1,721,066 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
Pigment epithelial-derived factor: a new player in dermal elastic fibre calcification?
Full text linkPigment epithelium-derived factor (PEDF) is an endogenously produced glycoprotein expressed in several organs during developmental stages and adulthood mainly acting on cell differentiation.(1) In vitro and in vivo studies have demonstrated that PEDF has neurotrophic and antioxidant activities as well as the ability to counteract angiogenesis, tumorigenesis, atherosclerosis, thrombosis and inflammation.(1,2) In addition, PEDF has been also related to bone metabolism, increasing alkaline phosphatase (ALP) expression and promoting osteoblast differentiation.(3) This article is protected by copyright. All rights reserved
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
Ageing of connective tissues
No full textAging, considered as a chronological and/or biological event, is an extremely complex and multifactorial process, which may represent the consequences of environmental noxae interfering with genetic and developmental programs.The extracellular matrix plays a crucial role in age-related degenerations, and the so-called "premature aging syndromes" give further evidence for the complexity of the relationships between connective tissue, age and diseases. Several reports already focused on the correlations between age and phenotypic expression of mesenchimal cells in vitro; moreover, it was also pointed out that morphological and functional alterations of connective tissues, at some extent, might be related in vivo to increasing age. Correlation between morphologic data and proteomic analyses sustains the hypothesis that senescence is the result of both genetic and epigenetic factors
Ultrastructural and morphometrical evaluations on normal human dermal connective tissue - The influence of age, sex and body region
No full textIn order to give detailed structural and quantitative evaluations for some of the most important dermal constituents such as collagen, elastic fibres and mesenchymal cells, and for the nonstructured extracellular matrix, we performed ultrastructural investigations on dermal biopsies from 50 healthy caucasian subjects aged from 6 fetal months to 83 years. Striking changes were observed, mainly in the perinatal period, for collagen, elastin and mesenchymal cells and, after 50 years of age, for collagen and elastin. Only slight or negligible differences were noted between males and females and in skin specimens taken from different parts of the body but similarly exposed to environmental factors (i.e. UV radiation). Modifications of the non-structured extracellular matrix appeared to be the consequence of changes affecting the other components. The results, therefore, emphasize the importance of the ageing factor in connective tissue metabolism and give further information on both qualitative and quantitative characteristics of normal human dermis
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
Impression Materials: A Comparison Of Their Biological Characteristics
No full textThe aim of this study was to assess the cytotoxicity of two types of impression dental materials: polyethers (Impregum Penta, Permadyne Penta Heavy and Light) and vinyl polysiloxanes (Elite Mono Tray, Medium, Low viscosity and Elite H-D Putty). Their cytotoxic effects were studied by indirect and direct tests. The indirect tests were performed by incubating impression materials in serum free cell culture medium to prepare the soluble extracts. Balb/c 3T3 cells were incubated with extract dilutions (25, 50, 75 and 100%) for 24 h. The extracts of polyether materials caused a decrease of cellular viability, evaluated by light microscopy, by cell counting and by MTT test. The extracts of vinyl polysiloxanes materials induced a slight effect on cellular number and viability. The direct tests were performed by placing the impression materials in the centre of Petri dishes while Balb/c 3T3 were settling. The cellular proliferation was drastically reduced by polyethers and it was unaffected by the presence of vinyl polysiloxanes. These results show that: (a) the polyether materials are more toxic than vinyl polysiloxanes in our experimental conditions, (b) the impression materials are cytotoxic to the same degree in all assay methods
The protein profile of fibroblasts: the role of proteomics.
No full textFibroblasts represent one of the most widely used cell types to investigate the biology of connective tissues in normal and pathologic conditions. Aim of the present review is to emphasize, in the light of the current literature, the importance of fibroblast proteomics as a powerful resource for functional genomics in health and disease. Only very recently, proteomic techniques has been applied to characterise human dermal fibroblasts, but few data are available concerning fibroblasts of various animal origins or derived from different tissues. Functional proteomic methods have been successfully used in order i) to investigate changes in protein synthesis resulting from stimulation of fibroblasts with exogenous and endogenous factors and in the presence of conditioned media; ii) to identify the underlying mechanisms that modulate fibroblast protein profile during senescence; iii) to obtain increased knowledge about the pathogenesis of diseases such as peribronchial fibrosis; iv) to better understand the molecular basis of biocompatibility. In addition, comparison of data obtained by proteome analysis, on in vitro aged human embryo fibroblasts and on in vitro cultured human fibroblasts from subjects of different ages, allowed differences and similarities of the aging process in different models to be highlighted. Although the number of proteomic studies has exponentially increased during the past couple of years, several proteins are still under-represented in most proteome maps, i.e. membrane, low abundant and basic proteins. Since a comprehensive proteomic approach must use a technology platform that is not biased against any protein class and is able to resolve co- and post-translationally modified forms of proteins, we exemplify here the major technical improvements in protein separation and identification. Moreover, glycosylation is the most common type of post-translational protein modification, and a special emphasis is therefore placed on the expanding role of glycomics
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
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
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