroblasts exhibit very scarce, if any, adhesion to matrix HA, either soluble or immobilised. Moreover, even at high concentration, HA molecules do not exert any visco-mechanical effect on lattice retraction and do not interfere with fibroblast-collagen interactions nor with focal adhesion contacts of fibroblasts with the substrate. This is probably relevant in organogenesis and wound repair. By contrast, HA greatly modifies the organisation of the actin cytoskeleton, suggesting that CD44-mediated signal transduction by HA may affect cell locomotion and orientation, as indicated by the fusiform shape of fibroblasts grown in the presence of immobilised HA. A role of HA in cell orientation could be relevant for the deposition of collagen fibrils in regeneration and tissue remodelling.

Functional analysis of the promoter of human sterol 27-hydroxylase gene in HepG2 cells.

Human sterol 27-hydroxylase catalyses the first step in the alternative pathway of bile acids biosynthesis in hepatocytes. However the gene encoding this enzyme (CYP27 gene) is expressed in every tissue and some evidence suggests that this enzyme plays a role in cholesterol homeostasis. Although modulation of CYP27 expression has been reported, the mechanisms underlying the regulation of this gene in human tissues is still poorly understood. To elucidate the mechanism governing CYP27 expression we cloned a 4.3 kb fragment of the 5' flanking region of the human CYP27 gene and constructed deletion mutants which were transfected into HepG2 cells. Functional assays showed that the -217/-10 nucleotide region from the translation start site (minimal promoter), devoid of TATA and CAAT boxes, contains all the elements for basal transcription. Foot-printing analysis of minimal promoter showed four protected regions (A-D). Regions A, B and D each contain one Sp1 binding site, and region C contains a HNF4 site. Electrophoretic mobility shift assays demonstrated that Sp1, Sp3 and HNF4 transcription factors bind these sites. Mutagenesis of any of these sites resulted in the loss of promoter activity. Co-transfection of the minimal promoter with Sp1 and Sp3 expression vectors transactivated CYP27 gene promoter in Drosophila SL2 cells, which lack endogenous Sp proteins. Transactivation of the minimal promoter was also observed in HeLa cells co-transfected with HNF4 expression vector. Therefore, Sp1, Sp3 and HNF4 co-operate in the expression of the human CYP27 gene in HepG2 cells.