1. Hypoxia influences the cellular cross-talk of human dermal fibroblasts. A proteomic approach.Naldini Antonella, Tiozzo Roberta, Sommer Pascal, Carraro Fabio, Annovi Giulia, Boraldi Federica, Quaglino Daniela, 2007, original scientific article Abstract: 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% CO2 and 21% O2 (pO2=140 mm Hg) or 2% O2 (pO2=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.
Found in: osebi
Keywords: Human fibroblast, Primary cell culture, Hypoxia, Connective tissue, Proteome, 2D gel electrophoresis, Mass-spectrometry
Published: 22.07.2019; Views: 2610; Downloads: 0
 Fulltext (919,07 KB) |
2. The effect of serum withdrawal on the protein profile of quiescent human dermal fibroblasts in primary cell culture.Quaglino Daniela, Tiozzo Roberta, Paolinelli Devincenzi Chiara, Annovi Giulia, Boraldi Federica, 2008, original scientific article Abstract: 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.
Found in: osebi
Keywords: Dermal fibroblasts / Primary cell culture / ROS production / Serum withdrawal
Published: 22.07.2019; Views: 2640; Downloads: 0
Fulltext (462,68 KB) |
3. Fibroblast protein profile analysis highlights the role of oxidative stress and vitamin K recycling in the pathogenesis of pseudoxanthoma elasticum.Ivonne Pasquali Ronchetti, Roberta Tiozzo, Giorgio De Santis, Fulvio Panico, Garcia Fernandez Maria Immaculada, Paolinelli Devincenzi Chiara, Deanna Guerra, Giulia Annovi, Federica Boraldi, Daniela Quaglino, 2009, original scientific article Abstract: 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.
Found in: osebi
Keywords: Ectopic calcification / Elastin / Fibroblast proteome / MRP6 / PXE
Published: 23.08.2019; Views: 2630; Downloads: 0
Fulltext (650,81 KB) |
4. Comparison of ex vivo and in vitro human fibroblast ageing models.Pascal Sommer, Roberta Tiozzo, Giulia Annovi, Federica Boraldi, Daniela Quaglino, 2010, original scientific article Abstract: Several 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.
Found in: osebi
Keywords: Ageing
Fibroblast
Connective tissue
Oxidative stress
Protein expression
Elastin
Published: 23.08.2019; Views: 2528; Downloads: 0
Fulltext (830,45 KB) |
5. Heparan Sulfate Affects Elastin Deposition in Fibroblasts Cultured from Donors of Different AgesBruna Parma, Roberta Tiozzo, Deanna Guerra, Pasquale Moscarelli, Federica Boraldi, Giulia Annovi, Pascal Sommer, Daniela Quaglino, 2012, original scientific article Abstract: 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 a-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.
Found in: osebi
Keywords: Heparan sulfate, Elastin, Fibroblasts
Published: 23.08.2019; Views: 2292; Downloads: 0
Fulltext (1,51 MB) |
6. Matrix Gla Protein and Alkaline Phosphatase Are Differently Modulated in Human Dermal Fibroblasts from PXE Patients and ControlsDeanna Guerra, Roberta Tiozzo, Tommaso Trenti, Leon J. Schurgers, Cees Vermeer, Giulia Annovi, Federica Boraldi, Daniela Quaglino, 2013, original scientific article Found in: osebi
Keywords: Fibroblast, Vitamin K, Elastin calcification, Proteomics
Published: 23.08.2019; Views: 2499; Downloads: 0
Fulltext (3,86 MB) |
