Low serum vitamin K in PXE results in defective carboxylation of mineralization inhibitors similar to the GGCX mutations in the PXE-like syndrome.

Soft-tissue mineralization is a tightly regulated process relying on the activity of systemic and tissue-specific inhibitors and promoters of calcium precipitation. Many of these, such as matrix gla protein (MGP) and osteocalcin (OC), need to undergo carboxylation to become active. This post-translational modification is catalyzed by the gammaglutamyl carboxylase GGCX and requires vitamin K (VK) as an essential co-factor. Recently, we described a novel phenotype characterized by aberrant mineralization of the elastic fibers resulting from mutations in GGCX. Because of the resemblance with pseudoxanthoma elasticum (PXE), a prototype disorder of elastic fiber mineralization, it was coined the PXE-like syndrome. As mutations in GGCX negatively affect protein carboxylation, it is likely that inactive inhibitors of calcification contribute to ectopic mineralization in PXE-like syndrome. Because of the remarkable similarities with PXE, we performed a comparative study of various forms of VK-dependent proteins in serum, plasma (using ELISA), and dermal tissues (using immunohistochemistry) of PXE-like and PXE patients using innovative, conformation-specific antibodies. Furthermore, we measured VK serum concentrations (using HPLC) in PXE-like and PXE samples to evaluate the VK status. In PXE-like patients, we noted an accumulation of uncarboxylated Gla proteins, MGP, and OC in plasma, serum, and in the dermis. Serum levels of VK were normal in these patients. In PXE patients, we found similar, although not identical results for the Gla proteins in the circulation and dermal tissue. However, the VK serum concentration in PXE patients was significantly decreased compared with controls. Our findings allow us to conclude that ectopic mineralization in the PXE-like syndrome and in PXE results from a deficient protein carboxylation of VK-dependent inhibitors of calcification. Although in PXE-like patients this is due to mutations in the GGCX gene, a deficiency of the carboxylation co-factor VK is at the basis of the decreased activity of calcification inhibitors in PXE.

Novel deletions causing pseudoxanthoma elasticum underscore the genomic instability of the ABCC6 region.

Mutations in ABCC6 cause pseudoxanthoma elasticum (PXE), a heritable disease that affects elastic fibers. Thus far, >200 mutations have been characterized by various PCR-based techniques (primarily direct sequencing), identifying up to 90% of PXE-causing alleles. This study wanted to assess the importance of deletions and insertions in the ABCC6 genomic region, which is known to have a high recombinational potential. To detect ABCC6 deletions/insertions, which can be missed by direct sequencing, multiplex ligation-dependent probe amplification (MLPA) was applied in PXE patients with an incomplete genotype. MLPA was performed in 35 PXE patients with at least one unidentified mutant allele after exonic sequencing and exclusion of the recurrent exon 23-29 deletion. Six multi-exon deletions and four single-exon deletions were detected. Using MLPA in addition to sequencing, we expanded the ABCC6 mutation spectrum with 9 novel deletions and characterized 25% of unidentified disease alleles. Our results further illustrate the instability of the ABCC6 genomic region and stress the importance of screening for deletions in the molecular diagnosis of PXE.

Parameters of oxidative stress are present in the circulation of PXE patients.

Pseudoxanthoma elasticum (PXE) is an inherited disorder characterized by calcification of elastic fibres leading to dermatological and vascular alterations associated to premature aged features and to life threatening clinical manifestations. The severity of the disease is independent from the type of mutation in the ABCC6 gene, and it has been suggested that local and/or systemic factors may contribute to the occurrence of clinical phenotype. The redox balance in the circulation of 27 PXE patients and of 50 healthy subjects of comparable age was evaluated by measuring the advanced oxidation protein products (AOPP), the lipid peroxidation derivatives (LOOH), the circulating total antioxidant status (TAS), the thiol content and the extracellular superoxide dismutase activity (EC-SOD). Patients were diagnosed by clinical, ultrastructural and molecular findings. Compared to control subjects, PXE patients exhibited significantly lower antioxidant potential, namely circulating TAS and free thiol groups, and higher levels of parameters of oxidative damage, as LOOH and of AOPP, and of circulating EC-SOD activity. Interestingly, the ratio between oxidant and antioxidant parameters was significantly altered in PXE patients and related to various score indices. This study demonstrates, for the first time, that several parameters of oxidative stress are modified in the blood of PXE patients and that the redox balance is significantly altered compared to control subjects of comparable age. Therefore, in PXE patients the circulating impaired redox balance may contribute to the occurrence of several clinical manifestations in PXE patients, and/or to the severity of disease, thus opening new perspectives for their management.

Exon 26-coded polypeptide: an isolated hydrophobic domain of human tropoelastin able to self-assemble in vitro.

Hydrophobic domains of human tropoelastin are able to aggregate in a variegated manner. Some aggregates have typical features of the whole protein while others show peculiar self-assembling profiles. Among these hydrophobic domains, an important role in the self-assembling properties of tropoelastin in vitro could be assigned to the peptide encoded by exon 26 of the human tropoelastin gene, that, although unstructured in solution, has great tendency to self-assemble in an ordered manner. The present report describes the aggregation properties of this hydrophobic domain of human tropoelastin analysed by different ultra-structural approaches. Transmission electron microscopy shows that the peptide is able to form different aggregation entities from short rods to very long and flexible fibers, depending on the temperature and on the incubation time. At a microm scale, very long fibers as well as fractal aggregation patterns were observed. Data show that the isolated domain encoded by exon 26 of the tropoelastin gene is able to aggregate in a manner very similar to the whole tropoelastin protein. The aggregation properties are due to the peculiar sequence of EX26, and not to its amino acid composition, as evidenced by the supramolecular analysis of a scrambled sequence of exon 26-coded domain of human tropoelastin, showing a quite different aggregation patterns. These findings confirm that specific sequences can play a driving role in the aggregation process of tropoelastin molecule, at least in vitro, and indicate exon 26-encoded domain among these sequences.

Pseudoxanthoma elasticum-like phenotype with cutis laxa and multiple coagulation factor deficiency represents a separate genetic entity.

Data on six patients with a Pseudoxanthoma Elasticum (PXE)-like phenotype, characterized by excessive skin folding (resembling cutis laxa) and a deficiency of the vitamin K-dependent clotting factors (II, VII, IX, and X) are presented. A comparison is made between the clinical, ultrastructural, and molecular findings in these patients and those seen in classic PXE and cutis laxa, respectively. Clinical overlap with PXE is obvious from the skin manifestations of yellowish papules or leathery plaques with dot-like depressions at presentation, angioid streaks and/or ocular peau d'orange, and fragmentation and calcification of elastic fibers in the dermis. Important phenotypic differences with PXE include much more severe skin laxity with spreading toward the trunk and limbs with thick, leathery skin folds rather than confinement to flexural areas, and no decrease in visual acuity. Moreover, detailed electron microscopic analyses revealed that alterations of elastic fibers as well as their mineralization were slightly different from those in classic PXE. Molecular analysis revealed neither causal mutations in the ABCC6 gene (ATP-binding cassette subfamily C member 6), which is responsible for PXE, nor in VKORC1 (vitamin K 2,3 epoxide reductase), known to be involved in vitamin K-dependent factor deficiency. However, the GGCX gene (gamma-glutamyl carboxylase), encoding an enzyme important for gamma-carboxylation of gla-proteins, harbored mutations in six out of seven patients analyzed. These findings all support the hypothesis that the disorder indeed represents a separate clinical and genetic entity, the molecular background of which remains to be unraveled.

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.

Dissection of human tropoelastin: supramolecular organization of polypeptide sequences coded by particular exons.

Polypeptide sequences encoded by some exons of the human tropoelastin gene (EDP, elastin-derived peptide) have been analysed for their ability to coacervate and to self-assembly. The great majority of them were shown to form organized structures, but only a few were indeed able to coacervate. Negative staining and rotary shadowing transmission electron microscopy showed the polypeptides to adopt a variety of supramolecular organization, from filaments, as those typical of tropoelastin, to amyloid-like fibers. The results obtained gave significant insight to the possible roles played by specific polypeptide sequences of tropoelastin.

Heparan sulphate interacts with tropoelastin, with some tropoelastin peptides and is present in human dermis elastic fibers.

A number of reports point to the presence of proteoglycans and/or glycosaminoglycans within elastic fibers in normal and in pathological conditions. We present data that heparan sulphate (HS)-containing proteoglycans are associated with normal elastic fibers in human dermis and that isolated HS chains interact in vitro with recombinant tropoelastin and with peptides encoded by distinct exons of the human tropoelastin gene (EDPs). By immunocytochemistry, HS chains were identified as associated with the amorphous elastin component in the human dermis and remained associated with the residual elastin in the partially degenerated fibers of old subjects. HS appeared particularly concentrated in the mineralization front of elastic fibers in the dermis of patients affected by pseudoxanthoma elasticum (PXE). In in vitro experiments, HS induced substantial changes in the coacervation temperature and in the aggregation properties of recombinant tropoelastin and of synthetic peptides (EDPs) corresponding to sequences encoded by exons 18, 20, 24 and 30 of the human tropoelastin gene. In particular, HS modified the coacervation temperature and favoured the aggregation into ordered structures of tropoelastin molecules and of EDPs 18, 20 and 24, but not of EDP30. These data strongly indicate that HS-elastin interactions may play a role in tissue elastin fibrogenesis as well as modulating elastin stability with time and in diseases.

Acquired Pseudoxanthoma elasticum-like syndrome in beta-thalassaemia patients.

A significant number of patients diagnosed with beta-thalassaemia develop clinical and histopathological manifestations similar to those of an inherited disorder called Pseudoxanthoma elasticum (PXE). The inherited PXE is caused by mutations in the ATP-binding cassette, subfamily C (CFTR/MRP), member 6 (ABCC6) gene and is characterized by mineralized elastic fibres in dermal, vascular and ocular tissues. As no disease-causing variant was found in the ABCC6 gene of 10 beta-thalassaemia patients with a PXE-like phenotype, the present study suggests that the PXE-like symptoms in these beta-thalassaemic patients are not related to ABCC6 mutations.

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.

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.