1,720,996 research outputs found
A proteomic approach for investigating the aging process; the human fibroblast model.
No full textProteomic analysis of changes in protein expession of fibroblasts aged in vitro or isolated from aging donors highlights some characteristics of the aging process
Connective tissue and diseases: from morphology to proteomics towards the development of new therapeutic approaches.
No full textConnective tissue consists of cells separated by the extracellular matrix, whose composition and amount vary according to age, to functional requirements, and to the presence of pathologic conditions. Within this non-random macromolecular assembly, collagens, elastin, proteoglycans and structural glycoproteins are mutually interdependent and modifications of one component, by extrinsic (environmental) and/or intrinsic (systemic, genetic, age-related) factors, may have consequences on the tissue as a whole. Since decades, different microscopical techniques have been applied mainly for diagnostic purposes and for detailed descriptions of changes occurring in cells and in matrix components. More recently, in order to dissect the molecular complexity of the matrix network, to analyse the interactions between cells and matrix and to look for modulators of cell phenotype, histomorphologic investigations have been implemented with proteomic studies that allow to identify possible diagnostic markers, and to better understand patho-mechanisms enabling the design of novel therapeutic strategies. Therefore, the progressively expanding, although incomplete, knowledge on connective tissue biology, sheds new light on the pathogenesis of diseases affecting single molecules (i.e. collagenopathies, mucopolysaccharidoses, elastinopathies) and discloses the importance of matrix components as fundamental regulators of cell phenotype, in relation, for instance, to the aging process and/or to cancer development and progression. Few examples will be presented demonstrating the promises of proteomics as a technique leading to the discovery of new therapies and possibly to the development of individualized treatments for a better patient care
THE ROLE OF VITAMIN K-DEPENDENT PROTEINS IN THE PATHOGENESIS OF PSEUDOXANTHOMA ELASTICUM
No full textPseudoxanthoma elasticum (PXE) is a genetic disease characterized by progressive mineralization of the elastic component, which has been related to a reduced expression of the active form of matrix gla protein (MGP), a strong inhibitor of ectopic calcifications. To be fully effective, MGP has to be carboxylated (c-MGP) through a vitamin K-depend- ent pathway. The observation that in PXE, patients have lower levels of circulating vitamin K, arose the question whether elastic fiber mineralization is due to an insufficient amount of the vitamin reaching peripheral connective tissues, or to the inability of PXE mesenchymal cells to utilize the vitamin. Unexpectedly, vitamin K supplementation does not exert bene- ficial effects on soft connective tissue mineralization in the PXE animal model. For a better understanding of the role of vitamin K-dependent carboxylation of MGP in PXE patho- genesis, aim of the present study was to investigate the effects of vitamin K1 (phylloquinone) and vitamin K2 (menaquinone- 4, MK-4) supplementation on control and on PXE dermal fibroblasts cultured in standard conditions and in calcifying medium. Results demonstrate that vitamin K1 and K2 can be taken up and accumulated at similar levels by control and PXE fibroblasts, that the carboxylation process can be conse- quently similarly up-regulated, that both vitamins K1 and K2, independently from concentration, similarly down-regulate the expression of CALU in all cell strains, whereas changes are negligible in the case of PDI, indicating that the effect of vita- min K supplementation on the expression of ER-proteins involved in vitamin K cycle is not pathway-specific. Surprisingly, MGP cannot be adequately carboxylated, even at increased levels of vitamin K. It can be therefore excluded that PXE fibroblasts are not capable to utilize the vitamin, thus suggesting that altered MGP characteristics/properties could contribute to defective carboxylation. Moreover, the observa- tion that in an in vitro calcification model, both vitamin K1 and K2 are ineffective in inhibiting the mineralization process, also in control fibroblasts, i.e. in cells that do not exhibit reduced cMGP, may underline the importance and the com- plexity of the extracellular environment in mineral deposit for- mation and in regulating cell behavior
THE ROLE OF DERMAL FIBROBLASTS IN THE DEVELOPMENT OF ECTOPIC CALCIFICATIONS
No full textSoft connective tissues calcifications (i.e. ectopic calcifications) represent a deleterious consequence of diabetes, renal disorders and aging, being a key determinant of cardiovascular morbidity and mortality. Although the molecular pathways leading to this undesired mineralization have been largely investigated in smooth muscle cell cultures (SMC), to date no effective treatments are available. In order to further investigate the process of ectopic calcifications, an in vitro calcification assay has been established by isolating dermal fibroblasts (DF) from healthy adult individuals and from patients affected by Pseudoxanthoma elasticum, a disease characterized by progressive calcification of elastic fibres. Cells were grown up to 30 days in standard or in a calcifying medium. The degree of mineralization was evaluated after Von Kossa staining, whereas markers of calcification (ALP, ANKH, BMP2, ENPP1, MGP, SPP1) were assessed by RT-PCR and Western Blot. Results demonstrate that: 1) in contrast to SMC, DF do not develop a calcifying signature, 2) changes in the expression of some osteogenic markers are more related to the aging of cell cultures, 3) the development of a calcified matrix is tightly dependent on the characteristics of the extracellular environment, 4) increased ALP activity is necessary but not sufficient to have mineral deposit formation; 5) the complex balance between pro- and anti-calcifying factors, including circulating factors as MGP and fetuin, plays a significant role in the occurrence of ectopic calcifications in vivo
Fibroblasts from patients affected by Pseudoxanthoma elasticum exhibit an altered PPi metabolism and are more responsive to pro-calcifying stimuli
No full textBACKGROUND:
Pseudoxanthoma elasticum (PXE) is a genetic disorder characterized by progressive calcification of soft connective tissues. The pathogenesis is still hard to pin down. In PXE dermal fibroblasts, in addition to impaired carboxylation of the vitamin K-dependent inhibitor matrix Gla protein (MGP), we have also demonstrated an up-regulation of alkaline phosphatase activity. In the light of these data we have suggested that both calcium and phosphate metabolism might be locally altered, both pathways acting in synergy on the occurrence of matrix calcification.
OBJECTIVE:
This study aims to better explore if cultured PXE fibroblasts, compared to control cells, exhibit a modified inorganic pyrophosphate (PPi) metabolism and are more responsive to pro-calcifying stimuli.
METHODS:
Primary human dermal fibroblasts isolated from healthy individuals and from PXE patients were cultured for different time points in standard and in pro-calcifying media. The expression of ANKH/ANKH, ENPP1/PC1, ALPL/TNAP, SPP1/OPN was evaluated by qRT-PCR and Western blot, respectively. TNAP activity was measured by spectrophotometric analyses, whereas calcification was investigated by light and electron microscopy as well as by micro-analytical techniques.
RESULTS:
In the presence of pro-calcifying stimuli, dermal fibroblasts alter their phenotype favouring matrix mineralization. In particular, ENPP1/PC1 and SPP1/OPN expression, as well as TNAP activity, was differently expressed in control and in PXE fibroblasts. Moreover, in pathologic cells the ratio between factors favouring and reducing PPi availability exhibits a more pronounced shift towards a pro-calcifying balance.
CONCLUSION:
PXE fibroblasts are more susceptible to pro-calcifying stimuli and in these cells an altered PPi metabolism contributes to matrix calcification
New insights into autophagic cell death in the gypsy moth Lymantria dispar: a proteomic approach
No full textAutophagy is an evolutionary ancient process based on the activity of genes conserved from yeast to metazoan taxa. Whereas its role as a mechanism to provide energy during cell starvation is commonly accepted, debate continues about the occurrence of autophagy as a means specifically activated to achieve cell death. The IPLB-LdFB insect cell line, derived from the larval fat body of the lepidoptera Lymantria dispar, represents a suitable model to address this question, as both autophagic and apoptotic cell death can be induced by various stimuli. Using morphological and functional approaches, we have observed that the culture medium conditioned by IPLB-LdFB cells committed to death by the ATPase inhibitor oligomycin A stimulates autophagic cell death in untreated IPLB-LdFB cells. Moreover, proteomic analysis of the conditioned media suggests that, in IPLB-LdFB cells, oligomycin A promotes a shift towards lipid metabolism, increases oxidative stress and specifically directs the cells towards autophagic activity
AN IN-VITRO MODEL OF CALCIFICATION FOR THE STUDY OF THE OSTEOGENIC POTENTIAL OF ADULT HUMAN DERMAL FIBROBLASTS.
No full textIn order to investigate the calcification process in both physiological or pathological conditions, in vitro osteogenic assays are generally performed using bone-derived cells, bone-marrow-derived mesenchymal stromal cells or vascular smooth muscle cells. In normal healthy individuals, mineral formation is limited to specialized tissues as skeletal bone and teeth, however, there are many disorders (
i.e. diabetes, kidney diseases,atherosclerosis as well as genetic conditions) in which soft connective tissues undergo mineralization.
In the present study a calcification assay has been established by isolating dermal fibroblasts from adult individuals and by growing these cells in a calcifying medium in which DMEM has been supplemented with 10mM β-glycerophosphate, 50μg/ml ascorbic acid and 10 nM dexametasone. After different periods of culture, up to 40 days, fibroblast cell cultures were stained with the Von Kossa method and the activity of alkaline phosphatase (ALP) measured by a spectrophometric assay.
Results indicate that in-vitro human dermal fibroblasts, which are characterized by a limited life span in culture, are capable to mineralize their secreted extracellular matrix, when grown in the presence of an osteogenic medium. Moreover, the process of mineralization appeared to progresses with time, since areas of calcifications become visible after two weeks of culture. Consistently with the activation of the osteogenic phenotype, fibroblasts exhibited also an upregulation of ALP activity. However, we have observed a remarkable heterogeneity among cells from different individuals, supporting the hypothesis that ALP is not a unique marker of calcification and that the mineralization process is the result of a fine regulation of many inhibitors and stimulatory factors
Oxidative stress is a key regulator of ectopic calcifications in beta-Thalassemia patients
No full textA surprisingly high percentage of clinical complications affecting beta-thalassemia (beta-thal) patients is due to ectopic calcification. In these patients, elastic fiber calcification is associated with accumulation of anion superoxide and to increased levels of oxidized proteins and lipids. As a consequence, carboxylation of the calcification inhibitor Matrix Gla Protein (MGP) might be impaired. Independently from the gene defect, common pathogenic pathways are associated with ectopic calcification in PXE and in a number of beta-thal patients
The multifaceted complexity of genetic diseases: a lesson from pseudoxanthoma elasticum.
Full text linkPseudoxanthoma 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
No full textSeveral 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
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