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Federica BORALDI

Professore Associato
Dipartimento di Scienze della Vita sede ex-Scienze Biomediche

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Pubblicazioni

2023 - Aged gastrocnemius muscle of mice positively responds to a late onset adapted physical training [Articolo su rivista]
Cisterna, B.; Lofaro, F. D.; Lacavalla, M. A.; Boschi, F.; Malatesta, M.; Quaglino, D.; Zancanaro, C.; Boraldi, F.
abstract

Introduction: A regular physical training is known to contribute to preserve muscle mass and strength, maintaining structure and function of neural and vascular compartments and preventing muscle insulin resistance and inflammation. However, physical activity is progressively reduced during aging causing mobility limitations and poor quality of life. Although physical exercise for rehabilitation purposes (e.g., after fractures or cardiovascular events) or simply aiming to counteract the development of sarcopenia is frequently advised by physicians, nevertheless few data are available on the targets and the global effects on the muscle organ of adapted exercise especially if started at old age.Methods: To contribute answering this question for medical translational purposes, the proteomic profile of the gastrocnemius muscle was analyzed in 24-month-old mice undergoing adapted physical training on a treadmill for 12 weeks or kept under a sedentary lifestyle condition. Proteomic data were implemented by morphological and morphometrical ultrastructural evaluations.Results and Discussion: Data demonstrate that muscles can respond to adapted physical training started at old age, positively modulating their morphology and the proteomic profile fostering protective and saving mechanisms either involving the extracellular compartment as well as muscle cell components and pathways (i.e., mitochondrial processes, cytoplasmic translation pathways, chaperone-dependent protein refolding, regulation of skeletal muscle contraction). Therefore, this study provides important insights on the targets of adapted physical training, which can be regarded as suitable benchmarks for future in vivo studies further exploring the effects of this type of physical activity by functional/metabolic approaches.

2023 - Circulating and Tumor-Associated Neutrophils in the Era of Immune Checkpoint Inhibitors: Dynamics, Phenotypes, Metabolism, and Functions [Articolo su rivista]
Gibellini, L.; Borella, R.; Santacroce, E.; Serattini, E.; Boraldi, F.; Quaglino, D.; Aramini, B.; De Biasi, S.; Cossarizza, A.
abstract

Simple Summary Neutrophils are the most abundant leukocytes in the circulation, represent the first line of defense in the immune system and mediate inflammation. Increasing evidence suggests that neutrophils constitute a large population of cells with phenotypic and functional heterogeneity. In this review, we summarize and discuss new findings delineating that both circulating neutrophils and tumor-associated neutrophils have a role in tumor prognosis and resistance to immune checkpoint inhibitors. Neutrophils are the most abundant myeloid cells in the blood and are a considerable immunological component of the tumor microenvironment. However, their functional importance has often been ignored, as they have always been considered a mono-dimensional population of terminally differentiated, short-living cells. During the last decade, the use of cutting-edge, single-cell technologies has revolutionized the classical view of these cells, unmasking their phenotypic and functional heterogeneity. In this review, we summarize the emerging concepts in the field of neutrophils in cancer, by reviewing the recent literature on the heterogeneity of both circulating neutrophils and tumor-associated neutrophils, as well as their possible significance in tumor prognosis and resistance to immune checkpoint inhibitors.

2023 - Validation of airway porcine epithelial cells as an alternative to human in vitro preclinical studies [Articolo su rivista]
Genna, V. G.; Adamo, D.; Galaverni, G.; Lepore, F.; Boraldi, F.; Quaglino, D.; Lococo, F.; Pellegrini, G.
abstract

Animal models are currently used in several fields of biomedical research as useful alternatives to human-based studies. However, the obtained results do not always effectively translate into clinical applications, due to interspecies anatomical and physiological differences. Detailed comparability studies are therefore required to verify whether the selected animal species could be a representative model for the disease or for cellular process under investigation. This has proven to be fundamental to obtaining reliable data from preclinical studies. Among the different species, swine is deemed an excellent animal model in many fields of biological research, and has been largely used in respiratory medicine, considering the high homology between human and swine airways. In the context of in vitro studies, the validation of porcine airway epithelial cells as an alternative to human epithelial cells is crucial. In this paper, porcine and human tracheal and bronchial epithelial cells are compared in terms of in vivo tissue architecture and in vitro cell behaviour under standard and airlifted conditions, analyzing the regenerative, proliferative and differentiative potentials of these cells. We report multiple analogies between the two species, validating the employment of porcine airway epithelial cells for most in vitro preclinical studies, although with some limitations due to species-related divergences.

2022 - Inhibition of the DNA Damage Response Attenuates Ectopic Calcification in Pseudoxanthoma Elasticum [Articolo su rivista]
Huang, Jianhe; Ralph, Douglas; Boraldi, Federica; Quaglino, Daniela; Uitto, Jouni; Li, Qiaoli
abstract

Pseudoxanthoma elasticum (PXE) is a heritable ectopic calcification disorder with multiorgan clinical manifestations. The gene at default, ABCC6, encodes an efflux transporter, ABCC6, which is a critical player regulating the homeostasis of inorganic pyrophosphate, a potent endogenous anticalcification factor. Previous studies suggested that systemic inorganic pyrophosphate deficiency is the major but not the exclusive cause of ectopic calcification in PXE. In this study, we show that the DNA damage response (DDR) and poly(ADP-ribose) (PAR) pathways are involved locally in PXE at sites of ectopic calcification. Genetic inhibition of PAR polymerase 1 gene PARP1, the predominant PAR-producing enzyme, showed a 54% reduction of calcification in the muzzle skin in Abcc6-/-Parp1-/- mice, compared with that of age-matched Abcc6-/-Parp1+/+ littermates. Subsequently, oral administration of minocycline, an inhibitor of DDR/PAR signaling, resulted in an 86% reduction of calcification in the muzzle skin of Abcc6-/- mice. Minocycline treatment also attenuated the DDR/PAR signaling and reduced the calcification of dermal fibroblasts derived from patients with PXE. The anticalcification effect of DDR/PAR inhibition was not accompanied by alterations in plasma inorganic pyrophosphate concentrations. These results suggest that local DDR/PAR signaling in calcification-prone tissues contributes to PXE pathogenesis and that its inhibition might provide a promising treatment strategy for ectopic calcification in PXE, a currently intractable disease.

2022 - Metabolic reprograming shapes neutrophil functions in severe COVID-19 [Articolo su rivista]
Borella, Rebecca; De Biasi, Sara; Paolini, Annamaria; Boraldi, Federica; Tartaro, Domenico Lo; Mattioli, Marco; Fidanza, Lucia; Neroni, Anita; Caro-Maldonado, Alfredo; Meschiari, Marianna; Franceschini, Erica; Quaglino, Daniela; Guaraldi, Giovanni; Bertoldi, Carlo; Sita, Marco; Busani, Stefano; Girardis, Massimo; Mussini, Cristina; Cossarizza, Andrea; Gibellini, Lara
abstract

: To better understand the mechanisms at the basis of neutrophil functions during SARS-CoV-2 we studied patients with severe COVID-19 pneumonia. They had high blood proportion of degranulated neutrophils and elevated plasma levels of myeloperoxidase (MPO), elastase and MPO-DNA complexes, which are typical markers of neutrophil extracellular traps (NET). Their neutrophils display dysfunctional mitochondria, defective oxidative burst, increased glycolysis, glycogen accumulation in the cytoplasm, and increase glycogenolysis. Hypoxia-inducible factor 1α (ΗΙF-1α) is stabilized in such cells, and it controls the level of glycogen phosphorylase L (PYGL), a key enzyme in glycogenolysis. Inhibiting PYGL abolishes the ability of neutrophils to produce NET. Patients displayed significant increases of plasma levels of molecules involved in the regulation of neutrophils' function, including CCL2, CXCL10, CCL20, IL-18, IL-3, IL-6, G-CSF, GM-CSF, IFN-γ. Our data suggest that metabolic remodelling is vital for the formation of NET and for boosting neutrophil inflammatory response, thus suggesting that modulating ΗΙF-1α or PYGL could represent a novel approach for innovative therapies. This article is protected by copyright. All rights reserved.

2022 - Mitochondrial and Endoplasmic Reticulum Alterations in a Case of Amyotrophic Lateral Sclerosis Caused by TDP-43 A382T Mutation [Articolo su rivista]
Zanini, Giada; Selleri, Valentina; Nasi, Milena; De Gaetano, Anna; Martinelli, Ilaria; Gianferrari, Giulia; Lofaro, Francesco Demetrio; Boraldi, Federica; Mandrioli, Jessica; Pinti, Marcello
abstract

Amyotrophic lateral sclerosis is the most common form of motor neuron disease. Mutations in TARDBP, the gene encoding the RNA-binding protein TDP-43, are responsible for about 5% of familial ALS. Here we report the clinical and biological features of an ALS patients with pA382T mutation in TPD-43 protein. Disease began with right hand muscles weakness, and equally involved upper and lower motor neuron with a classic phenotype, without cognitive impairment. While a family history of neurological diseases was reported, there was no evidence of familial frontotemporal dementia. Cultured fibroblasts from the patient were characterized by profound alterations of cell proteome, which impacts particularly the mitochondrial metabolic pathways and the endoplasmic reticulum. TDP-43 levels were similar to control, healthy fibroblasts, but a higher fraction localized in mitochondria. Mitochondrial network appeared fragmented, and the organelles smaller and more spheric. In agreement with impaired proteome and morphology of mitochondria, basal cell respiration was reduced. Mitochondrial DNA levels appeared normal. However, a higher amount of mitochondrial DNA was present in the cytosol, suggesting a pronounced mitochondrial DNA misplacement which can promote a pro-inflammatory response mediating by cGAS/STING. Thus, this case report further expands the clinical and pathological phenotype of A382T mutation.

2022 - The pathogenic c.1171A>G (p.Arg391Gly) and c.2359G>A (p.Val787Ile) ABCC6 variants display incomplete penetrance causing pseudoxanthoma elasticum in a subset of individuals [Articolo su rivista]
Szeri, Flora; Miko, Agnes; Navasiolava, Nastassia; Kaposi, Ambrus; Verschuere, Shana; Molnar, Beatrix; Li, Qiaoli; Terry, Sharon F; Boraldi, Federica; Uitto, Jouni; van de Wetering, Koen; Martin, Ludovic; Quaglino, Daniela; Vanakker, Olivier M; Tory, Kalman; Aranyi, Tamas
abstract

ABCC6 promotes ATP efflux from hepatocytes to bloodstream. ATP is metabolized to pyrophosphate, an inhibitor of ectopic calcification. Pathogenic variants of ABCC6 cause pseudoxanthoma elasticum, a highly variable recessive ectopic calcification disorder. Incomplete penetrance may initiate disease heterogeneity, hence symptoms may not, or differently manifest in carriers. Here, we investigated whether incomplete penetrance is a source of heterogeneity in pseudoxanthoma elasticum. By integrating clinical and genetic data of 589 patients, we created the largest European cohort. Based on allele frequency alterations, we identified two incomplete penetrant pathogenic variants, c.2359G>A (p.Val787Ile) and c.1171A>G (p.Arg391Gly), with 6.5% and 2% penetrance, respectively. However, when penetrant, the c.1171A>G (p.Arg391Gly) manifested a clinically unaltered severity. After applying in silico and in vitro characterization, we suggest that incomplete penetrant variants are only deleterious if a yet unknown interacting partner of ABCC6 is mutated simultaneously. The low penetrance of these variants should be contemplated in genetic counseling.

2022 - The “Elastic Perspective” of SARS-CoV-2 Infection and the Role of Intrinsic and Extrinsic Factors [Articolo su rivista]
Boraldi, F.; Lofaro, F. D.; Cossarizza, A.; Quaglino, D.
abstract

Elastin represents the structural component of the extracellular matrix providing elastic recoil to tissues such as skin, blood vessels and lungs. Elastogenic cells secrete soluble tropoelastin monomers into the extracellular space where these monomers associate with other matrix proteins (e.g., microfibrils and glycoproteins) and are crosslinked by lysyl oxidase to form insoluble fibres. Once elastic fibres are formed, they are very stable, highly resistant to degradation and have an almost negligible turnover. However, there are circumstances, mainly related to inflammatory conditions, where increased proteolytic degradation of elastic fibres may lead to consequences of major clinical relevance. In severely affected COVID-19 patients, for instance, the massive recruitment and activation of neutrophils is responsible for the profuse release of elastases and other proteolytic enzymes which cause the irreversible degradation of elastic fibres. Within the lungs, destruction of the elastic network may lead to the permanent impairment of pulmonary function, thus suggesting that elastases can be a promising target to preserve the elastic component in COVID-19 patients. Moreover, intrinsic and extrinsic factors additionally contributing to damaging the elastic component and to increasing the spread and severity of SARS-CoV-2 infection are reviewed.

2021 - A case report of pseudoxanthoma elasticum with rare sequence variants in genes related to inherited retinal diseases [Articolo su rivista]
Lofaro, F. D.; Mucciolo, D. P.; Murro, V.; Pavese, L.; Quaglino, D.; Boraldi, F.
abstract

A case of a patient with an early and severe visual impairment is described. Due to the occurrence of skin papules a suspect of pseudoxanthoma elasticum (PXE) was posed. PXE is a rare autosomal recessive disease clinically characterized by skin, cardiovascular and ocular manifestations, these last being those that most severely affect patients’ quality of life. A whole exome sequencing approach focusing on 340 genes related to the calcification process and/or to inherited retinal diseases (IRDs) was performed. Rare monoallelic sequence variants in ABCA4, ABCC6, IMPG1, POC1B and RAX2 were found. The presence of calcified elastic fibers was assessed by ultrastructural analysis on a skin biopsy. Diagnosis of PXE was based on clinical, biomolecular and morphological results, although the additional involvement of several IRD genes is important to explain the unexpectedly severe ophthalmological phenotype of the patient also in prognostic and therapeutic perspectives. Data indicate that genetic screening using a wide‐spectrum analysis approach is essential to assist ophthalmologists in improving patient counseling.

2021 - Adaptive Optics Imaging in Patients Affected by Pseudoxanthoma Elasticum [Articolo su rivista]
Murro, V.; Mucciolo, D. P.; Giorgio, D.; Pavese, L.; Boraldi, F.; Quaglino, D.; Finocchio, L.; Sodi, A.; Virgili, G.; Giansanti, F.
abstract

Purpose: To describe the retinal findings of patients affected by pseudoxanthoma elasticum (PXE) using a multimodal imaging approach including flood-illumination adaptive optics ophthalmoscopy (AO). Design: Retrospective case series. Materials and Methods: Patients affected by PXE were retrospectively studied. Clinical data, color, infrared and autofluorescence fundus imaging, optical coherence tomographic scans, and AO examinations were collected. Furthermore, the photoreceptor count was assessed. PXE diagnosis was confirmed by a positive skin biopsy and/or genetic testing. Results: Twenty-one eyes of 18 patients (11 females and 7 males) were included in the study. In 3 patients, both eyes were studied. The mean age at examination was 37 ± 16.4 years (range 11-66) and the mean best-corrected visual acuity (BCVA) was 0.1 ± 0.2 logMAR (range 0-1). We identified 3 types of angioid streaks (AS) using AO: “crack,” “band,” and “hypopigmented.” The first 2 were very similar and they differed in size; the third type showed specific clinical features. Comet lesions appeared as hyper-reflective round lesions on AO imaging. In all eyes, the cone mosaic appeared reduced inside the streaks compared to the neighboring areas (13,532.8 ± 1,366.5 cones/mm2 vs 16,817.1 ± 1,263.0 cones/mm2 respectively). Conclusion: Using AO imaging in PXE-related retinopathy, we were able to observe the presence of the photoreceptors within the angioid streaks, differentiate 3 types of angioid streaks, based on size and reflective features, and identify the very small crystalline bodies not identifiable using other retinal imaging techniques.

2021 - Age-related changes in the matrisome of the mouse skeletal muscle [Articolo su rivista]
Lofaro, F. D.; Cisterna, B.; Lacavalla, M. A.; Boschi, F.; Malatesta, M.; Quaglino, D.; Zancanaro, C.; Boraldi, F.
abstract

Aging is characterized by a progressive decline of skeletal muscle (SM) mass and strength which may lead to sarcopenia in older persons. To date, a limited number of studies have been performed in the old SM looking at the whole, complex network of the extracellular matrix (i.e., matrisome) and its aging-associated changes. In this study, skeletal muscle proteins were isolated from whole gastrocnemius muscles of adult (12 mo.) and old (24 mo.) mice using three sequential extractions, each one analyzed by liquid chromatography with tandem mass spectrometry. Muscle sections were investigated using fluorescence-and transmission electron microscopy. This study provided the first characterization of the matrisome in the old SM demonstrating several statisti-cally significantly increased matrisome proteins in the old vs. adult SM. Several proteomic findings were confirmed and expanded by morphological data. The current findings shed new light on the mutually cooperative interplay between cells and the extracellular environment in the aging SM. These data open the door for a better understanding of the mechanisms modulating myocellular behavior in aging (e.g., by altering mechano-sensing stimuli as well as signaling pathways) and their contribution to age-dependent muscle dysfunction.

2021 - Apoptosis in the Extraosseous Calcification Process [Articolo su rivista]
Boraldi, F.; Lofaro, F. D.; Quaglino, D.
abstract

Extraosseous calcification is a pathologic mineralization process occurring in soft connective tissues (e.g., skin, vessels, tendons, and cartilage). It can take place on a genetic basis or as a consequence of acquired chronic diseases. In this last case, the etiology is multifactorial, including both extra- and intracellular mechanisms, such as the formation of membrane vesicles (e.g., matrix vesicles and apoptotic bodies), mitochondrial alterations, and oxidative stress. This review is an overview of extraosseous calcification mechanisms focusing on the relationships between apoptosis and mineralization in cartilage and vascular tissues, as these are the two tissues mostly affected by a number of age-related diseases having a progressively increased impact in Western Countries.

2021 - Dermal Alterations in Clinically Unaffected Skin of Pseudoxanthoma elasticum Patients [Articolo su rivista]
Boraldi, F; Lofaro, Fd; Losi, L; Quaglino, D
abstract

Background: Pseudoxanthoma elasticum (PXE), due to rare sequence variants in the ABCC6 gene, is characterized by calcification of elastic fibers in several tissues/organs; however, the pathomechanisms have not been completely clarified. Although it is a systemic disorder on a genetic basis, it is not known why not all elastic fibers are calcified in the same patient and even in the same tissue. At present, data on soft connective tissue mineralization derive from studies performed on vascular tissues and/or on clinically affected skin, but there is no information on patients' clinically unaffected skin. Methods: Skin biopsies from clinically unaffected and affected areas of the same PXE patient (n = 6) and from healthy subjects were investigated by electron microscopy. Immunohistochemistry was performed to evaluate p-SMAD 1/5/8 and p-SMAD 2/3 expression and localization. Results: In clinically unaffected skin, fragmented elastic fibers were prevalent, whereas calcified fibers were only rarely observed at the ultrastructural level. p-SMAD1/5/8 and p-SMAD2/3 were activated in both affected and unaffected skin. Conclusion: These findings further support the concept that fragmentation/degradation is necessary but not sufficient to cause calcification of elastic fibers and that additional local factors (e.g., matrix composition, mechanical forces and mesenchymal cells) contribute to create the pro-osteogenic environment.

2021 - From Clinical Diagnosis to the Discovery of Multigene Rare Sequence Variants in Pseudoxanthoma elasticum: A Case Report [Articolo su rivista]
Lofaro, F. D.; Mucciolo, D. P.; Murro, V.; Pavese, L.; Quaglino, D.; Boraldi, F.
abstract

Pseudoxanthoma elasticum (PXE) is a rare autosomal recessive disease clinically characterised by early cutaneous alterations, and by late clinically relevant ocular, and cardiovascular manifestations. ABCC6 genetic tests are used to confirm clinical PXE diagnosis, but this strategy may be rather challenging when only one ABCC6 pathogenic variant is found. A next-generation sequencing approach focusing on 362 genes related to the calcification process and/or to inherited retinal diseases was performed on a patient with clinical PXE diagnosis (skin papules and laxity, angioid streaks, and atrophy) who was carrier of only one ABCC6 rare sequence variant. Beside ABCC6, several rare sequence variants were detected which can contribute either to the occurrence of calcification (GGCX and SERPINF1 genes) and/or to ophthalmological manifestations (ABCA4, AGBL5, CLUAP1, and KCNV2 genes). This wide-spectrum analysis approach facilitates the identification of rare variants possibly involved in PXE, thus avoiding invasive skin biopsy as well as expensive and time-consuming diagnostic odyssey and allows to broaden and to deepen the knowledge on this complex rare disease and to improve patients' counselling, also with a future perspective of personalised medicine.

2021 - Impact of glucocorticoid on a cellular model of parkinson’s disease: Oxidative stress and mitochondrial function [Articolo su rivista]
Claros, S.; Gil, A.; Martinelli, M.; Valverde, N.; Lara, E.; Boraldi, F.; Pavia, J.; Martin-Montanez, E.; Garcia-Fernandez, M.
abstract

Stress seems to contribute to the neuropathology of Parkinson’s disease (PD), possibly by dysregulation of the hypothalamic–pituitary–adrenal axis. Oxidative distress and mitochondrial dysfunction are key factors involved in the pathophysiology of PD and neuronal glucocorticoid-induced toxicity. Animal PD models have been generated to study the effects of hormonal stress, but no in vitro model has yet been developed. Our aim was to examine the impact of corticosterone (CORT) administration on a dopaminergic neuronal cell model of PD induced by the neurotoxin MPP+, as a new combined PD model based on the marker of endocrine response to stress, CORT, and oxidative-mitochondrial damage. We determined the impact of CORT, MPP+ and their co-incubation on reactive oxygen species production (O2−• ), oxidative stress cellular markers (advanced-oxidation protein products and total antioxidant status), mitochondrial function (mitochondrial membrane potential and mitochondrial oxygen consumption rate) and neurodegeneration (Fluoro-Jade staining). Accordingly, the administration of MPP+ or CORT individually led to cell damage compared to controls (p < 0.05), as determined by several methods, whereas their co-incubation produced strong cell damage (p < 0.05). The combined model described here could be appropriate for investigating neuropathological hallmarks and for evaluating potential new therapeutic tools for PD patients suffering mild to moderate emotional stress.

2021 - Insulin-like growth factor II prevents oxidative and neuronal damage in cellular and mice models of Parkinson's disease [Articolo su rivista]
Martin-Montanez, E.; Valverde, N.; Ladron de Guevara-Miranda, D.; Lara, E.; Romero-Zerbo, Y. S.; Millon, C.; Boraldi, F.; Avila-Gamiz, F.; Perez-Cano, A. M.; Garrido-Gil, P.; Labandeira-Garcia, J. L.; Santin, L. J.; Pavia, J.; Garcia-Fernandez, M.
abstract

Oxidative distress and mitochondrial dysfunction, are key factors involved in the pathophysiology of Parkinson's disease (PD). The pleiotropic hormone insulin-like growth factor II (IGF-II) has shown neuroprotective and antioxidant effects in some neurodegenerative diseases. In this work, we demonstrate the protective effect of IGF-II against the damage induced by 1-methyl-4-phenylpyridinium (MPP+) in neuronal dopaminergic cell cultures and a mouse model of progressive PD. In the neuronal model, IGF-II counteracts the oxidative distress produced by MPP + protecting dopaminergic neurons. Improved mitochondrial function, increased nuclear factor (erythroid-derived 2)-like2 (NRF2) nuclear translocation along with NRF2-dependent upregulation of antioxidative enzymes, and modulation of mammalian target of rapamycin (mTOR) signalling pathway were identified as mechanisms leading to neuroprotection and the survival of dopaminergic cells. The neuroprotective effect of IGF-II against MPP + -neurotoxicity on dopaminergic neurons depends on the specific IGF-II receptor (IGF-IIr). In the mouse model, IGF-II prevents behavioural dysfunction and dopaminergic nigrostriatal pathway degeneration and mitigates neuroinflammation induced by MPP+. Our work demonstrates that hampering oxidative stress and normalising mitochondrial function through the interaction of IGF-II with its specific IGF-IIr are neuroprotective in both neuronal and mouse models. Thus, the modulation of the IGF-II/IGF-IIr signalling pathway may be a useful therapeutic approach for the prevention and treatment of PD.

2021 - Phenotypic Features and Genetic Findings in a Cohort of Italian Pseudoxanthoma Elasticum Patients and Update of the Ophthalmologic Evaluation Score [Articolo su rivista]
Boraldi, F; Murro, V; Lofaro, Fd; Mucciolo, Dp; Costa, S; Pavese, L; Quaglino, D
abstract

Background: Pseudoxanthoma elasticum (PXE) is a rare ectopic calcification genetic disease mainly caused by ABCC6 rare sequence variants. The clinical phenotype is characterized by typical dermatological, ophthalmological and cardiovascular manifestations, whose frequency and severity are differently reported in the literature. Methods: A retrospective study was performed on 377 PXE patients of Italian origin, clinically evaluated according to the Phenodex Index, who underwent ABCC6 biomolecular analyses. Moreover, 53 PXE patients were further characterized by in-depth ophthalmological examinations. Results: A total of 117 different ABCC6 rare sequence variants were detected as being spread through the whole gene. The severity of the clinical phenotype was dependent on age, but it was not influenced by gender or by the type of sequence variants. In-depth ophthalmological examinations focused on the incidences of coquille d'oeuf, comet lesions, pattern dystrophy-like lesions, optic disk drusen and posterior-pole atrophy. Conclusion: Given the large number of patients analyzed, we were able to better evaluate the occurrence of less frequent alterations (e.g., stroke, myocardial infarction, nephrolithiasis). A more detailed description of ophthalmological abnormalities allowed us to stratify patients and better evaluate disease progression, thus suggesting a further update of the PXE score system.

2021 - Pomacea canaliculata ampullar proteome: A nematode-based bio-pesticide induces changes in metabolic and stress-related pathways [Articolo su rivista]
Boraldi, F.; Lofaro, F. D.; Bergamini, G.; Ferrari, A.; Malagoli, D.
abstract

Pomacea canaliculata is a freshwater gastropod known for being both a highly invasive species and one of the possible intermediate hosts of the mammalian parasite Angiostrongylus cantonensis. With the aim of providing new information concerning P. canaliculata biology and adaptability, the first proteome of the ampulla, i.e., a small organ associated with the circulatory system and known as a reservoir of nitrogen-containing compounds, was obtained. The ampullar proteome was derived from ampullae of control snails or after exposure to a nematode-based molluscicide, known for killing snails in a dose-and temperature-dependent fashion. Proteome analysis revealed that the composition of connective ampulla walls, cell metabolism and oxidative stress response were affected by the biopesticide. Ultrastructural investigations have highlighted the presence of rhogocytes within the ampullar walls, as it has been reported for other organs containing nitrogen storage tissue. Collected data suggested that the ampulla may belong to a network of organs involved in controlling and facing oxidative stress in different situations. The response against the nematode-based molluscicide recalled the response set up during early arousal after aestivation and hibernation, thus encouraging the hypothesis that metabolic pathways and antioxidant defences promoting amphibiousness could also prove useful in facing other challenges stimulating an oxidative stress response, e.g., immune challenges or biocide exposure. Targeting the oxidative stress resistance of P. canaliculata may prove helpful for increasing its susceptibility to bio-pesticides and may help the sustainable control of this pest’s diffusion.

2020 - Altered bioenergetics and mitochondrial dysfunction of monocytes in patients with COVID-19 pneumonia [Articolo su rivista]
Gibellini, L.; De Biasi, S.; Paolini, A.; Borella, R.; Boraldi, F.; Mattioli, M.; Lo Tartaro, D.; Fidanza, L.; Caro-Maldonado, A.; Meschiari, M.; Iadisernia, V.; Bacca, E.; Riva, G.; Cicchetti, L.; Quaglino, D.; Guaraldi, G.; Busani, S.; Girardis, M.; Mussini, C.; Cossarizza, A.
abstract

In patients infected by SARS-CoV-2 who experience an exaggerated inflammation leading to pneumonia, monocytes likely play a major role but have received poor attention. Thus, we analyzed peripheral blood monocytes from patients with COVID-19 pneumonia and found that these cells show signs of altered bioenergetics and mitochondrial dysfunction, had a reduced basal and maximal respiration, reduced spare respiratory capacity, and decreased proton leak. Basal extracellular acidification rate was also diminished, suggesting reduced capability to perform aerobic glycolysis. Although COVID-19 monocytes had a reduced ability to perform oxidative burst, they were still capable of producing TNF and IFN-γ in vitro. A significantly high amount of monocytes had depolarized mitochondria and abnormal mitochondrial ultrastructure. A redistribution of monocyte subsets, with a significant expansion of intermediate/pro-inflammatory cells, and high amounts of immature monocytes were found, along with a concomitant compression of classical monocytes, and an increased expression of inhibitory checkpoints like PD-1/PD-L1. High plasma levels of several inflammatory cytokines and chemokines, including GM-CSF, IL-18, CCL2, CXCL10, and osteopontin, finally confirm the importance of monocytes in COVID-19 immunopathogenesis.

2020 - Impaired Mitochondrial Morphology and Functionality in Lonp1wt/- Mice [Articolo su rivista]
De Gaetano, Anna; Gibellini, Lara; Bianchini, Elena; Borella, Rebecca; De Biasi, Sara; Nasi, Milena; Boraldi, Federica; Cossarizza, Andrea; Pinti, Marcello
abstract

LONP1 is a nuclear-encoded mitochondrial protease crucial for organelle homeostasis; mutations ofLONP1have been associated with Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies (CODAS) syndrome. To clarify the role of LONP1 in vivo, we generated a mouse model in whichLonp1was ablated. The homozygousLonp(-/-)mouse was not vital, while the heterozygousLonp1(wt/-)showed similar growth rate, weight, length, life-span and histologic features as wild type. Conversely, ultrastructural analysis of heterozygous enterocytes evidenced profound morphological alterations of mitochondria, which appeared increased in number, swollen and larger, with a lower complexity. Embryonic fibroblasts (MEFs) fromLonp1(wt/-)mice showed a reduced expression ofLonp1andTfam, whose expression is regulated by LONP1. Mitochondrial DNA was also reduced, and mitochondria were swollen and larger, albeit at a lesser extent than enterocytes, with a perinuclear distribution. From the functional point of view, mitochondria from heterozygous MEF showed a lower oxygen consumption rate in basal conditions, either in the presence of glucose or galactose, and a reduced expression of mitochondrial complexes than wild type. In conclusion, the presence of one functional copy of theLonp1gene leads to impairment of mitochondrial ultrastructure and functions in vivo.

2020 - Neuronal Metabolism and Neuroprotection: Neuroprotective Effect of Fingolimod on Menadione-Induced Mitochondrial Damage [Articolo su rivista]
Gil, A.; Martin-Montanez, E.; Valverde, N.; Lara, E.; Boraldi, F.; Claros, S.; Romero-Zerbo, S. -Y.; Fernandez, O.; Pavia, J.; Garcia-Fernandez, M.
abstract

Imbalance in the oxidative status in neurons, along with mitochondrial damage, are common characteristics in some neurodegenerative diseases. The maintenance in energy production is crucial to face and recover from oxidative damage, and the preservation of different sources of energy production is essential to preserve neuronal function. Fingolimod phosphate is a drug with neuroprotective and antioxidant actions, used in the treatment of multiple sclerosis. This work was performed in a model of oxidative damage on neuronal cell cultures exposed to menadione in the presence or absence of fingolimod phosphate. We studied the mitochondrial function, antioxidant enzymes, protein nitrosylation, and several pathways related with glucose metabolism and glycolytic and pentose phosphate in neuronal cells cultures. Our results showed that menadione produces a decrease in mitochondrial function, an imbalance in antioxidant enzymes, and an increase in nitrosylated proteins with a decrease in glycolysis and glucose-6-phosphate dehydrogenase. All these effects were counteracted when fingolimod phosphate was present in the incubation media. These effects were mediated, at least in part, by the interaction of this drug with its specific S1P receptors. These actions would make this drug a potential tool in the treatment of neurodegenerative processes, either to slow progression or alleviate symptoms.

2020 - Pattern dystrophy-like changes and coquille d’oeuf atrophy in elderly patients affected by pseudoxanthoma elasticum [Articolo su rivista]
Murro, V.; Mucciolo, D. P.; Giorgio, D.; Sodi, A.; Boraldi, F.; Quaglino, D.; Virgili, G.; Giansanti, F.
abstract

Purpose: To evaluate the retinal features of elderly patients affected by pseudoxanthoma elasticum (PXE). Materials and methods: This is a retrospective case series of 62 eyes of 31 elderly PXE patients (age > 50 years). Clinical data, ultra-widefield fundus imaging (color, red-free (RF), infra-red imaging (IR), fundus autofluorescence (FAF)), and OCT examinations were collected. Diagnosis was confirmed by genetic testing or skin biopsy. Results: Thirty-one patients (10 males and 21 females (mean age 61.3 years, range 50–74 years)) were included in our study. Visual acuity ranged from 20/20 Snellen equivalent to 20/200. The mean follow-up was 66.4 ± 20.7 months (range 10–88). Pattern dystrophy-like changes (PD) (52 eyes of 26 patients, 83.8%) and atrophy resembling the “diffuse trickling” pattern described in geographic atrophy were present in the majority of patients. Twenty-three eyes of 12 patients (67.6%) had peripapillary atrophy, 9 eyes of 5 patients (26.4%) macular atrophy, 6 eyes of 3 patients (17.6%) displayed posterior pole atrophy and in 6 eyes of 3 patients (17.6%), atrophy could be detected beyond the vascular arcades (mid-peripheral atrophy). End-stage atrophy covered the entire area indicated as “coquille d’oeuf” (eggshell). Choroidal neovascularization occurred in 49 eyes of 26 patients (94.2%) with PD and in 6 eyes of 3 patients (60%) without PD. Genetic examinations were available for 29 patients (29/31, 93.5%). Conclusions: The elderly PXE patients were characterized by pattern dystrophy-like changes with more or less extensive atrophy, progressive over time, which in some cases affected the whole area of the coquille d’oeuf during the course of the disease.

2020 - Rare Co-occurrence of Beta-Thalassemia and Pseudoxanthoma elasticum: Novel Biomolecular Findings [Articolo su rivista]
Boraldi, F.; Lofaro, F. D.; Costa, S.; Moscarelli, P.; Quaglino, D.
abstract

A number of beta-thalassemia patients, independently from the type of beta-thalassemia (β0 or β+) and blood transfusion requirements, may develop, after puberty, dermal, cardiovascular, and ocular complications associated with an ectopic mineralization phenotype similar to that observed in another rare genetic disorder, namely, Pseudoxanthoma elasticum (PXE). To date, the causes of these alterations in beta-thalassemia patients are not known, but it has been suggested that they could be the consequence of oxidative stress-driven epigenetic regulatory mechanisms producing an ABCC6 down-regulation. Since, in the last years, several genes have been associated to the ectopic mineralization phenotype, this study, for the first time, applied, on beta-thalassemia patients with ectopic mineralization phenotype, a multigene testing strategy. Selection of genes to be analyzed was done on the basis of (i) their genetic involvement in calcification diseases or (ii) their role in calcium-phosphate equilibrium. Although, due to the rarity of these conditions, a limited number of patients was analyzed, the detection of pathogenic variants represents the proof of concept that PXE and beta-thalassemia traits co-occur on a genetic basis and that, in addition to causative mutations, functional polymorphisms may further influence connective tissue manifestations. The use of a multigene-based next-generation sequencing represents a useful time- and cost-effective approach, allowing to identify sequence variants that might improve prognostic assessment and better management of these patients, especially in the current era of precision medicine aiming to identify individual optimal care based on a unique personal profile.

2020 - Relationship Between Mitochondrial Structure and Bioenergetics in Pseudoxanthoma elasticum Dermal Fibroblasts [Articolo su rivista]
Lofaro, F. D.; Boraldi, F.; Garcia-Fernandez, M.; Estrella, L.; Valdivielso, P.; Quaglino, D.
abstract

Pseudoxanthoma elasticum (PXE) is a genetic disease considered as a paradigm of ectopic mineralization disorders, being characterized by multisystem clinical manifestations due to progressive calcification of skin, eyes, and the cardiovascular system, resembling an age-related phenotype. Although fibroblasts do not express the pathogenic ABCC6 gene, nevertheless these cells are still under investigation because they regulate connective tissue homeostasis, generating the “arena” where cells and extracellular matrix components can promote pathologic calcification and where activation of pro-osteogenic factors can be associated to pathways involving mitochondrial metabolism. The aim of the present study was to integrate structural and bioenergenetic features to deeply investigate mitochondria from control and from PXE fibroblasts cultured in standard conditions and to explore the role of mitochondria in the development of the PXE fibroblasts’ pathologic phenotype. Proteomic, biochemical, and morphological data provide new evidence that in basal culture conditions (1) the protein profile of PXE mitochondria reveals a number of differentially expressed proteins, suggesting changes in redox balance, oxidative phosphorylation, and calcium homeostasis in addition to modified structure and organization, (2) measure of oxygen consumption indicates that the PXE mitochondria have a low ability to cope with a sudden increased need for ATP via oxidative phosphorylation, (3) mitochondrial membranes are highly polarized in PXE fibroblasts, and this condition contributes to increased reactive oxygen species levels, (4) ultrastructural alterations in PXE mitochondria are associated with functional changes, and (5) PXE fibroblasts exhibit a more abundant, branched, and interconnected mitochondrial network compared to control cells, indicating that fusion prevail over fission events. In summary, the present study demonstrates that mitochondria are modified in PXE fibroblasts. Since mitochondria are key players in the development of the aging process, fibroblasts cultured from aged individuals or aged in vitro are more prone to calcify, and in PXE, calcified tissues remind features of premature aging syndromes; it can be hypothesized that mitochondria represent a common link contributing to the development of ectopic calcification in aging and in diseases. Therefore, ameliorating mitochondrial functions and cell metabolism could open new strategies to positively regulate a number of signaling pathways associated to pathologic calcification.

2020 - The biology of vascular calcification [Capitolo/Saggio]
Quaglino, D.; Boraldi, F.; Lofaro, F. D.
abstract

Vascular calcification (VC), characterized by different mineral deposits (i.e., carbonate apatite, whitlockite and hydroxyapatite) accumulating in blood vessels and valves, represents a relevant pathological process for the aging population and a life-threatening complication in acquired and in genetic diseases. Similarly to bone remodeling, VC is an actively regulated process in which many cells and molecules play a pivotal role. This review aims at: (i) describing the role of resident and circulating cells, of the extracellular environment and of positive and negative factors in driving the mineralization process; (ii) detailing the types of VC (i.e., intimal, medial and cardiac valve calcification); (iii) analyzing rare genetic diseases underlining the importance of altered pyrophosphate-dependent regulatory mechanisms; (iv) providing therapeutic options and perspectives.

2020 - The mineralization process of insoluble elastin fibrillar structures: Ionic environment vs degradation [Articolo su rivista]
Boraldi, F.; Moscarelli, P.; Lofaro, F. D.; Sabia, C.; Quaglino, D.
abstract

Despite its long half-life and physiological role, elastin undergoes irreversible changes (i.e elastolysis and/or calcification) impairing resilience of soft connective tissues. At present, it is still undefined: 1) to which extent elastin fibers have to be fragmented in order to increase their susceptibility to calcify; 2) which is the contribution of ionic environment on elastin mineralization; 3) why, in the same tissue area, mineralized coexist with non-mineralized fibers. The in vitro mineralization process was investigated on insoluble elastin, hydrolyzed or not-hydrolyzed, and incubated in different cell-free ionic environments. Mineral deposition is favored on hydrolyzed fibrillar structures due to exposure of multiple charged sites increasing the adsorption of Ca2+ that can attract phosphate and increase the local ion concentration over the point of supersaturation, representing the minimum requirement for hydroxyapatite nucleation sites. At physiological pH, the degree of elastin mineralization is influenced by hydrolysis and complexity of medium composition, since ionic species, as sodium, potassium, magnesium, in addition to calcium and phosphorus, interfere with the calcification process. These findings broaden the knowledge on the factors controlling hydroxyapatite deposition on insoluble elastin and can also explain why, in vivo, calcified and non-calcified fibers can be observed within the same tissue.

2019 - Coquille d’oeuf in young patients affected with Pseudoxantoma elasticum [Articolo su rivista]
Murro, V.; Mucciolo, D. P.; Giorgio, D.; Sodi, A.; Boraldi, F.; Quaglino, D.; Virgili, G.; Rizzo, S.
abstract

Purpose: To evaluate the fundus phenotype of young patients affected with Pseudoxantoma Elasticum (PXE). Materials and Methods: Retrospective case series of five young PXE patients. Clinical data, ultra-widefield imaging (color, red-free (RF), choroidal (Ch) and fundus autofluorescence (FAF)) and OCT examination were collected. Diagnosis was confirmed by the characteristic histopathological abnormalities in skin biopsies and genetic testing results. Results: Five patients, 2 males and 3 females (mean age 16 years, range 12–20 years) were included in our study. The visual acuity was 20/20 in all subjects. Fundus evaluation revealed peau d’orange in all patients: multiple, yellowish/white round lesions, scattered from the posterior pole to the mid-peripheral retina of each eye. Ultra-wide field imaging allows us to capture and describe the entire area of coquille d’oeuf/peau d’orange in a single picture, facilitating their identification and discrimination. Angiod streaks were visible in both eyes of four patients. In one patient optic disc drusen were detected in both eyes. All patients presented comet lesions. Conclusions: PXE-related retinopathy findings: peau d’orange/coquille d’oeuf, angioid streaks, comet lesions and drusen of the optic disc were present early in PXE patients. The early detection of coquille d’oeuf/peau d’orange revealed a preferable area into midperiphery where Bruch’s membrane will be more likely to be affected.

2019 - Exome sequencing and bioinformatic approaches reveals rare sequence variants involved in cell signalling and elastic fibre homeostasis: new evidence in the development of ectopic calcification [Articolo su rivista]
Boraldi, Federica; Lofaro, Francesco Demetrio; Romano, Oriana; Grilli, Andrea; Losi, Lorena; Moscarelli, Pasquale; Bicciato, Silvio; Quaglino, Daniela
abstract

Elastic fibres undergo aberrant mineralization in genetic as well as in acquired pathologic conditions causing severe impairment of tissue mechanical properties. Despite the number of investigations performed so far, the pathogenesis of these alterations is still elusive, due to both the complexity of the elastin network and the involvement of many genes and/or pro-osteogenic signalling pathways. Whole Exome Sequencing (WES) was performed on DNA from three patients affected by beta-thalassemia exhibiting soft connective tissue calcification. WES data were analysed with a bioinformatic approach, allowing to screen and to select genes carrying rare sequence variants. These genes were matched with those present in Extracellular Matrix DB. This approach enables to shed light on the involvement of the extracellular matrix in the occurrence of ectopic calcification. Results revealed a number of rare sequence variants in genes related to elastic fibre assembly and integrity. For instance, the involvement of fibrillins and collagen type VI in the formation of a modified microfibrillar scaffold may lead to elastic fibres less resilient and more prone to hydroxyapatite deposition. Moreover, data reveal that changes in mitochondrial metabolic pathways are sustained by a genetic background and emphasize that a persistent chronic oxidative stress can further influence extracellular matrix homeostasis and cell signalling through the TGFβ-BMP axis. Eventually, the presence of multiple rare sequence variants in the Solute Carrier Family 25 Member 5 (SLC25A5) gene is suggestive of the role of this gene as a key factor linking mitochondria metabolism, ADP/ATP ratio and oxidative stress thus affecting extracellular matrix homeostasis and activation of pro-osteogenic factors.

2019 - Intraretinal hyperreflective foci in PXE-related retinopathy with acquired vitelliform lesions: a long-term follow-up [Articolo su rivista]
Murro, V.; Mucciolo, D. P.; Giorgio, D.; Sodi, A.; Boraldi, F.; Quaglino, D.; Virgili, G.; Rizzo, S.
abstract

Purposes: To describe the long-term follow-up of a patient affected by Pseudoxanthoma Elasticum (PXE) and acquired macular vitelliform lesions in both eyes. Material and methods: Case report Results: We reported the 9-year follow-up of a patient affected by PXE. We described the onset and the resolution of the vitelliform macular lesions which lasted 5 years. The vitelliform lesion appeared almost simultaneously in both eyes with an initial increase in size, even though asymmetrical. We detected the intraretinal migration of hyper-reflective foci in both eyes during the follow-up. Choroidal neovascularization (CNV) occurred in her right eye during the follow-up. Visual acuity decreased from 20/20 to 20/32 in left eye; from 20/20 to 20/100 in her right eye. Conclusions: we reported the natural history of acquired vitelliform lesion in PXE-related retinopathy describing the Intraretinal hyperreflective foci migration.

2019 - The S1P mimetic fingolimod phosphate regulates mitochondrial oxidative stress in neuronal cells [Articolo su rivista]
Martin-Montanez, E.; Pavia, J.; Valverde, N.; Boraldi, F.; Lara, E.; Oliver, B.; Hurtado-Guerrero, I.; Fernandez, O.; Garcia-Fernandez, M.
abstract

Fingolimod is one of the few oral drugs available for the treatment of multiple sclerosis (MS), a chronic, inflammatory, demyelinating and neurodegenerative disease. The mechanism of action proposed for this drug is based in the phosphorylation of the molecule to produce its active metabolite fingolimod phosphate (FP) which, in turns, through its interaction with S1P receptors, triggers the functional sequestration of T lymphocytes in lymphoid nodes. On the other hand, part if not most of the damage produced in MS and other neurological disorders seem to be mediated by reactive oxygen species (ROS), and mitochondria is one of the main sources of ROS. In the present work, we have evaluated the anti-oxidant profile of FP in a model of mitochondrial oxidative damage induced by menadione (Vitk3) on neuronal cultures. We provide evidence that incubation of neuronal cells with FP alleviates the Vitk3-induced toxicity, due to a decrease in mitochondrial ROS production. It also decreases regulated cell death triggered by imbalance in oxidative stress (restore values of advanced oxidation protein products and total thiol levels). Also restores mitochondrial function (cytochrome c oxidase activity, mitochondrial membrane potential and oxygen consumption rate) and morphology. Furthermore, increases the expression and activity of protective factors (increases Nrf2, HO1 and Trx2 expression and GST and NQO1 activity), being some of these effects modulated by its interaction with the S1P receptor. FP seems to increase mitochondrial stability and restore mitochondrial dynamics under conditions of oxidative stress, making this drug a potential candidate for the treatment of neurodegenerative diseases other than MS.

2019 - Toward the Molecular Deciphering of Pomacea canaliculata Immunity: First Proteomic Analysis of Circulating Hemocytes [Articolo su rivista]
Boraldi, F.; Lofaro, FRANCESCO DEMETRIO; Accorsi, A.; Ross, E.; Malagoli, D.
abstract

Pomacea canaliculata is a freshwater snail with interesting biological features that include invasiveness, human parasite hosting, and adult regeneration. Its immune system may represent the target for strategies aimed at controlling the spread of the snail population and its hosting of the human parasite Angiostrongylus cantonensis. Moreover, immune functions likely have a role in the snail's ability to wound heal and regenerate. Despite its importance in multiple processes, very little is known about the molecular basis of P. canaliculata immunity. Aiming to contribute to filling this gap, the ultrastructure of circulating hemocytes in healthy snails is studied and the first proteomic analysis of these cells is performed, evidencing 83 unique proteins, 96% of which have identifiable homologs in other species. Fifteen proteins are retrieved as potentially involved in immune-related signaling pathways, such as hemocyanin, C1q-like protein, and HSP90 together with cytoskeleton and cytoskeleton-related proteins involved in cell motility and membrane dynamics. This first proteome study on non-stimulated hemocytes provides a valid reference for future investigations on the molecular changes under stressful circumstances, like pathogen exposure, wounding, or environmental changes.

2018 - Heparan sulfates facilitate harmless amyloidogenic fibril formation interacting with elastin-like peptides [Articolo su rivista]
Boraldi, Federica; Moscarelli, Pasquale; Bochicchio, Brigida; Pepe, Antonietta; Salvi, Anna M.; Quaglino, Daniela
abstract

Heparan sulfates (HSs) modulate tissue elasticity in physiopathological conditions by interacting with various matrix constituents as tropoelastin and elastin-derived peptides. HSs bind also to protein moieties accelerating amyloid formation and influencing cytotoxic properties of insoluble fibrils. Interestingly, amyloidogenic polypeptides, despite their supposed pathogenic role, have been recently explored as promising bio-nanomaterials due to their unique and interesting properties. Therefore, we investigated the interactions of HSs, obtained from different sources and exhibiting various degree of sulfation, with synthetic amyloidogenic elastin-like peptides (ELPs), also looking at the effects of these interactions on cell viability and cell behavior using in vitro cultured fibroblasts, as a prototype of mesenchymal cells known to modulate the soft connective tissue environment. Results demonstrate, for the first time, that HSs, with differences depending on their sulfation pattern and chain length, interact with ELPs accelerating aggregation kinetics and amyloid-like fibril formation as well as self-association. Furthermore, these fibrils do not negatively affect fibroblasts' cell growth and parameters of redox balance, and influence cellular adhesion properties. Data provide information for a better understanding of the interactions altering the elastic component in aging and in pathologic conditions and may pave the way for the development of composite matrix-based biomaterials.

2018 - High speed flow cytometry allows the detection of circulating endothelial cells in hemangioblastoma patients [Articolo su rivista]
De Biasi, Sara; Gibellini, Lara; Feletti, Alberto; Pavesi, Giacomo; Bianchini, Elena; Lo Tartaro, Domenico; Pecorini, Simone; De Gaetano, Anna; Pullano, Rosalberta; Nasi, Milena; Pinti, Marcello; Cossarizza, Andrea; Boraldi, Federica
abstract

Circulating endothelial cells (CECs) detach from the intima monolayer after endothelial damages. Their circulating endothelial progenitors (CEPs) represent less than 0.01% of nucleated blood cells. Increased levels of CECs and CEPs have been detected in patients with several types of cancer, suggesting that they could be a useful blood-based marker for detecting a tumor, or for monitoring its clinical course. However, their routine monitoring is time consuming and technically challenging. Here, we present a flow cytometry method for quantifying such cells in a cohort of patients with hemangioblastoma (HB). HB is a rare benign tumor, responsible for 1-2.5% of primary intracranial tumors and up to 10% of spinal cord tumors, and for which no tools are available to predict the onset or recurrence in patients undergoing surgical removal of tumor mass. This method allowed us to accurately quantifying CEC and CEP before and after surgery. CEPs are present at high levels in HB patients than control before intervention, and decrease after tumor removal, suggesting that their percentage could represent a valid tool to monitor the disease onset and recurrence.

2018 - Interactions between elastin-like peptides and an insulating poly(ortho-aminophenol) membrane investigated by AFM and XPS [Articolo su rivista]
Carbone, Maria Elvira; Ciriello, Rosanna; Moscarelli, Pasquale; Boraldi, Federica; Bianco, Giuliana; Guerrieri, Antonio; Bochicchio, Brigida; Pepe, Antonietta; Quaglino, Daniela; Salvi, Anna Maria
abstract

This investigation was undertaken to explore the mutual recognition of the pentapeptide (ValGlyGlyValGly)n, a hydrophobic elastin-like peptide (ELP), suspended in deionized water in monomer (n = 1) and trimer (n = 3) forms and the outer surface of a very thin, insulating polymer, poly(ortho-aminophenol) (PoAP), electrochemically grown on a platinum foil by cyclic voltammetry in a neutral medium (phosphate-buffered saline, I = 0.1M) immersed in the suspension. As a prior task, the proved propensity of the ValGlyGlyValGly sequence, at the given minimal length (three or more repeats), to self-assemble into amyloid-like fibrils when solubilized in an aqueous environment was considered within the framework of testing PoAP surfaces for the specific detection of amyloid precursors. From our knowledge of the chemical structure and physical properties of both biomacromolecule families obtained in previous studies, we focused on the efficacy of the binding sites offered to ELP fibrils by PoAP in its as-prepared form or properly modified either by postsynthesis oxidation or by adsorption/entrapping of ELP monomer(s) with or without protecting terminal groups. Consistent with all methods of preparation, the best surfaces, recognizable by the trimer fibrils, are those modified to carry a larger number of carbonyls, particularly by entrapment of ELP monomer(s) during PoAP electrosynthesis using an imprinting-inspired method. The degree of attachment of fibrillar aggregates, detected by atomic force microscopy and X-ray photoelectron spectroscopy, provides unequivocal evidence of the cooperative forces involving PoAP–ELP interactions. The results obtained suggest the prospect of using the proposed Pt/PoAP/ELP systems as biodetectors in Alzheimer disease.

2018 - Mineralization by mesenchymal stromal cells is variously modulated depending on commercial platelet lysate preparations [Articolo su rivista]
Boraldi, Federica; Burns, Jorge S.; Bartolomeo, Angelica; Dominici, Massimo; Quaglino, Daniela
abstract

Background aims: Numerous cellular models have been developed to investigate calcification for regenerative medicine applications and for the identification of therapeutic targets in various complications associated with age-related diseases. However, results have often been contradictory due to specific culture conditions, cell type ontogeny and aging status. Human platelet lysate (hPL) has been recently investigated as valuable alternative to fetal bovine serum (FBS) in cell culture and bone regeneration. A parallel comparison of how all these multiple factors may converge to influence mineralization has yet to be reported. Methods: To compare mineralization of human mesenchymal cell types known to differ in extracellular matrix calcification potency, bone marrow–derived mesenchymal stromal cells and dermal fibroblasts from neonatal and adult donors, at both low and high passages, were investigated in an ex vivo experimental model by supplementing the osteogenic induction medium with FBS or with hPL. Four commercial hPL preparations were profiled by liquid chromatography/electrospray ionization quadrupole time-of-flight spectrometry, and mineralization was visualized by von Kossa staining and quantified by morphometric evaluations after 9, 14 and 21 days of culture. Results: Data demonstrate that (i) commercial hPL preparations differ according to mass spectra profiles, (ii) hPL variously influences mineral deposition depending on cell line and possibly on platelet product preparation methods, (iii) donor age modifies mineral deposition in the presence of the same hPL and (iv) reduced in vitro proliferative capacity affects osteogenic induction and response to hPL. Conclusion: Despite the standardized procedures applied to obtain commercial hPL, this study highlights the divergent effects of different preparations and emphasizes the importance of cellular ontology, donor age and cell proliferative capacity to optimize the osteogenic induction capabilities of mesenchymal stromal cells and design more effective cell-based therapeutic protocols.

2018 - Peripapillary comet lesions and comet rain in PXE-related retinopathy [Articolo su rivista]
Murro, Vittoria; Mucciolo, Dario Pasquale; Sodi, Andrea; Boraldi, Federica; Quaglino, Daniela; Virgili, Gianni; Rizzo, Stanislao
abstract

Purpose: To study peripapillary comet lesions (PCL) in Italian patients affected with pseudoxanthoma elasticum (PXE). Methods: Retrospective review of fundoscopic and swept-source (SS) optical coherence tomography (OCT) images of patients with PXE examined at the Regional Reference Center for Hereditary Retinal Degenerations at the Careggi Teaching Hospital of Florence from 2012 to 2017. Results: From 148 eyes of 74 patients affected with PXE, we identified 24 eyes of 14 patients (11 were female) with a mean age of 39 years (range, 20–58 years) characterized by peripapillary comet lesions. Of these 24 eyes, 15 eyes (of 10 patients) were characterized by comet rain. The smallest comet lesion at the OCT examination appeared as a focal roundish hyper-reflective alteration at the level of the outer retinal segments and RPE-Bruch’s membrane complex; the larger lesions appeared as circular and ovoid structures with hyper-reflective borders in the outer nuclear layer. Conclusion: The comet lesion formation process involves the outer layers of the retina and RPE/Bruch’s membrane complex. It consists of a degenerative/rearrangement process of the photoreceptors which occurs in an area of focal altered RPE/Bruch’s membrane resembling the outer retinal tubulation.

2018 - The Effects of Parenteral K1 Administration in Pseudoxanthoma Elasticum Patients Versus Controls. A Pilot Study [Articolo su rivista]
Carrillo-Linares, Juan Luis; García-Fernández, María Inmaculada; Morillo, María José; Sánchez, Purificación; Rioja, José; Barón, Francisco Javier; Ariza, María José; Harrington, Dominic J.; Card, David; Boraldi, Federica; Quaglino, Daniela; Valdivielso, Pedro
abstract

Introduction: Pseudoxanthoma elasticum (PXE) is a rare disease caused by mutations in the ABCC6 gene. Vitamin K1 is involved in the posttranslational carboxylation of some proteins related to inhibition of the calcification process. Our aim was to investigate, in patients affected by PXE, baseline levels of vitamin K1-dependent proteins and -metabolites and whether parenteral administration of phytomenadione was effective in modulating their levels. Methods: We included eight PXE patients with typical clinical symptoms (skin, retina, and vascular calcification) and two ABCC6 causative mutations; 13 clinically unaffected first-degree patients' relatives (9 carrying one ABCC6 mutation and 4 non-carriers). We assessed urinary vitamin K1 metabolites and serum Glu- and Gla-OC, Gas6 and undercaboxylated prothrombin (PIVKA-II), at baseline and after 1 and 6 weeks after a single intramuscular injection of 10 mg vitamin K1. Results: Comparison of PXE patients, heterozygous, and non-carriers revealed differences in baseline levels of serum MK-4 and of urinary vitamin K metabolites. The response to phytomenadione administration on vitamin K-dependent proteins was similar in all groups. Conclusion: The physiological axis between vitamin K1 and vitamin K-dependent proteins is preserved; however, differences in the concentration of vitamin K metabolites and of MK-4 suggest that vitamin K1 metabolism/catabolism could be altered in PXE patients.

2017 - IGF-II promotes neuroprotection and neuroplasticity recovery in a long-lasting model of oxidative damage induced by glucocorticoids [Articolo su rivista]
Martín Montañez, E; Millon, C; Boraldi, Federica; Garcia Guirado, F; Pedraza, C; Lara, E; Santin, L. J; Pavia, J; Garcia Fernandez, M.
abstract

Insulin-like growth factor-II (IGF-II) is a naturally occurring hormone that exerts neurotrophic and neuroprotective properties in a wide range of neurodegenerative diseases and ageing. Accumulating evidence suggests that the effects of IGF-II in the brain may be explained by its binding to the specific transmembrane receptor, IGFII/M6P receptor (IGF-IIR). However, relatively little is known regarding the role of IGF-II through IGF-IIR in neuroprotection. Here, using adult cortical neuronal cultures, we investigated whether IGF-II exhibits long-term antioxidant effects and neuroprotection at the synaptic level after oxidative damage induced by high and transient levels of corticosterone (CORT). Furthermore, the involvement of the IGF-IIR was also studied to elucidate its role in the neuroprotective actions of IGF-II. We found that neurons treated with IGF-II after CORT incubation showed reduced oxidative stress damage and recovered antioxidant status (normalized total antioxidant status, lipid hydroperoxides and NAD(P) H:quinone oxidoreductase activity). Similar results were obtained when mitochondria function was analysed (cytochrome c oxidase activity, mitochondrial membrane potential and subcellular mitochondrial distribution). Furthermore, neuronal impairment and degeneration were also assessed (synaptophysin and PSD-95 expression, presynaptic function and FluoroJade B® stain). IGF-II was also able to recover the long-lasting neuronal cell damage. Finally, the effects of IGF-II were not blocked by an IGF-IR antagonist, suggesting the involvement of IGF-IIR. Altogether these results suggest that, in or model, IGF-II through IGF-IIR is able to revert the oxidative damage induced by CORT. In accordance with the neuroprotective role of the IGF-II/IGF-IIR reported in our study, pharmacotherapy approaches targeting this pathway may be useful for the treatment of diseases associated with cognitive deficits (i.e., neurodegenerative disorders, depression, etc.).

2017 - Pigment epithelial-derived factor: a new player in dermal elastic fibre calcification? [Articolo su rivista]
Boraldi, Federica; Losi, Lorena; Quaglino, Daniela
abstract

Pigment 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.

2016 - Innovative Flow Cytometry Allows Accurate Identification of Rare Circulating Cells Involved in Endothelial Dysfunction [Articolo su rivista]
Boraldi, Federica; Bartolomeo, Angelica; De Biasi, Sara; Orlando, Stefania; Costa, Sonia; Cossarizza, Andrea; Quaglino, Daniela
abstract

Introduction Although rare, circulating endothelial and progenitor cells could be considered as markers of endothelial damage and repair potential, possibly predicting the severity of cardiovascu- lar manifestations. A number of studies highlighted the role of these cells in age-related dis- eases, including those characterized by ectopic calcification. Nevertheless, their use in clinical practice is still controversial, mainly due to difficulties in finding reproducible and accurate methods for their determination. Methods Circulating mature cells (CMC, CD45-, CD34+, CD133-) and circulating progenitor cells (CPC, CD45dim, CD34bright, CD133+) were investigated by polychromatic high-speed flow cytometry to detect the expression of endothelial (CD309+) or osteogenic (BAP+) differentia- tion markers in healthy subjects and in patients affected by peripheral vascular manifesta- tions associated with ectopic calcification. Results This study shows that: 1) polychromatic flow cytometry represents a valuable tool to accu- rately identify rare cells; 2) the balance of CD309+ on CMC/CD309+ on CPC is altered in patients affected by peripheral vascular manifestations, suggesting the occurrence of vas- cular damage and low repair potential; 3) the increase of circulating cells exhibiting a shift towards an osteoblast-like phenotype (BAP+) is observed in the presence of ectopic calcification. Conclusion Differences between healthy subjects and patients with ectopic calcification indicate that this approach may be useful to better evaluate endothelial dysfunction in a clinical context.

2016 - Magnesium modifies the structural features of enzymatically mineralized collagen gels affecting the retraction capabilities of human dermal fibroblasts embedded within this 3D system [Articolo su rivista]
Boraldi, Federica; Bartolomeo, Angelica; Annovi, Giulia; Debret, Romain; Quaglino, Daniela
abstract

Mineralized collagen gels have been developed as in vitro models to better understand the mechanisms regulating the calcification process and the behavior of a variety of cell types. The vast majority of data are related to stem cells and to osteoblast-like cells, whereas little information is available for dermal fibroblasts, although these cells have been associated with ectopic calcification and consequently to a number of pathological conditions. Therefore, we developed and characterized an enzymatically mineralized collagen gel in which fibroblasts were encapsulated within the 3D structure. MgCl2 was also added during gel polymerization, given its role as (i) modulator of ectopic calcification; (ii) component of biomaterials used for bone replacement; and (iii) constituent of pathological mineral deposits. Results demonstrate that, in a short time, an enzymatically mineralized collagen gel can be prepared in which mineral deposits and viable cells are homogeneously distributed. MgCl2 is present in mineral deposits and significantly affects collagen fibril assembly and organization. Consequently, cell shape and the ability of fibroblasts to retract collagen gels were modified. The development of three-dimensional (3D) mineralized collagen matrices with both different structural features and mineral composition together with the use of fibroblasts, as a prototype of soft connective tissue mesenchymal cells, may pave new ways for the study of ectopic calcification.

2015 - Donor's age and replicative senescence favour the in-vitro mineralization potential of human fibroblasts [Articolo su rivista]
Boraldi, Federica; Bartolomeo, Angelica; Di Bari, Caterina; Cocconi, Andrea; Quaglino, Daniela
abstract

Aberrant mineralization of soft connective tissues (ectopic calcification) may occur as a frequent age-related complication. Still, it remains unclear the role of mesenchymal cell donor's age and of replicative senescence on ectopic calcification. Therefore, the ability of cells to deposit in-vitro hydroxyapatite crystals and the expression of progressive ankylosis protein homolog (ANKH), ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), tissue non specific alkaline phosphatase (TNAP) and osteopontin (OPN) have been evaluated in human dermal fibroblasts derived from neonatal (nHDF) and adult (aHDF) donors (ex-vivo ageing model) or at low and high cumulative population doublings (CPD) up to replicative senescence (in-vitro ageing model). This study demonstrates that: 1) replicative senescence favours hydroxyapatite formation in cultured fibroblasts; 2) donor's age acts as a major modulator of the mineralizing potential of HDF, since nHDF are less prone than aHDF to induce calcification; 3) donor's age and replicative senescence play in concert synergistically increasing the calcification process; 4) the ANKH+ENPP1/TNAP ratio, being crucial for pyrophosphate/inorganic phosphate balance, is greatly influenced by donor's age, as well as by replicative senescence, and regulates mineral deposition; 5) OPN is only modulated by replicative senescence.

2015 - Histology-directed and imaging mass spectrometry: An emerging technology in ectopic calcification [Articolo su rivista]
Taverna, Domenico; Boraldi, Federica; De Santis, Giorgio; Caprioli, Richard M.; Quaglino, Daniela
abstract

The present study was designed to demonstrate the potential of an optimized histology directed protein identification combined with imaging mass spectrometry technology to reveal and identify molecules associated to ectopic calcification in human tissue. As a proof of concept, mineralized and non-mineralized areas were compared within the same dermal tissue obtained from a patient affected by Pseudoxanthoma elasticum, a genetic disorder characterized by calcification only at specific sites of soft connective tissues. Data have been technically validated on a contralateral dermal tissue from the same subject and compared with those from control healthy skin. Results demonstrate that this approach 1) significantly reduces the effects generated by techniques that, disrupting tissue organization, blend data from affected and unaffected areas; 2) demonstrates that, abolishing differences due to inter-individual variability, mineralized and non-mineralized areas within the same sample have a specific protein profile and have a different distribution of molecules; and 3) avoiding the bias of focusing on already known molecules, reveals a number of proteins that have been never related to the disease nor to the calcification process, thus paving the way for the selection of new molecules to be validated as pathogenic or as potential pharmacological targets.

2015 - Pseudoxanthoma elasticum and Reflectance confocal microscopy: report of two affected young sisters [Relazione in Atti di Convegno]
Mandel, Victor Desmond; Boraldi, Federica; Pellacani, Giovanni; Ciardo, Silvana; Mazzaglia, Giovanna; Farnetani, Francesca
abstract

Introduction & Objectives: Pseudoxanthoma elasticum (PXE) is a rare inherited multisystem disorder that mainly affects skin, eyes and cardiovascular system. The associated clinical signs are due to progressive calcification of elastic fibres and blood vessels, although normal levels of calcium and phosphorus in blood and urine. The first clinical description of the disease was done in 1881 by Rigal, and in 1896 was named PXE by Darier. Transmission of the disease is autosomal recessive. PXE is caused by homozygous or compound heterozygous mutations in the ATP-binding cassette subfamily C member 6 (ABCC6) gene, which encodes a transmembrane transport ADP dependent protein (MRP6). The gene is expressed predominantly in the liver and kidney, and found in low level in the tissue involved by PXE. The clinical expression of PXE is heterogeneous with considerable variation in age of onset, progression and severity of the disease, even in individuals of the same family with identical mutations. We present the case of two young sisters of 13 and 14 years old affected by PXE and the correlation between the histopathology and the reflectance confocal microscopy (RCM). Material & Methods: In our two patients we made diagnosis of PXE using clinical assessment and RCM. RCM (Vivascope 1500®: Caliber I.D., Rochester, USA) is a novel technique, based on a laser light of 830 nm of wavelength (near-infrared), which allows the visualization of skin structures at a nearly histological resolution. A depth of 250  μm can be reached, enabling the examination of the skin up to the upper dermis or to the reticular dermis, in relation of the thickness of the epidermis. The clinical and instrumental diagnosis was also confirmed by histopathological examination. The parents and the brother of 20 years old are also investigated. Moreover, we performed genetic test and counseling to all the family. Results: At dermatological examination of both patients we observed that the skin in the cervical region appeared soft, lax, wrinkled and redundant hanging in folds, while in the axillae and cubital fossae there were coalescent yellowish papules forming plaques with a symmetrically distribution. Ophthalmoscopy identified the presence of angioid streaks in both eyes of the two sisters, confirmed by retinograpy and angiography. Cardiovascular evaluation didn’t show changes in the younger patient while mild mitral valve regurgitation was identified in the older patient. Histopathological examination revealed calcification and fragmentation of elastic fibers in the dermis that appeared distorted. The parents and the brother of 20 years old didn’t show signs and symptoms of the disorder. We found a mutation of the ABCC6 gene in the two sisters while parents and brother carried one copy of the mutated gene. We performed RCM on the skin surface of the neck of our two young patients and we observed curled and fragmented thick fibers of the connective tissue in reticular dermis on both patients, which correspond to the short and curled elastic fibers of the reticular dermis in histopathology. Conclusions: Dermatological, ophthalmological and cardiological evaluation and monitoring is essential for an early diagnosis of PXE, adequate handling and better management of the associated complications when these are present. We suggest that the use of RCM it may be usefool to detect PXE in an early stage.

2015 - Pseudoxanthoma elasticum and reflectance confocal microscopy: report of two affected young sisters [Articolo su rivista]
Mandel, Victor Desmond; Boraldi, Federica; Pellacani, Giovanni; Ciardo, Silvana; Mazzaglia, Giovanna; Farnetani, Francesca
abstract

Pseudoxanthoma elasticum (PXE) is a rare inherited multisystem disorder that mainly affects skin, eyes and cardiovascular system. The associated clinical signs are due to progressive calcification of elastic fibres and blood vessels, despite normal levels of calcium and phosphorus in blood and urine. The first clinical description of the disease was done in 1881 by Rigal, and in 1896 it was named PXE by Darier. Transmission of the disease is autosomal recessive. PXE is caused by homozygous or compound heterozygous mutations in the ATP-binding cassette subfamily C member 6 (ABCC6) gene, which encodes a transmembrane transport ADP-dependent protein (MRP6). The gene is expressed predominantly in the liver and kidney, and found in low level in the tissue involved by PXE. The clinical expression of PXE is heterogeneous with considerable variation in age of onset, progression and severity of the disease, even in individuals of the same family with identical mutations. We present the case of two young sisters affected by PXE and the correlation between the histopathology and the reflectance confocal microscopy (RCM). Parents and brother carry one copy of the mutated gene, without showing signs and symptoms of the disorder. We report the main clinical aspects of PXE and we highlight the importance of early diagnosis of the disease for adequate therapeutical management of associated complications.

2014 - Can APOE and MTHFR polymorphisms have an influence on the severity of cardiovascular manifestations in Italian Pseudoxanthoma elasticum affected patients? [Articolo su rivista]
Boraldi, Federica; Costa, Sonia; Rabacchi, Claudio; Ciani, Miriam; Vanakker, Olivier; Quaglino, Daniela
abstract

Background: The clinical phenotype of Pseudoxanthoma elasticum (PXE) affected patients, although progressive with age, is very heterogeneous, even in the presence of identical ABCC6 mutations, thus suggesting the occur- rence of modifier genes. Beside typical skin manifestations, the cardiovascular (CV) system, and especially the peripheral vasculature, is frequently and prematurely compromised. Methods and results: A cohort of 119 Italian PXE patients has been characterized for apolipoprotein E (APOE) and methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms by PCR. The severity of the clinical phe- notype has been quantified according to the Phenodex PXE International score system. Statistical analysis (chi2 test, odd ratio, regression analysis, analysis of variance) were done by GraphPad. Data demonstrate that the fre- quency of APOE alleles is similar in PXE patients and in healthy subjects and that the allelic variant E2 confers a protection against the age-related increase of CV manifestations. By contrast, PXE patients are characterized by high frequency of the MTHFR-T677T polymorphism. With age, CV manifestations in T677T, but also in C677T, pa- tients are more severe than those associated with the C677C genotype. Interestingly, compound heterozygosity for C677T and A1298C polymorphisms is present in 70% of PXE patients. Conclusions: PXE patients may be screened for these polymorphisms in order to support clinicians for a better management of disease-associated CV complications.

2014 - Changes in dermal fibroblasts from Abcc6-/- mice are present before and after the onset of ectopic tissue mineralization [Articolo su rivista]
Boraldi, Federica; Bartolomeo, Angelica; Li, Q.; Uitto, J.; Quaglino, Daniela
abstract

Pseudoxanthoma elasticum (PXE), a rare genetic disease caused by mutations in the ABCC6 gene, is characterized by progressive calcification of elastic fibers in the skin, eyes, and the cardiovascular system. The pathomechanism of the mineralization is still obscure. Several hypotheses have been proposed, one of them suggesting a role for fibroblasts in controlling the amount and the quality of the calcified extracellular matrix. This hypothesis raises the question whether changes in mesenchymal cells are the cause and/or the consequences of the calcification process. In this study, fibroblasts were isolated and cultured from Abcc6+/+ and Abcc6-/- mice of different ages to investigate parameters known to be associated with the phenotype of fibroblasts from PXE patients. Results demonstrate that a few changes (Ank and Opn downregulation) are already present before the occurrence of calcification.By contrast, a modification of other parameters (intracellularO2- content, Tnap activity, and Bmp2 upregulation) can be observed inAbcc6-/- mice after the onset of tissue mineralization.These data suggest that in the Abcc6 -/- genotype, dermal fibroblasts actively contribute to changes that promote matrix calcification and that these cells can be further modulated with time by the calcified environment, thus contributing to the age-dependent progression of the disease.

2014 - Fibroblasts from patients affected by Pseudoxanthoma elasticum exhibit an altered PPi metabolism and are more responsive to pro-calcifying stimuli [Articolo su rivista]
Boraldi, Federica; Annovi, Giulia; Bartolomeo, Angelica; Quaglino, Daniela
abstract

BACKGROUND: 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.

2014 - Involvement of IGF-II receptors in the antioxidant and neuroprotective effects of IGF-II on adult cortical neuronal cultures [Articolo su rivista]
Martin Montañez, E.; Pavia, J.; Santin, L. J.; Boraldi, Federica; Estivill Torrus, G.; Aguirre, J. A.; Garcia Fernandez, M.
abstract

Insulin-like growth factor-II (IGF-II) is a naturally occurring peptide that exerts known pleiotropic effects ranging from metabolic modulation to cellular development, growth and survival. IGF-II triggers its actions by binding to and activating IGF (IGF-I and IGF-II) receptors. In this study, we assessed the neuroprotective effect of IGF-II on corticosterone-induced oxidative damage in adult cortical neuronal cultures and the role of IGF-II receptors in this effect. We provide evidence that treatment with IGF-II alleviates the glucocorticoid-induced toxicity to neuronal cultures, and this neuroprotective effect occurred due to a decrease in reactive oxygen species (ROS) production and a return of the antioxidant status to normal levels. IGF-II acts via not only the regulation of synthesis and/or activity of antioxidant enzymes, especially manganese superoxide dismutase, but also the restoration of mitochondrial cytochrome c oxidase activity and mitochondrial membrane potential. Although the antioxidant effect of IGF-I receptor activation has been widely reported, the involvement of the IGF-II receptor in these processes has not been clearly defined. The present report is the first evidence describing the involve- ment of IGF-II receptors in redox homeostasis. IGF-II may therefore contribute to the mechanisms of neuropro- tection by acting as an antioxidant, reducing the neurodegeneration induced by oxidative insults. These results open the field to new pharmacological approaches to the treatment of diseases involving imbalanced redox homeostasis. In this study, we demonstrated that the antioxidant effect of IGF-II is at least partially mediated by IGF-II receptors.

2014 - Silencing of mitochondrial Lon protease deeply impairs mitochondrial proteome and function in colon cancer cells. [Articolo su rivista]
Gibellini, Lara; Pinti, Marcello; Boraldi, Federica; Giorgio, Valentina; Bernardi, Paolo; Bartolomeo, Regina; Nasi, Milena; DE BIASI, Sara; Missiroli, Sonia; Carnevale, Gianluca; Losi, Lorena; Tesei, Anna; Pinton, Paolo; Quaglino, Daniela; Cossarizza, Andrea
abstract

Lon is a nuclear-encoded, mitochondrial protease that assists protein folding, degrades oxidized/damaged proteins, and participates in maintaining mtDNA levels. Here we show that Lon is up-regulated in several human cancers and that its silencing in RKO colon cancer cells causes profound alterations of mitochondrial proteome and function, and cell death. We silenced Lon in RKO cells by constitutive or inducible expression of Lon shRNA. Lon-silenced cells displayed altered levels of 39 mitochondrial proteins (26% related to stress response, 14.8% to ribosome assembly, 12.7% to oxidative phosphorylation, 8.5% to Krebs cycle, 6.3% to β-oxidation, and 14.7% to crista integrity, ketone body catabolism, and mtDNA maintenance), low levels of mtDNA transcripts, and reduced levels of oxidative phosphorylation complexes (with >90% reduction of complex I). Oxygen consumption rate decreased 7.5-fold in basal conditions, and ATP synthesis dropped from 0.25 ± 0.04 to 0.03 ± 0.001 nmol/mg proteins, in the presence of 2-deoxy-d-glucose. Hydrogen peroxide and mitochondrial superoxide anion levels increased by 3- and 1.3-fold, respectively. Mitochondria appeared fragmented, heterogeneous in size and shape, with dilated cristae, vacuoles, and electrondense inclusions. The triterpenoid 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid, a Lon inhibitor, partially mimics Lon silencing. In summary, Lon is essential for maintaining mitochondrial shape and function, and for survival of RKO cells.-Gibellini, L., Pinti, M., Boraldi, F., Giorgio, V., Bernardi, P., Bartolomeo, R., Nasi, M., De Biasi, S., Missiroli, S., Carnevale, G., Losi, L., Tesei, A., Pinton, P., Quaglino, D., Cossarizza, A. Silencing of mitochondrial Lon protease deeply impairs mitochondrial proteome and function in colon cancer cells.

2014 - Sirtuin 3 interacts with Lon protease and regulates its acetylation status. [Articolo su rivista]
Gibellini, Lara; Pinti, Marcello; Beretti, Francesca; Pierri, Cl; Onofrio, A; Riccio, Massimo; Carnevale, Gianluca; DE BIASI, Sara; Nasi, Milena; Torelli, F; Boraldi, Federica; DE POL, Anto; Cossarizza, Andrea
abstract

Lon is a mitochondrial protease that degrades oxidized damaged proteins, assists protein folding and participates in maintaining mitochondrial DNA levels. Changes in Lon mRNA levels, protein levels and activity are not always directly correlated, suggesting that Lon could be regulated at post translational level. We found that Lon and SIRT3, the most important mitochondrial sirtuin, colocalize and coimmunoprecipitate in breast cancer cells, and silencing or inhibition of Lon did not alter SIRT3 levels. Silencing of SIRT3 increased the levels of Lon protein and of its acetylation, suggesting that Lon is a target of SIRT3, likely at K917.

2014 - Structural characterization and biological properties of the amyloidogenic elastin-like peptide (VGGVG)3 [Articolo su rivista]
Moscarelli, P.; Boraldi, Federica; Bochicchio, B.; Pepe, A.; Salvi, A. M.; Quaglino, Daniela
abstract

The peculiar and unique properties of elastin are due to the abundance of hydrophobic residues and of repetitive sequences as XGGZG (X, Z = V, L or A). Unexpectedly, these sequences not only provide elasticity to the whole protein, but are also able to form amyloid-like fibrils. Even though amyloid fibrils have been associated for a long time to the development of serious disorders as Alzheimer's disease, recent evidence suggests that toxicity may be related to oligomeric species or to pre-fibrillar intermediates, rather than to mature fibrils. In addition, a number of studies highlighted the potential of “bio-inspired” materials based on amyloid-like nanostructures. The present study has been undertaken with the aim to characterize a chemically synthesized elastin-like peptide (VGGVG)3. Structural and biological features were compared with those of peptides as poly(VGGVG) and VGGVG that, having the same amino acid sequence, but different length and supramolecular structure have been previously investigated for their amyloidogenic properties. Results demonstrate that a minimum sequence of 15 amino acids is sufficient to aggregate into short amyloid-like fibrils, whose formation is however strictly dependent on the specific VGGVG repeated sequence. Moreover, in the attempt to elucidate the relationship among aggregation properties, fibers morphology and biocompatibility, 3T3 fibroblasts were grown in the presence of VGGVG-containing elastin-like peptides (ELPs) and analyzed for their ability to proliferate, attach and spread on ELPs-coated surfaces. Data clearly show that amyloid-like fibrils made of (VGGVG)3 are not cytotoxic at least up to the concentration of 100 μg/ml, even after several days of culture, and are a good support for cell attachment and spreading.

2014 - Thymic maturation and programmed cell death [Capitolo/Saggio]
Quaglino, Daniela; Accorsi, Alice; Boraldi, Federica; Ottaviani, Enzo
abstract

The thymus plays a crucial role in the development and maintenance of the immune system, being the main site of T cell differentiation and maturation throughout life. Associated to dramatic structural changes, its function seems to markedly diminish with time, never the less, there are several data indicating that, despite organ atrophy, at least part of the thymus remains active throughout one's lifetime. In the last decades, several studies, aiming to understand the significance of age-dependent changes in thymic structure and function, highlighted the concept that developmental and maturational stages strongly depend on the balanced and coordinated occurrence of life and death options. In particular, programmed cell death represents a fundamental requirement in order to assure a proper functionality of the immune response and to avoid the formation of uncontrolled and potentially self-damaging lymphocytic clones. By contrast, the time-dependent thymic atrophy is due to progressive replacing of lymphoid with adipose tissue. In the light of the increased knowledge on the factors/mechanisms controlling the process of adipogenesis, it could be suggested that fat accumulation in the thymic stroma might not be considered a passive, deleterious consequence of aging, but instead a potential source of molecules with various biological functions. Therefore, thymus represents a very interesting model in terms of energy expenditure and trade off, tissue homeostasis, immune defence and disease escape. The implications of changes in thymic structure, in the ratio of proliferation and programmed cell death as well as the occurrence of fat involution still represent an open question and will be discussed in the present chapter.

2013 - DIFFERENTIALLY EXPRESSED PROTEINS IN FIBROBLASTS FROM Abcc6-/- AND Abcc6+/+ MICE HIGHLIGHT THE ROLE OF A LOCALLY ALTERED PHOSPHATE METABOLISM IN THE OCCURRENCE OF ECTOPIC CALCIFICATION [Abstract in Rivista]
Bartolomeo, Angelica; Boraldi, Federica; Uitto, J.; Quaglino, Daniela
abstract

Progressive calcification of elastic fibers is typical of Pseudoxanthoma elasticum (PXE), a rare genetic disease due to mutations in the ABCC6 gene.The pathogenesis of mineral- ization is only partially known.1 Previous studies on dermal fibroblasts from PXE patients demonstrated that the calcifica- tion process is associated to impaired carboxylation of matrix- gla-protein that, in its active form, binds to calcium, therefore inhibiting mineralization.2 However, the recent observation that PXE fibroblasts exhibit also a significant upregulation of alka- line phosphatase (TNAP) activity suggested that an abnormal phosphate metabolism may take place within soft connective tis- sues, thus contributing to ectopic calcification.3 To improve the understanding of PXE it was developed a transgenic mouse model by specifically inactivating the Abcc6 gene.4 Consistently, Abcc6-/- mice recapitulate several histopathological findings typ- ical of PXE, including the slow progression of the disease.5 Aim of the present study was to isolate fibroblasts from Abcc6+/+ and Abcc6-/- mice of different ages (i.e. before and after the devel- opment of ectopic calcification) and to investigate proteins con- trolling phosphate levels in the extracellular matrix. Results demonstrate a down-regulation of pyrophosphatase/phosphodi- esterase 1, of progressive ankylosis protein and of osteopontin, whereas bone morphogenetic protein 2 and TNAP activity were up-regulated in fibroblasts from Abcc6-/- animals. These data support the hypothesis that in PXE the unbalanced ratio between factors locally controlling both calcium and phosphate homeostasis are crucial in triggering tissue calcification.

2013 - Ectopic calcification in β-thalassemia patients is associated with increased oxidative stress and lower MGP carboxylation [Articolo su rivista]
Boraldi, Federica; Garcia Fernandez, M.; PAOLINELLI DEVINCENZI, Chiara; Annovi, Giulia; Schurgers, L.; Vermeer, C.; Cianciulli, P.; Ronchetti, I.; Quaglino, Daniela
abstract

A number of beta-thalassemia (β-thal) patients in the course of the disease exhibit ectopic calcification affecting skin, eyes and the cardiovascular system. Clinical and histopathological features have been described similar to those in pseudoxanthoma elasticum (PXE), although different genes are affected in the two diseases. Cultured dermal fibroblasts from β-thal patients with and without PXE-like clinical manifestations have been compared for parameters of redox balance and for the expression of proteins, which have been already associated with the pathologic mineralisation of soft connective tissues. Even though oxidative stress is a well-known condition of β-thal patients, our results indicate that the occurrence of mineralized elastin is associated with a more pronounced redox disequilibrium, as demonstrated by the intracellular increase of anion superoxide and of oxidized proteins and lipids. Moreover, fibroblasts from β-thal PXE-like patients are characterized by decreased availability of carboxylated matrix Gla protein (MGP), as well as by altered expression of proteins involved in the vitamin K-dependent carboxylation process. Results demonstrate that elastic fibre calcification is promoted when redox balance threshold levels are exceeded and the vitamin K-dependent carboxylation process is affected decreasing the activity of MGP, a well-known inhibitor of ectopic calcification. Furthermore, independently from the primary gene defect, these pathways are similarly involved in fibroblasts from PXE and from β-thal PXE-like patients as well as in other diseases leading to ectopic calcification, thus suggesting that can be used as markers of pathologic mineralisation.

2013 - Fibroblast involvement in soft connective tissue calcification [Articolo su rivista]
Ronchetti, I.; Boraldi, Federica; Annovi, Giulia; Cianciulli, P.; Quaglino, Daniela
abstract

Soft connective tissue calcification is not a passive process, but the consequence of metabolic changes of local mesenchymal cells that, depending on both genetic and environmental factors, alter the balance between pro- and anti-calcifying pathways. While the role of smooth muscle cells and pericytes in ectopic calcifications has been widely investigated, the involvement of fibroblasts is still elusive. Fibroblasts isolated from the dermis of pseudoxanthoma elasticum (PXE) patients and of patients exhibiting PXE-like clinical and histopathological findings offer an attractive model to investigate the mechanisms leading to the precipitation of mineral deposits within elastic fibers and to explore the influence of the genetic background and of the extracellular environment on fibroblast-associated calcifications, thus improving the knowledge on the role of mesenchymal cells on pathologic mineralization

2013 - Matrix gla protein and alkaline phosphatase are differently modulated in human dermal fibroblasts from PXE patients and controls [Articolo su rivista]
Boraldi, Federica; Annovi, Giulia; Vermeer, C.; Schurgers, L. J.; Trenti, T.; Tiozzo, Roberta; Guerra, Deanna; Quaglino, Daniela
abstract

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.

2013 - Oxidative stress is a key regulator of ectopic calcifications in beta-Thalassemia patients [Abstract in Rivista]
Quaglino, Daniela; Boraldi, Federica; Annovi, Giulia; Cianciulli, Paolo
abstract

A 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.

2012 - Heparan sulfate affects elastin deposition in fibroblasts cultured from donors of different ages [Articolo su rivista]
Annovi, Giulia; Boraldi, Federica; Moscarelli, P.; Guerra, Deanna; Tiozzo, Roberta; Parma, B.; Sommer, P.; Quaglino, Daniela
abstract

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 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

2012 - THE ROLE OF DERMAL FIBROBLASTS IN THE DEVELOPMENT OF ECTOPIC CALCIFICATIONS [Abstract in Rivista]
Boraldi, Federica; Annovi, Giulia; Tiozzo, Roberta; Quaglino, Daniela
abstract

Soft 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.

2012 - THE ROLE OF VITAMIN K-DEPENDENT PROTEINS IN THE PATHOGENESIS OF PSEUDOXANTHOMA ELASTICUM [Abstract in Rivista]
Boraldi, Federica; Annovi, Giulia; Schurgers, L.; Vermeer, C.; Quaglino, Daniela
abstract

Pseudoxanthoma 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.

2011 - AN IN-VITRO MODEL OF CALCIFICATION FOR THE STUDY OF THE OSTEOGENIC POTENTIAL OF ADULT HUMAN DERMAL FIBROBLASTS. [Abstract in Rivista]
Boraldi, Federica; Annovi, Giulia; Tiozzo, Roberta; Quaglino, Daniela
abstract

In 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.

2011 - The multifaceted complexity of genetic diseases: a lesson from pseudoxanthoma elasticum. [Capitolo/Saggio]
Quaglino, Daniela; Boraldi, Federica; Annovi, Giulia; I., Ronchetti
abstract

Pseudoxanthoma 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.

2010 - Comparison of ex vivo and in vitro human fibroblast ageing models [Articolo su rivista]
Boraldi, Federica; Annovi, Giulia; Tiozzo, Roberta; Sommer, P.; Quaglino, Daniela
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.

2009 - Connective tissue and diseases: from morphology to proteomics towards the development of new therapeutic approaches. [Articolo su rivista]
Quaglino, Daniela; Boraldi, Federica; Annovi, Giulia; Guerra, Deanna; Ronchetti, Ivonne
abstract

Connective 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.

2009 - Cytotoxic Evaluation of Elastomeric Dental Impression Materials on a Permanent Mouse Cell Line and on a Primary Human Gingival Fibroblast Culture [Articolo su rivista]
Boraldi, Federica; Chiara, Coppi; Bortolini, Sergio; Consolo, Ugo; Tiozzo, Roberta
abstract

The need for clinically relevant in vitro tests of dental materials is widely recognized. Nearly all dental impression materials are introduced into the mouth just after mixing and allowed to set in contact with the oral tissues. Under these conditions, the materials may be toxic to cells or may sensitize the tissues. The aim of the present study is to evaluate the potential cytotoxicity of new preparations of elastomeric dental impression materials: A) four vinylpolysiloxanes: Elite H-D Putty and Elite H-D Light Body (Zhermack, Badia Polesine, Rovigo, Italy); Express Putty and Express Light Body (3M ESPE AG Seefeld, Germany) and B) two polyethers: Impregum Penta and Permadyne Penta L (3M ESPE AG Seefeld, Germany). The cytotoxicity of these impression materials were examined using two different cell lines: Balb/c 3T3 (permanent cell line) and human gingival fibroblasts (primary cell line) and their effects were studied by indirect and direct tests. The direct tests are performed by placing one sample of the impression materials in the centre of the Petri dishes at the time of the seeding of cells. The cell growth was evaluated at the 12th and 24th hours by cell number. The indirect tests were performed by incubating a square of 1 cm diameter impression material in 5 mL of medium at 37 °C for 24 hours (“eluates”). Subconfluent cultures are incubated with “eluates” for 24 hours. The MTT-formazan production is the method used for measuring the cell viability. The results indicate that: a) polyether materials are cytotoxic under both experimental conditions; b) among vinylpolysiloxanes, only Express Light Body (3M ESPE AG Seefeld, Germany) induces clear inhibition of cellular viability of Balb/c 3T3 evaluated by direct and indirect tests and c) the primary cell line is less sensitive to the toxic effect than the permanent cell line.

2009 - Fibroblast protein profile analysis highlights the role of oxidative stress and vitamin K recycling in the pathogenesis of pseudoxanthoma elasticum [Articolo su rivista]
Boraldi, Federica; Annovi, Giulia; Guerra, Deanna; Paolinelli Devincenzi, Chiara; Maria Inmaculada Garcia, Fernandez; Panico, Fulvio; De Santis, Giorgio; Tiozzo, Roberta; Ronchetti, Ivonne; Quaglino, Daniela
abstract

Pseudoxanthoma elasticum (PXE) is a genetic disorder associated to mutations in the ABCC6 gene, however the pathogenetic mechanisms leading to elastic fibre calcification 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. In conclusion, these data shed new light on pathogenetic pathways in PXE, by identifying a network of proteins affecting elastic fibre calcification through inefficient vitamin K recycling, and highlighting the role of several differentially expressed proteins that could be regarded as targets for validating the efficacy of future therapeutic strategies aiming to delay and/or revert the pathologic phenotype of PXE fibroblasts. Moreover, these data open new perspectives for investigating PXE-like phenotypes in the absence of ABCC6 mutations.

2009 - New insights into autophagic cell death in the gypsy moth Lymantria dispar: a proteomic approach [Articolo su rivista]
Malagoli, Davide; Boraldi, Federica; Annovi, Giulia; Quaglino, Daniela; Ottaviani, Enzo
abstract

Autophagy 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.

2008 - A long-term study on female mice fed on a genetically modified soybean: effects on liver ageing. [Articolo su rivista]
M., Malatesta; Boraldi, Federica; Annovi, Giulia; B., Baldelli; S., Battistelli; M., Biggiogera; Quaglino, Daniela
abstract

Liver represents a suitable model for monitoring the effects of a diet, due to its key role in controlling the whole metabolism. Although no direct evidence has been reported so far that genetically modified (GM) food may affect health, previous studies on hepatocytes from young female mice fed on GM soybean demonstrated nuclear modifications involving transcription and splicing pathways. In this study, the effects of this diet were studied on liver of old female mice in order to elucidate possible interference with ageing. The morpho-functional characteristics of the liver of 24-month-old mice, fed from weaning on control or GM soybean, were investigated by combining a proteomic approach with ultrastructural, morphometrical and immunoelectron microscopical analyses. Several proteins belonging to hepatocyte metabolism, stress response, calcium signalling and mitochondria were differentially expressed in GM-fed mice, indicating a more marked expression of senescence markers in comparison to controls. Moreover, hepatocytes of GM-fed mice showed mitochondrial and nuclear modifications indicative of reduced metabolic rate. This study demonstrates that GM soybean intake can influence some liver features during ageing and, although the mechanisms remain unknown, underlines the importance to investigate the long-term consequences of GM-diets and the potential synergistic effects with ageing, xenobiotics and/or stress conditions.

2008 - A proteomic approach for investigating the aging process; the human fibroblast model. [Relazione in Atti di Convegno]
Quaglino, Daniela; Boraldi, Federica; Annovi, Giulia
abstract

Proteomic analysis of changes in protein expession of fibroblasts aged in vitro or isolated from aging donors highlights some characteristics of the aging process.

2008 - Comparison of Fibroblasts from Patients Affected by Pseudoxanthoma elasticum (PXE) or by β-Thalassemia with (β-thal-PXE+) and without PXE-Like Clinical Manifestations (β-thal-PXE-) [Abstract in Rivista]
Boraldi, Federica; PAOLINELLI DEVINCENZI, Chiara; M. I., GARCIA FERNANDEZ; Annovi, Giulia; Quaglino, Daniela; Tiozzo, Roberta; P., Cianciulli; Ronchetti, Ivonne
abstract

The role of altered redox balance in the pathogenesis of PXE-like clinical manifestations in patients affected by beta-thalassemia is discussed.

2008 - New insights into Pseudoxanthoma elasticum pathogenesis by proteome analysis. [Abstract in Rivista]
Quaglino, Daniela; Boraldi, Federica; Annovi, Giulia; Guerra, Deanna; Panico, Fulvio; Spaggiari, Antonio; DE SANTIS, Giorgio; Tiozzo, Roberta; Ronchetti, Ivonne
abstract

Proteome analysis on fibroblasts from PXE patients allowed to investigate on pathways involved in the pathogenesis of the disorder, thus highlighting the importance of oxidative stress and vitamin K recycling.

2008 - Parameters of oxidative stress are present in the circulation of PXE patients [Articolo su rivista]
M. I., Garcia Fernandez; Gheduzzi, Dealba; Boraldi, Federica; PAOLINELLI DEVINCENZI, Chiara; P., Sanchez; P., Valdivielso; M. J., Morilla; Quaglino, Daniela; Guerra, Deanna; Casolari, Sara; L., Bercovitch; Ronchetti, Ivonne
abstract

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.

2008 - The effect of serum withdrawal on the protein profile of quiescent human dermal fibroblasts in primary cell culture [Articolo su rivista]
Boraldi, Federica; Annovi, Giulia; PAOLINELLI DEVINCENZI, Chiara; Tiozzo, Roberta; Quaglino, Daniela
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.

2007 - Antioxidant status in the circulation of patients with Pseudoxanthoma elasticum (PXE). [Abstract in Rivista]
Boraldi, Federica; M. I., GARCIA FERNANDEZ; Gheduzzi, Dealba; PAOLINELLI DEVINCENZI, Chiara; P., Sanchez; P., Valdivielso; M. J., Morilla; Guerra, Deanna; Casolari, Sara; Quaglino, Daniela; Ronchetti, Ivonne
abstract

Several markers of redox balance have been measured in the plasma of PXE patients. Data demonstrate the presence of an altered redox balance not only at a local level (connective tissue fibroblasts), but also in the circulation. A correlation with clinical manifestations is provided.

2007 - Fibroblasts from patients with beta-Thalassemia and Pseudoxanthoma elasticum-like clinical manifestations. [Abstract in Rivista]
PAOLINELLI DEVINCENZI, Chiara; Boraldi, Federica; M. I., GARCIA FERNANDEZ; Annovi, Giulia; Quaglino, Daniela; Tiozzo, Roberta; Ceccarelli, Daniela; P., Cianciulli; P. L., Forni; Ronchetti, Ivonne
abstract

In vitro studies on the phenotype of dermal fibroblasts from patients with beta-thalassemia and PXE-like clinical manifestations are described and the the role of altered redox balance is discussed

2007 - Hypoxia influences the cellular cross-talk of human dermal fibroblasts. A proteomic approach [Articolo su rivista]
Boraldi, Federica; Annovi, Giulia; Carraro, F.; Naldini, A.; Tiozzo, Roberta; Sommer, P.; Quaglino, Daniela
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% 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.

2007 - Is matrix GLA Protein (MGP) a key regulator of elastic fiber calcification? [Abstract in Rivista]
Annovi, Giulia; Gheduzzi, Dealba; Boraldi, Federica; L. J., Schurgers; Tiozzo, Roberta; Quaglino, Daniela; Ronchetti, Ivonne
abstract

The role of vitamin k dependent carboxylation of Matrix Gla protein in PXE patogenesis is described and discussed.

2007 - Matrix Gla Protein involved in elastic fiber calcification in the dermis of pseudoxanthoma elasticum patients. [Articolo su rivista]
Gheduzzi, Dealba; Boraldi, Federica; Annovi, Giulia; Paolinelli Devincenzi, Chiara; L., Schurgers; C., Vermeer; Quaglino, Daniela; Ronchetti, Ivonne
abstract

Mature MGP (Matrix gamma-carboxyglutamic acid protein) is known to inhibit soft connective tissues calcification. We investigated its possible involvement in pseudoxanthoma elasticum (PXE), a genetic disorder whose clinical manifestations are due to mineralization of elastic fibers. PXE patients have lower serum concentration of total MGP compared to controls (P<0.001). Antibodies specific for the noncarboxylated (Glu-MGP) and for the gamma-carboxylated (Gla-MGP) forms of MGP were assayed on ultrathin sections of dermis from controls and PXE patients. Normal elastic fibers in controls and patients were slightly positive for both forms of MGP, whereas Gla-MGP was more abundant within control's than within patient's elastic fibers (P<0.001). In patients' calcified elastic fibers, Glu-MGP intensively colocalized with mineral precipitates, whereas Gla-MGP precisely localized at the mineralization front. Data suggest that MGP is present within elastic fibers and is associated with calcification of dermal elastic fibers in PXE. To investigate whether local cells produce MGP, dermal fibroblasts were cultured in vitro and MGP was assayed at mRNA and protein levels. In spite of very similar MGP mRNA expression, cells from PXE patients produced 30% less of Gla-MGP compared to controls. Data were confirmed by immunocytochemistry on ultrathin sections. Normal fibroblasts in vitro were positive for both forms of MGP. PXE fibroblasts were positive for Glu-MGP and only barely positive for Gla-MGP (P<0.001). In conclusion, MGP is involved in elastic fiber calcification in PXE. The lower ratio of Gla-MGP over Glu-MGP in pathological fibroblasts compared to controls suggests these cells may play an important role in the ectopic calcification in PXE.

2007 - New insights on the pathogenesis of Pseudoxanthoma elasticum by proteome analysis. [Abstract in Rivista]
Boraldi, Federica; Annovi, Giulia; PAOLINELLI DEVINCENZI, Chiara; Gheduzzi, Dealba; Tiozzo, Roberta; Ronchetti, Ivonne; Quaglino, Daniela
abstract

Data are presented on the protein profile of dermal fibroblasts cultured in vitro from PXE patients, with the aim to elucidate molecular pathways involved in the pathogenesis of the disease

2007 - Response of confluent human dermal fibroblasts to serum deprivation investigated by proteome analysis. [Abstract in Rivista]
Boraldi, Federica; Annovi, Giulia; PAOLINELLI DEVINCENZI, Chiara; Guidetti, Rita; Tiozzo, Roberta; Quaglino, Daniela
abstract

A preliminary characterization of the protein profile of human dermal fibroblstats cultured in vitro after confluence, in the absence of serum factors, is provided.

2006 - Oxidative stress in fibroblasts from patients with pseudoxanthoma elasticum: possible role in the pathogenesis of clinical manifestations [Articolo su rivista]
Ronchetti, Ivonne; MI Garcia, Fernandez; Boraldi, Federica; Quaglino, Daniela; Gheduzzi, Dealba; De Vincenzi Paolinelli, C.; Tiozzo, Roberta; Bergamini, Stefania; D., Ceccarelli; U., Muscatello
abstract

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 (Delta Psi(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.

2005 - Ageing of connective tissues [Relazione in Atti di Convegno]
Quaglino, Daniela; Gheduzzi, Dealba; Boraldi, Federica; Ronchetti, Ivonne
abstract

Aging, 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.

2005 - Dermal fibroblasts from pseudoxanthoma elasticum patients have raised MMP-2 degradative potential [Articolo su rivista]
Quaglino, Daniela; Sartor, L; Garbisa, S; Boraldi, Federica; Croce, Maria Antonietta; Passi, A; De Luca, G; Tiozzo, Roberta; Ronchetti, Ivonne
abstract

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.

2005 - Identification of mineralized elastic fibers on wet samples by SEM [Articolo su rivista]
Boraldi, Federica; Tonelli, Massimo; Gheduzzi, Dealba; Ronchetti, Ivonne; Quaglino, Daniela
abstract

A method is described that could be of potential use for the rapid ultrastructural identification of abnormal and fragmented elastic fibers in very small wet samples of dermal biopsies from patients affected by Pseudoxanthoma elasticum (PXE). Moreover, the method, which consists of the use of sealed capsules transparent to electrons, allows the rapid and accurate localization and detection of mineralized areas in PXE patients and of their ion composition by X-ray microanalysis. This methodology could be of great help in any tissue disorder, especially in connective tissue disorders, characterized by structural alterations associated with ion precipitation.

2005 - Impression Materials: A Comparison Of Their Biological Characteristics [Articolo su rivista]
Coppi, C; Bortolini, Sergio; Boraldi, Federica; Consolo, Ugo; Tiozzo, Roberta
abstract

The 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.

2005 - New insights on the pathogenesis of clinical manifestations in Pseudoxathoma elasticum. [Abstract in Rivista]
Ronchetti, Ivonne; M. I., GARCIA FERNANDEZ; Boraldi, Federica; Quaglino, Daniela; Gheduzzi, Dealba; PAOLINELLI DEVINCENZI, Chiara; Tiozzo, Roberta; Bergamini, Stefania; Ceccarelli, Daniela; Muscatello, Umberto
abstract

Data are presented on the presence of oxidative stress in fibroblasts from PXE patients and the potential role in PXE pathogenesis is discussed.

2005 - On the pathogenesis of PXE-like clinical manifestations in Beta-thalassemic patients [Abstract in Rivista]
Boraldi, Federica; PAOLINELLI DEVINCENZI, Chiara; M. I., Garcia Fernandez; Quaglino, Daniela; Tiozzo, Roberta; Croce, Maria Antonietta; P., Cianciulli; F., Sorrentino; G. L., Forni; Ronchetti, Ivonne
abstract

Altered fibroblast phenotype and membrane transport properties are described in patients affected by beta-thalassemia with elastic fiber mineralization and PXE-like clinical manifestations.

2005 - The role of hypoxic condition on the protein profile profile of human fibroblasts [Abstract in Rivista]
Boraldi, Federica; Annovi, Giulia; Carraro, F; Ronchetti, Ivonne; Tiozzo, Roberta; Quaglino, Daniela
abstract

Proteome analysis of normal human dermal fibroblasts culture in normoxic and hypoxic conditions

2004 - Adhesion and proliferation of human dermal fibroblasts on collagen matrix [Articolo su rivista]
Croce, Maria Antonietta; Silvestri, C; Guerra, Deanna; Carnevali, E; Boraldi, Federica; Tiozzo, Roberta; Parma, B.
abstract

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.

2004 - Analisi della biocompatibilità dei materiali dentari da impronta [Abstract in Rivista]
Tiozzo, Roberta; Boraldi, Federica; Bortolini, Sergio; A., Natali; Consolo, Ugo
abstract

Vedasi l'allegato

2004 - Functional genomics: the PXE model [Abstract in Atti di Convegno]
Ronchetti, Ivonne; Quaglino, Daniela; Gheduzzi, Dealba; Croce, Maria Antonietta; Boraldi, Federica; Tiozzo, Roberta
abstract

Pseudoxanthoma elasticum represent a disease model for investigating the relationships bettween altered fibroblast phenotype and elastic fiber degeneration/calcification.

2004 - The protein profile of fibroblasts: the role of proteomics. [Articolo su rivista]
Quaglino, Daniela; Boraldi, Federica; L., Bini; Volpi, Nicola
abstract

Fibroblasts 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.

2004 - The skin equivalent model: developments and perspectives. [Articolo su rivista]
Croce, Maria Antonietta; Sammarco, Rita; PAOLINELLI DEVINCENZI, Chiara; Boraldi, Federica; Gheduzzi, Dealba; Damour, O.; Sommer, P.; Ronchetti, Ivonne; Tiozzo, Roberta; Quaglino, Daniela
abstract

Three-dimensional culture systems have been developed to mimic natural interactions among cells and between cells and the extracellular matrix. The “skin equivalent ” is a 3-D co-culture system of fibroblasts and keratinocytes used as a covering surface in extended wounds, and as a model for studying the influence of each cell type on the synthesis of the extracellular matrix. In the present study, a three-dimensional scaffold made of chitosan, glycosaminoglycans and collagen has been colonised by keratinocytes from newborn foreskin an by fibroblasts isolated from donors of different ages. In the skin equivalent, the neo-synthesized connective tissue is characterized by the presence of amorphous elastic fibers, strengthening the usefulness of this three-dimensional model as a powerful tool for investigating connective tissue metabolism in different physiological (i.e. aging) and pathological conditions. Interestingly, when fibroblasts isolated from aged donors were cultured in vitro, even though in the presence of abundant nutrients and growth factors, their old phenotype appeared not to be completely overcome, indicating that aging is the result of a continuous remodelling controlled by epigenetic as well as genetic factors. Finally, the aged phenotype of fibroblasts seems to exert a stronger influence on keratinocytes compared to that of keratinocytes on fibroblasts.

2004 - VALUTAZIONE DELLA CITOTOSSICITA’ DEI MATERIALI DENTALI DA IMPRONTA [Articolo su rivista]
Tiozzo, Roberta; Boraldi, Federica; Magagna, F; Bortolini, Sergio; Consolo, Ugo
abstract

THE AIM OF THIS STUDY WAS TO ASSESS THE CYTOTOXICITY OF SOME IMPRESSION MATERIALS AND OF THEIR COMPONENTS (PASTE AND CATALYST). THE FOLLOWING MATERIALS WERE TESTED: ELITE H-D + PUTTY SOFT NORMAL SETTING, ELITE H-D + LIGHT BODY NORMAL SETTING, ELITE H-D+ MAXI TRAY MATERIALS; EXPRESS PUTTY, FIRMER SET, EXPRESS FAST SET, LIGHT BODY, PERMADYNE PENTA L, IMPREGUM PENTA AND AQUASIL FS DECA MONOPHASE.THEI CYTOTOXIC EFFECTS WERE STUDIED BY INDIRECT AND DIRECT TESTING ACCORDING TO ISO 10993-PART 5. DIRECT TESTS WERE PERFORMED BY PLACING IMPRESSION MATERIALS IN THE CENTER OF PETRI DISHES CONTAINIG HUMAN GINGIVAL FIBROBLASTS.

2003 - Cell-matrix interactions of in vitro human skin fibroblasts upon addition of hyaluronan [Articolo su rivista]
Boraldi, Federica; Croce, Maria Antonietta; Quaglino, Daniela; Sammarco, Rita; Carnevali, Elena; Tiozzo, Roberta; Ronchetti, Ivonne
abstract

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 1-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. (C) 2003 Elsevier Science Ltd. All rights reserved.

2003 - Hyaluronan uptake by adult human skin fibroblasts in vitro [Articolo su rivista]
Croce, M. A.; Boraldi, F.; Quaglino, D.; Tiozzo, R.; Pasquali-Ronchetti, I.
abstract

Low and high molecular weight hyaluronan (HA) was added to adult human fibroblasts grown in monolayer to assess its influence on CD44 expression, its internalisation and effect on cell growth. CD44 expression on the surface of in vitro fibroblasts was not modified by different concentrations of FCS, whereas it was sensitive to cell cycle, being higher in the growing than in the resting phase. Independently from molecular weight, upon addition of exogenous HA (from 0.1 up to 1 mg/mL) to fibroblasts in the growing phase, a slight but constant decrease of the expression of CD44 on the surface of fibroblasts was observed; moreover, HA induced a rearrangement of CD44 into patches in close relationship with the terminal regions of stress fibers, which became thicker and more rigid after a few hours from the addition of HA to the medium. Fluorescent HA, added to the culture medium, rapidly attached to the plasma membrane and in less than two minutes was observed within cells, partly in association with its receptor CD44. By the contemporary use of neutral red, which accumulates into functional lysosomes, the great majority of internalised HA was found within lysosomes. HA receptor RHAMM-IHABP was rather homogeneously localised within the cytoplasm of normal growing fibroblasts. Upon addition of HA, the RHAMM-IHABP distribution became discontinuous around the nucleus. Addition of HA to fibroblasts induced a significant inhibition of cell growth, which was dependent on HA concentration and irrespective of HA molecular weight, at least in the ranges tested. Results show that extra-cellular HA is rapidly taken up by human dermal fibroblasts together with its CD44 receptor, and transported mostly to the lysosomes. Both low and high molecular weight HA induced down-regulation of cell proliferation, which would seem to be mediated by HA catabolism.

2003 - Multidrug resistance protein-6 (MRP6) in human dermal fibroblasts. Comparison between cells from normal subjects and from Pseudoxanthoma elasticum patients [Articolo su rivista]
Boraldi, Federica; Quaglino, Daniela; Croce, Maria Antonietta; M. I. G., Fernandez; Tiozzo, Roberta; Gheduzzi, Dealba; Bacchelli, Barbara; Ronchetti, Ivonne
abstract

Multidrug resistance protein-6 (MRP6) is a membrane transporter whose deficiency leads to the connective tissue disorder Pseudoxanthoma elasticum (PXE). In vitro dermal fibroblasts from normal and PXE subjects, homozygous for the R1141X mutation, were compared for their ability to accumulate and to release fluorescent calcein, in the absence and in the presence of inhibitors and competitors of the MDR-multidrug resistance protein (MRP) systems, such as 3-(3-(2-(7-choro-2 quinolinyl) ethenyl)phenyl ((3-dimethyl amino-3-oxo-propyl)thio) methyl) propanoic acid (MK571), verapamil (VPL), vinblastine (VBL), chlorambucil (CHB), benzbromarone (BNZ) and indomethacin (IDM). In the absence of chemicals, calcein accumulation was significantly higher and the release significantly slower in PXE cells compared to controls. VBL and CHB reduced calcein release in both cell strains, without affecting the differences between PXE and control fibroblasts. VPL, BNZ and IDM consistently delayed calcein release from both control and PXE cells; moreover, they abolished the differences between normal and MRP6-deficient fibroblasts observed in the absence of chemicals. These findings suggest that VPL, BNZ and IDM interfere with MRP6-dependent calcein extrusion in in vitro human normal fibroblasts. Interestingly, MK571 almost completely abolished calcein release from PXE cells, whereas it induced a strong but less complete inhibition in control fibroblasts, suggesting that MRP6 is not inhibited by MK571. Data show that MRP6 is active in human fibroblasts, and that its sensitivity to inhibitors and competitors of MDR-MRPs' membrane transporters is different from that of other translocators, namely, MRP1. It could be suggested that MRP1 and MRP6 transport different physiological substances and that MRP6 deficiency cannot be overcome by other membrane transporters, at least in fibroblasts. These data further support the hypothesis that MRP6 deficiency may be relevant for fibroblast metabolism and responsible for the metabolic alterations of these cells at the basis of connective tissue clinical manifestations of PXE. (C) 2003 Elsevier B.V./Intemational Society of Matrix Biology. All rights reserved.

2003 - Normal human dermal fibroblasts: Proteomic analysis of cell layer and culture medium [Articolo su rivista]
Boraldi, Federica; L., Bini; S., Liberatori; A., Armini; V., Pallini; Tiozzo, Roberta; Ronchetti, Ivonne; Quaglino, Daniela
abstract

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.

2003 - Proteome analysis of dermal fibroblasts cultured in vitro from human healthy subjects of different ages [Articolo su rivista]
Boraldi, Federica; Bini, L.; Liberatori, S.; Armini, A.; Pallini, V.; Tiozzo, Roberta; Ronchetti, Ivonne; Quaglino, Daniela
abstract

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.

2003 - Study of the potential citotoxicity of dental impression materials. [Relazione in Atti di Convegno]
Tiozzo, Roberta; F., Magagna; Boraldi, Federica; Croce, Maria Antonietta; Bortolini, Sergio; Consolo, Ugo
abstract

The aim of this study was to assess the cytotoxicity of tow 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.

2001 - Characterization of pseudoxanthoma elasticum-like lesions in the skin of patients with beta-thalassemia [Articolo su rivista]
Contri, Miranda; Bacchelli, Barbara; Boraldi, Federica; Quaglino, Daniela; Ronchetti, Ivonne; Taparelli, Francesca; Carnevali, Elena; Seidenari, Stefania; A., Francomano; V., Bettoli; V., De Sanctis
abstract

Background: Pseudoxanthoma elasticum (PXE), an inherited disorder of unknown pathogenesis, is characterized by elastic fiber mineralization, collagen fibril alterations, and accumulation of thread material in the extracellular space. PXE-like clinical lesions have been described in patients with beta -thalassemia. Objective and Methods: Dermal lesions in these two genetic disorders were compared by light and electron microscopy and by immunocytochemistry. Results: In both disorders, elastic fiber polymorphism, fragmentation, and mineralization were structurally identical. Elastic fiber mineralization in beta -thalassemia was associated with vitronectin, bone sialoprotein, and alkaline phosphatase, similar to what was observed in inherited PXE. Furthermore, abnormalities of collagen fibrils and filament aggregates were identical in both disorders. In both inherited and beta -thalassemia-associated PXE, unrelated gene defects seem to induce cell metabolic abnormalities that lead to identical clinical and structural phenotypes. Conclusion: Data indicate that patients with beta -thalassemia may undergo important alterations of connective tissues, a better understanding of which may help in preventing clinical complications.

2001 - Hyaluronan affects protein and collagen synthesis by in vitro human skin fibroblasts [Articolo su rivista]
Croce, Maria Antonietta; K., Dyne; Boraldi, Federica; Quaglino, Daniela; G., Cetta; Tiozzo, Roberta; Ronchetti, Ivonne
abstract

Given the importance of hyaluronan (HA) for the homeostasis of connective tissues during embryogenesis and aging and its role in tissue repair, the aim of the present study was to examine the effect of exogenous HA on the synthesis of total protein, collagen and HA by in vitro human dermal fibroblasts. With differences between different cell strains, HA, at concentrations between 0.5 and 1 muM, induced a significant decrease in total protein synthesised and secreted into the medium compared to controls (P < 0.05), and particularly in collagen (- 40%; P < 0.05). The ratios between collagen types I and III and between collagen types V and I were normal. Pulse and chase experiments showed that protein degradation was normal. The presence of exogenous HA did not affect HA synthesis. Data strongly indicate that a relatively high concentration of HA in the extracellular space, such as during development and in the first phases of tissue repair, would partially limit the deposition of the extracellular matrix, and of collagen in particular. This would suggest a role for HA in delaying tissue differentiation during embryogenesis and in preventing fibrosis and scar formation in fetus and in the early phases of wound healing.

2000 - Abnormal phenotype of in vitro dermal fibroblasts from patients with pseudoxanthoma elasticum (PXE) [Articolo su rivista]
Quaglino, Daniela; Boraldi, Federica; Barbieri, D; Croce, Maria Antonietta; Tiozzo, Roberta; Ronchetti, Ivonne
abstract

Pseudoxanthoma elasticum (PXE) is a genetic connective tissue disease, whose gene and pathogenesis are still unknown. Dermal fibroblasts from patients affected by PXE have been compared in vitro with fibroblasts taken from sex and age-matched normal individuals. Cells were grown and investigated in monolayer, into three-dimensional collagen gels and in suspension. Compared with normal cells, PXE fibroblasts cultured in monolayer entered more rapidly within the S phase and exhibited an increased proliferation index; on the contrary, similarly to normal fibroblasts, PXE cells did not grow in suspension. Furthermore, compared with normal fibroblasts, PXE cells exhibited lower efficiency in retracting collagen type I lattices and lower adhesion properties to collagen type I and to plasma fibronectin. This behavior was associated with higher expression of integrin subunits alpha 2, alpha 5, alpha v, whereas beta 1 subunit as well as alpha 2 beta 1 and alpha 5 beta 1 integrin expression was lower than in controls. Compared to controls, PXE fibroblasts had higher CAM protein expression in accordance with their high tendency to form cellular aggregates, when kept in suspension. The demonstration that PXE fibroblasts have altered cell-cell and cell-matrix interactions, associated with modified proliferation capabilities, is consistent with the hypothesis that the gene responsible for PXE might have a broad regulatory role on the cellular machinery. (C) 2000 Elsevier Science B.V. All rights reserved.

1999 - Coordinate changes of polyamine metabolism regulatory proteins during the cell cycle of normal human dermal fibroblasts [Articolo su rivista]
S., Bettuzzi; Davalli, Pierpaola; Astancolle, Serenella; C., Pinna; R., Roncaglia; Boraldi, Federica; Tiozzo, Roberta; M., Sharrard; Corti, Arnaldo
abstract

In human dermal fibroblasts, brought to quiescence (G(0)) by serum starvation, the S phase peaked 24 h and G(2)/M phases 36 h after serum re-addition, Under the same conditions, ornithine decarboxylase mRNA peaked at 12 h, decreased markedly in S phase and remained low until 48 h, Conversely, ornithine decarboxylase antizyme transcript dropped to its lowest level at 12 h, while reaching its highest values between 24 and 48 h. Ornithine decarboxylase activity followed essentially the pattern of its mRNA, but relative changes were much greater, S-Adenosylmethionine decarboxylase transcript and enzyme activity also peaked at around 12 h, decreasing thereafter, Spermidine/spermine N-1-acetyltransferase mRNA and activity reached the highest values at 36-48 h, Putrescine concentration increased up to 18 h and fell dramatically in the S phase, remaining low thereafter. Both spermidine and spermine reached peaks at 18 h and decreased in the S phase, but not nearly as much as putrescine, We discuss hom this comprehensive study may help Co understand the involvement of polyamines in the control of cell proliferation, (C) 1999 Federation of European Biochemical Societies.

1999 - Identification of heterozygote carriers in families with a recessive form of pseudoxanthoma elasticum (PXE) [Articolo su rivista]
Bacchelli, Barbara; Quaglino, Daniela; Gheduzzi, Dealba; Taparelli, Francesca; Boraldi, Federica; B., Trolli; O., Le Saux; Cd, Boyd; Ronchetti, Ivonne
abstract

Skin biopsies of 18 healthy relatives of patients with pseudoxanthoma elasticum (PXE), belonging to six different recessive families, have been examined by optical and electron microscopy in order to determine morphologic alterations potentially useful for the identification of carriers of this genetic disorder. These morphologic features have been compared with those observed in the same tissue areas of eight PXE patients belonging to the same families, with six normal subjects, and to the carrier status of these apparently unaffected relatives as determined by haplotype analysis using informative markers surrounding the locus of the PXE gene on chromosome 16p. The dermis of all the relatives of PXE patients, established by haplotype analysis to be heterozygote carriers of a mutation in the PXE gene, exhibited several alterations very similar, although less severe, to those typical in PXE patients. Alterations were present in the reticular dermis and consisted of irregular-sized collagen bundles and elastic fibers; elastic fibers fragmented, cribriform, and mineralized; numerous fibroblasts, larger than normal, and subendothelial elastin in small vessels. Strikingly, none of these dermal changes were noted in an unaffected relative in one family who was identified as a noncarrier by haplotype analysis. Although many of these alterations are not specific for PXE, the presence of these morphologic changes in unaffected relatives of PXE patients indicates alterations in skin that could be diagnostic for carriers of a subclinical phenotype of PXE.

1997 - Cell behaviour and cell-matrix interactions of human palmar aponeurotic cells in vitro [Articolo su rivista]
Quaglino, Daniela; G., Bergamini; Croce, Maria Antonietta; Boraldi, Federica; D., Barbieri; Caroli, Alessandro; A., Marcuzzi; Tiozzo, Roberta; Ronchetti, Ivonne
abstract

The present investigation has been performed to better characterize, in vitro, normal aponeurotic cells in comparison with dermal fibroblasts and with cells derived from Dupuytren´s affected aponeuroses. Cells were cultured in monolayer and/or into three-dimensional collagen gels. Cell structure, adhesion, and spreading capability on different substrates, as well as integrin expression were investigated by light and electron microscopy and by flow cytometry. Cell-matrix interactions were also analyzed by gel retraction experiments in the presence, or absence, of RGD peptides and anti-integrin antibodies. Normal aponeurotic cells, compared with dermal fibroblasts, exhibited in vitro peculiar structural features, which were substantially maintained in Dupuytren´s aponeurotic cells, irrespective of the substrate they were grown on. By contrast, the aponeurotic cell behavior was different in normal and diseased cells, these latter approaching that of dermal fibroblasts. Normal aponeurotic cells, in fact, were characterized by low efficiency in retracting the collagen gel, low alpha(2), alpha(1) and alpha(5) integrin subunit expression and low adhesion properties onto collagen and fibronectin, whereas cells isolated from the aponeuroses of Dupuytren´s patients exhibited higher capability of retracting the collagen gel, increased adhesion properties toward collagen and fibronectin, and higher levels of integrin expression. No differences were observed between dermal fibroblasts from Dupuytren´s patients or from normal subjects. These in vitro results are consistent with those previously obtained in situ, suggesting that palmar aponeurotic cells have a peculiar phenotype and that changes in cell-matrix interactions occur in Dupuytren´s contracture. Moreover, by comparing data obtained from the retracted fibrotic cords and the still clinically unaffected aponeuroses of the same patients, it may be noted that Dupuytren´s disease is not only confined to the clinically involved branches, but includes the whole aponeurosis of the affected hand.

1997 - Time-course of protective effects on the aorta wall by treatment with delapril, indapamide and their combination in stroke-prone spontaneously hypertensive rats (SHRsp) [Abstract in Rivista]
Contri, Miranda; Taparelli, Francesca; Boraldi, Federica; Biagini, Giuseppe
abstract

The investigation was aimed at characterizing long term changes in the aorta wall structure in hypertensive rats.

1996 - Connective tissue in skin biopsies from patients suffering systemic sclerosis [Articolo su rivista]
Quaglino, Daniela; Bergamini, G; Boraldi, Federica; Manzini, E; Davidson, Jm; Ronchetti, Ivonne
abstract

Little information is available on elastin during systemic sclerosis since biochemical and morphological data have primarily focused on collagen and glycosaminoglycan alterations of connective tissues in this pathological process. We performed ultrastructural, morphometric, biochemical and in situ hybridisation analyses on skin biopsies from patients affected by scleroderma and from site and age-matched control subjects. Affected skin revealed alterations in the distribution and organisation of collagen bundles and fibrils together with zonal increase of the microfibrillar component. Elastic fibres were significantly more numerous than in control shin, were more frequently vacuolated and characterised by electron-dense inclusions; moreover, morphometric analysis provided evidence for a significant increase of the percentage of both collagen bundles and elastin fibres in the measured tissue, compared to normal skin. Biochemical analysis seemed to confirm the increased elastin content per unit of dried weight tissue in sclerodermic skin. Differences observed among patients were only partially associated with disease duration and/or to severity of clinical manifestations. The abnormal amount of elastic fibres observed in skin biopsies from patients, and data from in situ hybridisation suggest the presence of a deregulation of the whole extracellular matrix that might be related to the role of cytokines such as TGF-beta, which has already been suggested to be involved in systemic sclerosis and in enhanced collagen and elastin production.

1996 - Matrix proteins with high affinity for calcium ions are associated with mineralization within the elastic fibers of pseudoxanthoma elasticum dermis [Articolo su rivista]
Contri, Miranda; F., Boraldi; F., Taparelli; A., De Paepe; Ronchetti, Ivonne
abstract

Ultrathin sections from the dermis of five normal subjects and from 10 patients suffering from pseudoxanthoma elasticum (PXE) were analyzed by immunoelectron microscopy with the aim of identifying and localizing proteins associated with the mineral precipitates within the altered elastic fibers. Serial sections were processed by indirect immunogold cytochemistry using primary antibodies against human fibronectin, vitronectin, bone sialoprotein, alkaline phosphatase, osteonectin, and osteopontin. In the latter two cases, antibodies against synthetic peptides were also used. The results indicate that normal elastic fibers contained osteopontin, and that this protein was associated with the apparently normal elastin as well as with the needle-shaped mineral precipitates in the elastic fibers of patients. On the contrary, significant amounts of vitronectin, alkaline phosphatase and, less, of bone sialoprotein were associated with the polymorphous mineral precipitates inside the elastic fibers. Large amounts of osteonectin and fibronectin, together with vitronectin, were localized on the microfilament aggregates, which were often associated with altered elastic fibers in PXE dermis and were never visualized in the dermis of control subjects. The results seem to indicate once more that PXE is a complex disorder of the fibroblast synthetic control. Elastic fiber mineralization might be considered a secondary event, which could depend on the abnormal synthesis and accumulation within the elastic fibers of proteins that are normally involved in mineralization processes.

1996 - Ultrastructural and morphometrical evaluations on normal human dermal connective tissue - The influence of age, sex and body region [Articolo su rivista]
Quaglino, Daniela; G., Bergamini; Boraldi, Federica; Ronchetti, Ivonne
abstract

In 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.