Fibroblasts from patients affected by Pseudoxanthoma elasticum exhibit an altered PPi metabolism and are more responsive to pro-calcifying stimuli

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

Donor's age and replicative senescence favour the in-vitro mineralization potential of human fibroblasts

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

Mineralization by mesenchymal stromal cells is variously modulated depending on commercial platelet lysate preparations

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 marrowâderived 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

Integrated CGH/WES Analyses Advance Understanding of Aggressive Neuroblastoma Evolution: A Case Study

Neuroblastoma (NB) is the most common extra-cranial malignancy in preschool children. To portray the genetic landscape of an overly aggressive NB leading to a rapid clinical progression of the disease, tumor DNA collected pre- and post-treatment has been analyzed. Array comparative genomic hybridization (aCGH), whole-exome sequencing (WES), and pharmacogenetics approaches, respectively, have identified relevant copy number alterations (CNAs), single nucleotide variants (SNVs), and polymorphisms (SNPs) that were then combined into an integrated analysis. Spontaneously formed 3D tumoroids obtained from the recurrent mass have also been characterized. The results prove the power of combining CNAs, SNVs, and SNPs analyses to assess clonal evolution during the disease progression by evidencing multiple clones at disease onset and dynamic genomic alterations during therapy administration. The proposed molecular and cytogenetic integrated analysis empowers the disease follow-up and the prediction of tumor recurrence

SARS-CoV-2 infection and replication kinetics in different human cell types: The role of autophagy, cellular metabolism and ACE2 expression

Aims: This study evaluated SARS-CoV-2 replication in human cell lines derived from various tissues and inves­ tigated molecular mechanisms related to viral infection susceptibility and replication. Main methods: SARS-CoV-2 replication in BEAS-2B and A549 (respiratory tract), HEK-293 T (kidney), HuH7 (liver), SH-SY5Y (brain), MCF7 (breast), Huvec (endothelial) and Caco-2 (intestine) was evaluated by RT-qPCR. Concomitantly, expression levels of ACE2 (Angiotensin Converting Enzyme) and TMPRSS2 were assessed through RT-qPCR and western blot. Proteins related to autophagy and mitochondrial metabolism were moni­tored in uninfected cells to characterize the cellular metabolism of each cell line. The effect of ACE2 over­ expression on viral replication in pulmonary cells was also investigated. Key findings: Our data show that HuH7, Caco-2 and MCF7 presented a higher viral load compared to the other cell lines. The increased susceptibility to SARS-CoV-2 infection seems to be associated not only with the differential levels of proteins intrinsically related to energetic metabolism, such as ATP synthase, citrate synthase, COX and NDUFS2 but also with the considerably higher TMPRSS2 mRNA expression. The two least susceptible cell types, BEAS-2B and A549, showed drastically increased SARS-CoV-2 replication capacity when ACE2 was overex­pressed. These modified cell lines are relevant for studying SARS-CoV-2 replication in vitro. Significance: Our data not only reinforce that TMPRSS2 expression and cellular energy metabolism are important molecular mechanisms for SARS-CoV-2 infection and replication, but also indicate that HuH7, MCF7 and Caco-2 are suitable models for mechanistic studies of COVID-19. Moreover, pulmonary cells overexpressing ACE2 can be used to understand mechanisms associated with SARS-CoV-2 replication.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP: 2016/20796-2FAPESP: 2020/04709-8FAPESP: 2020/13480-4FAPESP: 2020/06153-7FAPESP: 2020/08943-5FAPESP: 2006/60402-1FAPESP: 2019/10922-9CAPES: code 001CNPq: 303035/2018-8CNPq: 405691/2018-

Calcificazioni ectopiche: dalla patogenesi verso prospettive terapeutiche

La calcificazione patologica dei tessuti connettivi lassi o calcificazione ectopica (eC) è una frequente complicanza di patologie associate all’aumentare dell’età. Recenti studi hanno dimostrato che la deposizione di idrossiapatite non è il risultato di un processo passivo, ma di una dinamica interazione di fattori pro- e anti-calcificanti, anche se i meccanismi patogenetici non sono del tutto noti e non vi sono terapie efficaci. In questo contesto, patologie genetiche come lo Pseudoxanthoma elasticum (PXE), dovuta a mutazioni del gene ABCC6, rappresentano un modello interessante per studiare il ruolo di specifiche pathway molecolari nella progressiva mineralizzazione della componente elastica connettivale. Lo scopo dell’attività di ricerca svolta nell’ambito del Dottorato è stato quello di contribuire ad una migliore caratterizzazione e comprensione del ruolo delle cellule mesenchimali nella patogenesi delle eC. In primo luogo, i fibroblasti dermici isolati da soggetti sani e affetti da PXE sono stati coltivati in vitro, in presenza di stimoli pro-calcificanti. In questo ambiente, i fibroblasti modificano il loro fenotipo, alterando l’espressione di ENNP1/PC1 e di SPP1/OPN e l’attività di TNAP, inducendo la mineralizzazione. Nel PXE il rapporto tra fattori promuoventi e inibenti la disponibità di PPi (importante inibitore del processo di mineralizzazione) é spostasto in favore della calcificazione. Tuttavia, utilizzando cellule isolate da pazienti, dove la eC è già un processo in atto, resta da chiarire se i cambiamenti osservati siano la causa e/o la conseguenza della mineralizzazione. Uno studio su fibroblasti dermici ottenuti da topi transgenici Abcc6+/+ e Abcc6 -/-, di diversa età, indica che queste cellule mostrano modificazioni legate al genotipo, ossia rilevate già prima della comparsa delle eC (Ank e Opn), e modificazioni che possono rappresentare una risposta all’ambiente calcificato e/o una conseguenza del processo d’invecchiamento (O2-, Tnap e Bmp2). Per comprendere quindi se le cellule, con l’invecchiamento, sono più prone alla eC, fibroblasti isolati da neonati (nHDF) e da adulti (aHDF) (modello d’invecchiamento ex vivo ) sono stati coltivati fino alla senescenza replicativa (modello d’invecchiamento in vitro) e mantenuti in coltura sia in mezzo standard che calcificante. I risultati dimostrano che l’età del donatore e la senescenza replicativa agiscono di concerto favorendo il processo di eC alterando il rapporto tra ANKH+ENPP1/TNAP e quindi la disponibilità di PPi. È stato ipotizzato che le alterazioni morfo-funzionali età-dipendenti dei tessuti connettivi, incluse le eC, possono essere la conseguenza dell’ “inflammaging” in cui fattori solubili e cellule circolanti giocano un ruolo importante. Fibroblasti (nHDF e aHDF) sono stati coltivati in un mezzo pro-calcificante in presenza di lisati piastrinici umani (hPL). I risultati ottenuti indicano che hPL promuove la mineralizzazione, sebbene con differenze legate al donatore e/o ai metodi utilizzati per preparare il lisato. Inoltre, in pazienti affetti da eC, le cellule endoteliali mature e le cellule progenitrici (coinvolte nel processo di danno vascolare e di riparo, rispettivamente), identificate mediante un citofluorimetro di nuova generazione, mostrano uno “shift” verso un fenotipo osteoblasto-simile, suggerendo che queste cellule potrebbero essere utilizzate come marker di eC. Infine, i fibroblasti sono stati coltivati all’interno di un gel di collagene arricchito in precipitati minerali ottenuti per via enzimatica. I gel sono stati caratterizzati mediante SEM-FEG e la morfologia/vitalità cellulare sono state valutate mediante microscopia a fluorescenza. Sono in corso ulteriori studi per valutare la risposta delle cellule mesenchimali in questo sistema 3D.Pathologic calcification of soft connective tissues is a frequent complication of age-related disorders (i.e. atherosclerosis and kidney diseases). Recent studies have demonstrated that mineral precipitates are the result of a dynamic process due to an altered production of pro- and anti-calcifying factors by mesenchymal cells. Due to the complex interactions between cells, structural molecules, soluble factors and genes, pathomechanisms of ectopic calcification are still elusive and no specific therapies are available. Within this context, inherited diseases such as Pseudoxanthoma elasticum (PXE), due to ABCC6 gene mutations, represent interesting models to investigate specific molecular pathways. Aim of the present PhD dissertation was to better understand the role of mesenchymal cells in ectopic calcification. Firstly, dermal fibroblasts from PXE and healthy subjects were cultured in vitro in the presence of pro-calcifying stimuli. Under these environmental conditions, fibroblasts alter their phenotype changing ENPP1/PC1 and SPP1/OPN expression, as well as TNAP activity, thus favouring matrix mineralization. Interestingly, in PXE cells, the ratio between factors favouring and reducing PPi availability (i.e. one of the most powerful inhibitor of the mineralization process) exhibits a more pronounced shift towards a pro-calcifying balance. A still unsolved question is whether changes in fibroblasts’ behaviour are the cause and/or the consequences of the calcification process. Investigation on dermal fibroblasts from Abcc6 transgenic mice of different ages indicated that these cells exhibit modifications related to the genotype, being detected well before the development of calcification (Ank and Opn down-regulation), whereas other changes (O2- content, Tnap activity, and Bmp2 up-regulation) may represent a cellular response to the calcified environment and/or the consequence of the ageing process per se. To understand if cells are more responsive to ectopic calcification with ageing, thus contributing to the incidence of aberrant mineralization in the ageing population, fibroblasts from neonatal (nHDF) and adult (aHDF) subjects (ex-vivo aging model) were cultured up to replicative senescence (in vitro aging model) in standard and in calcifying media. Results demonstrated that donor's age and replicative senescence play in concert increasing the calcification process and altering the ANKH+ENPP1/TNAP ratio, thus affecting PPi availability. It could be suggested that age-related morpho-funcional alterations of soft connective tissues, including ectopic calcification, may be an additional consequence of “inflammaging”, where soluble factors as well as circulating cells play a role. Studies in which human platelet lysate was added to cultured nHDF and aHDF indicate that platelet-released factors promote mineralization, although with differences due to individual variability/susceptibility and/or to the methods used to prepare the lysate. In addition, in patients affected by vascular calcification, circulating endothelial and progenitor cells (i.e. cells involved in vascular damage and repair, respectively), being detected by high performance flow cytometer, exhibit a shift toward an osteoblast-like phenotype, thus paving the way for the clinical application of these cells as markers of vascular calcification. Finally, in order to provide a 3D substrate for the growth of cells in the presence of small dispersed mineral precipitates, an enzymatically calcified collagen gel has been developed and characterized by SEM-FEG, whereas cell viability and morphology were evaluated by fluorescence microscopy. Further studies are in progress to evaluate the response of mesenchymal cells in these culture conditions

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

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

Magnesium Modifies the Structural Features of Enzymatically Mineralized Collagen Gels Affecting the Retraction Capabilities of Human Dermal Fibroblasts Embedded within This 3D System

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