Generation of human PDGFRα-transgenic mouse: a novel experimental model of skin fibrosis

ABSTRACT (Ita) Introduzione: Il recettore del PDGFα (fattore di crescita derivato dalle piastrine) è un target della risposta autoimmune nella sclerosi sistemica (SSc). Sia le IgG totali purificate da siero (SSc-IgG) che gli anticorpi anti-PDGFRα clonati da cellule B della memoria di pazienti sclerodermici (SSc-Mabs) [Moroncini G. et al., A&R 2015] hanno mostrato la capacità di incrementare la trascrizione del gene del collagene in fibroblasti ottenuti dalla cute di donatori sani e di indurre fibrosi ex vivo, su cute trapiantata in topi SCID [Luchetti M. et al., A&R 2016]. Al fine di riprodurre questi risultati in vivo, abbiamo generato topi transgenici per il PDGFRα umano. Materiali e Metodi: L’intera sequenza cDNA del PDGFRα umano è stata clonata nel locus ad espressione ubiquitaria Rosa26 a livello del cromosoma 6 murino ed inserita in cellule staminali embrionali (ES) prelevate da topi C57BL/6. I cloni cellulari geneticamente modificati sono stati selezionati per la microiniezione nella blastocisti, con successiva produzione di topi chimerici. Topi eterozigoti C57BL/6 transgenici per il PDGFRα umano della generazione F2 sono stati utilizzati per stabilire la colonia. Topi maschi di dodici settimane sono stati inoculati a livello della cute del dorso, al giorno 0, 3, 6 e 9, con 0.02 mg/ml di SSc-Mabs (VHPAM-Vκ16F4 or VHPAM-Vκ13B8), o con 2 mg/ml di SSc-IgG o di IgG purificate dal siero di donatori sani (HD-IgG). Parallelamente sono stati inoculati topi controllo con la soluzione veicolo (PBS). Topi C57BL/6 wild type della stessa età e sesso sono stati usati come controlli. Tutti gli animali sono stati sacrificati al giorno 14. Nei tessuti di cute prelevati sono stati analizzati l’espressione del transgene PDGFRα umano e la quantità di collagene. Allo scopo di indurre fibrosi sistemica, sono state inoltre impiantate a livello della cute del dorso degli animali per 28 giorni minipompe osmotiche contenenti 200 μl di PBS come soluzione veicolo o di anticorpo monoclonale SSc-Mab VHPAM-Vκ16F4 (100 μg) o SSc-Mab VHPAM-Vλ16F4 (100 μg) o della miscela di SSc-Mabs VHPAM-Vκ16F4 e VHPAM-Vλ16F4 (50 μg + 50 μg). I topi sono stati sacrificati al giorno 28. Sui tessuti di cute e polmone prelevati sono stati valutati i livelli di collageno, i macrofagi a fenotipo M2 e la deposizione di actina del muscolo liscio (α-SMA). Risultati: I topi transgenici sono risultati fenotipicamente normali, fertili e non hanno mostrato apparenti caratteristiche patologiche. L’espressione del PDGFRα umano sia come mRNA che come proteina è stata rilevata nella cute di tutti i topi transgenici. L’iniezione intradermica dell’anticorpo monoclonale umano stimolatorio SSc-Mab VHPAM-Vκ16F4 o di SSc-IgG ha determinato un ispessimento del derma ed una aumentata deposizione di collagene, mentre l’anticorpo monoclonale umano non stimolatorio SSc-Mab VHPAM-Vκ13B8 o le HD-IgG non hanno indotto alcuna significativa alterazione della cute rispetto al PBS. I topi C57BL/6 wild type non hanno mostrato alcun cambiamento significativo della cute con tutti gli anticorpi inoculati. La somministrazione continua a livello sottocutaneo dell’anticorpo monoclonale SSc-Mab VHPAM-Vκ16F4 o SSc-Mab VHPAM-Vλ16F4 o della miscela dei due ha determinato un ispessimento del derma ed una aumentata deposizione di collagene a livello della cute. Inoltre nel tessuto polmonare è stato evidenziato un aumento di collagene a livello perivascolare e peribronchiolare, con aree di infiammazione caratterizzate dalla presenza di macrofagi a fenotipo M2 e piccoli capillari con cellule endoteliali α-SMA positive. Conclusioni: Abbiamo generato un nuovo modello murino umanizzato di fibrosi cutanea grazie alla concomitante espressione del PDGFRα umano e all’inoculo di anticorpi stimolatori anti-PDGFRα umano. Questo modello murino può essere utile per l’identificazione e la validazione preclinica di nuovi approcci terapeutici per la cura della sclerodermia.ABSTRACT (Eng) Background: Platelet Derived Growth Factor (PDGF) Receptor α (PDGFRα) is a target of the autoimmune response in scleroderma (SSc). Both total serum IgG (SSc-IgG) and anti-PDGFRα antibodies cloned from memory B cells of SSc patients (SSc-Mabs) [Moroncini G. et al., A&R 2015] demonstrated the ability to increase collagen gene transcription in healthy donor skin fibroblasts and to induce fibrosis ex vivo, in skin grafts in SCID mice [Luchetti M. et al., A&R 2016]. In order to replicate these findings in vivo, we generated human PDGFRα-transgenic mice. Materials and Methods: Full length human PDGFRα cDNA was knocked-in into the ubiquitously expressed Rosa26 locus on mouse chromosome 6. Correctly targeted C57BL/6 ES cell clones were selected for blastocyst microinjection, followed by chimera production. F2 heterozygous C57BL/6-hPDGFRα transgenic mice were used to establish the colony. Twelve weeks-old male mice were injected into the back skin at days 0, 3, 6 and 9, either with 0.02 mg/ml of SSc-Mabs (VHPAM-Vκ16F4 or VHPAM-Vκ13B8), or with 2 mg/ml of SSc-IgG or IgG purified from serum of healthy donors (HD-IgG). Vehicle only injection control was included. Age- and sex- matched C57BL/6 wild type mice were used as controls. Animals were sacrificed at day 14. Human PDGFRα transgene expression and collagen amount were assessed in explanted skin tissue. To induce systemic fibrosis, osmotic minipumps containing either 200 μl PBS as vehicle or SSc-MabVHPAM-Vκ16F4 (100 μg) or SSc-MabVHPAM-Vλ16F4 (100 μg) or a combination of SSc-Mabs VHPAM-Vκ16F4 and VHPAM-Vλ16F4 (50 μg + 50 μg) were implanted for 28 days on the back skin of animals. Mice were sacrificed at day 28. Collagen amount, M2 macrophages and alpha smooth muscle (α-SMA) deposition were assessed in explanted skin and lung tissue. Results: Transgenic mice were phenotypically normal, fertile, and did not display any apparent pathological features.Human PDGFRα mRNA and protein were detectable in the skin of all examined transgenic mice. Intradermal injection of stimulatory human SSc-Mab VHPAM-Vκ16F4 or SSc-IgG resulted in dermal thickening and increased collagen deposition, whereas non-stimulatory human SSc-Mab VHPAM-Vκ13B8 or HD-IgG did not induce any significant skin tissue alterations compared to vehicle control. C57BL/6 wild type mice did not show any significant skin tissue changes with any antibodies. Subcutaneous continuous administration of SSc-MabVHPAM-Vκ16F4 or SSc-MabVHPAM-Vλ16F4 or the combination of SSc-Mabs VHPAM-Vκ16F4 and VHPAM-Vλ16F4 resulted in dermal thickening and increased collagen deposition in skin tissue. Moreover perivascular and peribronchiolar increased collagen amount were detected in lung tissue, with some inflammatory areas characterized by M2 macrophages and α-SMA endothelial positive cells in small vessels. Conclusions: We generated a novel humanized mouse model of skin fibrosis based on the concomitant expression of human PDGFRα and injection of stimulatory anti-PDGFRα antibodies. This mouse model may be useful for identification and preclinical validation of new therapeutic strategies for SSc

Systemic Sclerosis: From Pathophysiology to Novel Therapeutic Approaches

Systemic sclerosis (SSc) is a systemic, immune-mediated chronic disorder characterized by small vessel alterations and progressive fibrosis of the skin and internal organs. The combination of a predisposing genetic background and triggering factors that causes a persistent activation of immune system at microvascular and tissue level is thought to be the pathogenetic driver of SSc. Endothelial alterations with subsequent myofibroblast activation, excessive extracellular matrix (ECM) deposition, and unrestrained tissue fibrosis are the pathogenetic steps responsible for the clinical manifestations of this disease, which can be highly heterogeneous according to the different entity of each pathogenic step in individual subjects. Although substantial progress has been made in the management of SSc in recent years, disease-modifying therapies are still lacking. Several molecular pathways involved in SSc pathogenesis are currently under evaluation as possible therapeutic targets in clinical trials. These include drugs targeting fibrotic and metabolic pathways (e.g., TGF-β, autotaxin/LPA, melanocortin, and mTOR), as well as molecules and cells involved in the persistent activation of the immune system (e.g., IL4/IL13, IL23, JAK/STAT, B cells, and plasma cells). In this review, we provide an overview of the most promising therapeutic targets that could improve the future clinical management of SSc

Skin Gene Expression Profiles in Systemic Sclerosis: From Clinical Stratification to Precision Medicine

Systemic sclerosis, also known as scleroderma or SSc, is a condition characterized by significant heterogeneity in clinical presentation, disease progression, and response to treatment. Consequently, the design of clinical trials to successfully identify effective therapeutic interventions poses a major challenge. Recent advancements in skin molecular profiling technologies and stratification techniques have enabled the identification of patient subgroups that may be relevant for personalized treatment approaches. This narrative review aims at providing an overview of the current status of skin gene expression analysis using computational biology approaches and highlights the benefits of stratifying patients upon their skin gene signatures. Such stratification has the potential to lead toward a precision medicine approach in the management of SSc

Analysis of M1 and M2 tumor associated macrophages in tongue squamous cell carcinomas.

none6Tumor associated macrophages (TAMs) are the dominant leukocyte population found in the tumor microenvironment and their mobilization into tumor tissues is a critical event in tumor initiation, growth, and development. TAMs are a heterogeneous population of innate myeloid cells and take on various phenotypes depending on the context of the molecular stimuli from the microenvironment. Basically, there are two main recognized categories: classically-activated pro-inflammatory M1 macrophages (expressing the CD11c antigen) and alternatively-activated anti-inflammatory M2 macrophages (expressing the CD163 antigen). Although TAMs have been detected in head and neck cancers, little is known about their phenotype in the context of the tongue squamous cell carcinoma (TSCC), which have the highest incidence in maxillofacial malignant tumors. The aim of the present retrospective study was to characterize the macrophage polarization in low grade (G1) and high grade (G3) TSCC, and to examine the importance of their relative localization (tumor stroma, inflammation area or tumor nest) on tumor-promoting capabilities and tumor-prognostic relevance.noneSilvia Agarbati1, Marco Mascitti, Giuseppina Campisi, Lorenzo Lo Muzio, Corrado Rubini, Francesca FazioliAgarbati1, Silvia; Mascitti, Marco; Campisi, Giuseppina; LO MUZIO, Lorenzo; Rubini, Corrado; Fazioli, Francesc

Legislative Documents

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Circulating Inflamma-miRs as Potential Biomarkers of Cognitive Impairment in Patients Affected by Alzheimer's Disease

none17noAlzheimer's disease (AD), the most prevalent neurodegenerative disease in the growing population of elderly people, is still lacking minimally-invasive circulating biomarkers that could facilitate the diagnosis and the monitoring of disease progression. MicroRNAs (miRNAs) are emerging as tissue-specific and/or circulating biomarkers of several age-related diseases, but evidence on AD is still not conclusive. Since a systemic pro-inflammatory status was associated with an increased risk of AD development and progression, we focused our investigation on a subset of miRNAs modulating the inflammatory process, namely inflamma-miRNAs. The expression of inflamma-miR-17-5p, -21-5p, -126-3p, and -146a-5p was analyzed in plasma samples from 116 patients with AD compared with 41 age-matched healthy control (HC) subjects. MiR-17-5p, miR-21-5p, and miR-126-3p plasma levels were significantly increased in AD patients compared to HC. Importantly, a strong inverse relationship was observed between miR-21-5p and miR-126-3p, and the cognitive impairment, assessed by Mini-Mental State Examination (MMSE). Notably, miR-126-3p was able to discriminate between mild and severe cognitive impairment. Overall, our results reinforce the hypothesis that circulating inflamma-miRNAs could be assessed as minimally invasive tools associated with the development and progression of cognitive impairment in AD.restrictedGiuliani, Angelica; Gaetani, Simona; Sorgentoni, Giulia; Agarbati, Silvia; Laggetta, Maristella; Matacchione, Giulia; Gobbi, Mirko; Rossi, Tommaso; Galeazzi, Roberta; Piccinini, Gina; Pelliccioni, Giuseppe; Bonfigli, Anna Rita; Procopio, Antonio Domenico; Albertini, Maria Cristina; Sabbatinelli, Jacopo; Olivieri, Fabiola; Fazioli, FrancescaGiuliani, Angelica; Gaetani, Simona; Sorgentoni, Giulia; Agarbati, Silvia; Laggetta, Maristella; Matacchione, Giulia; Gobbi, Mirko; Rossi, Tommaso; Galeazzi, Roberta; Piccinini, Gina; Pelliccioni, Giuseppe; Bonfigli, Anna Rita; Procopio, Antonio Domenico; Albertini, Maria Cristina; Sabbatinelli, Jacopo; Olivieri, Fabiola; Fazioli, Francesc

Protein expression of cytokines and matrix components in lung tissue assessed by Western blotting.

<p>Collagen Col1A1 (<b>A</b>), TGFβ (<b>B</b>), IL-1β (<b>C</b>), IL-2 (<b>D</b>), IL-6 (<b>E</b>) and IL-10 (<b>F</b>) protein levels in whole lung tissue lysates obtained from C57BL/6 mice 21 days after endotracheal injection of sterile saline (saline) or bleomycin (bleo), the latter also followed by intravenous infusion of hUC-MSC (bleo+hUC-MSC) or sterile saline (bleo+saline). Results are representative of three independent experiments. Densitometric analysis of protein bands is normalized to actin and expressed as mean ± SD (n = 5 per group). ** = P < 0.01, *** = P < 0.001 compared to bleomycin treated mice.</p

Time course of cytokines and matrix components in lung tissue assessed by quantitative real-time PCR.

<p>Gene expression analysis of Col1A1 (<b>A</b>), TGFβ (<b>B</b>), α-SMA (<b>C</b>), IL-1β (<b>D</b>), IL-2 (<b>E</b>), IL-6 (<b>F</b>) and IL-10 (<b>G</b>) in whole lung mRNA obtained at days 8, 14 and 21 after endotracheal injection of sterile saline (saline) or bleomycin (bleomycin), the latter also followed by intravenous infusion of hUC-MSC (bleomycin+hUC-MSC) or sterile saline (bleomycin+saline). Results are expressed as mean ± SD (n = 5 per group) and are representative of three independent experiments performed in triplicate. * = P < 0.05, ** = P < 0.01 compared to bleomycin treated mice.</p