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FRANCESCO DEMETRIO LOFARO

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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.

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 “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 - 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 - 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 - 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 - 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 - 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.