Simona Ceccarelli

Human Adipose-Derived Stromal Cells for Cell-Based Therapies in the Treatment of Systemic Sclerosis:

The present study was designed to evaluate the clinical outcome of cell-based therapy with cultured adipose-derived stromal cells (ASCs) for the treatment of cutaneous manifestations in patients affected by systemic sclerosis (SSc). ASCs have an extraordinary developmental plasticity, including the ability to undergo multilineage differentiation and self-renewal. Moreover, ASCs can be easily harvested from small volumes of liposuction aspirate, showing great in vitro viability and proliferation rate. Here we isolated, characterized, and expanded ASCs, assessing both their mesenchymal origin and their capability to differentiate towards the adipogenic, osteogenic, and chondrogenic lineage. We developed an effective method for ASCs transplantation into sclerodermic patients by means of a hyaluronic acid (HA) solution, which allowed us to achieve precise structural modifications. ASCs were isolated from subcutaneous adipose tissue of six sclerodermic patients and cultured in a chemical-defined medium before autologous transplantation to restore skin sequelae. The results indicated that transplantation of a combination of ASCs in HA solution determined a significant improvement in tightening of the skin without complications such as anechoic areas, fat necrosis, or infections, thus suggesting that ASCs are a potentially valuable source of cells for skin therapy in rare diseases such as SSc and generally in skin disorders.

Epstein-Barr virus latent membrane protein 1 promotes concentration in multivesicular bodies of fibroblast growth factor 2 and its release through exosomes

FGF‐2, a potent angiogenic factor that is involved in tumor invasion, is known to be released extracellularly by a nonclassical secretory pathway. Recently it has become clear that Epstein‐Barr virus, specifically its oncoprotein LMP1, can induce expression of angiogenic factors. Among these factors is FGF‐2. LMP1 not only promotes expression of FGF‐2, but also the release extracellularly of its 18‐kDa isoform. We analyzed the mechanism of FGF‐2 release induced by LMP1. Confocal immunofluorescence microscopy revealed colocalization of FGF‐2 with LMP1 in small dots also stained positively for CD63 and cathepsin D, markers of late endosomes or multivesicular bodies. Biochemical analysis and immunoelectron microscopy of purified exosomal fractions from cotransfected cells demonstrated increased release of exosomes and the concentration of LMP1 and FGF‐2 in these structures. Moreover, cotransfection appeared to induce partial redistribution of the Na+/K+‐ATPase, which participates in FGF‐2 release, from the plasma membrane to the intracellular LMP1/FGF‐2 positive dots. Treatment with ouabain, which inhibits Na+/K+‐ATPase activity, partially suppressed FGF‐2 secretion via exosomes in a dose‐dependent manner. The results suggest that exosomes may represent a previously unrecognized mechanism for FGF‐2 release mediated by LMP1, and that this pathway involves the activity of Na+/K+‐ATPase.

The clinical application of autologous bioengineered skin based on a hyaluronic acid scaffold

The aim of this work was to generate an in vitro skin substitute harbouring autologous fibroblasts, keratinocytes and melanocytes, to establish a new one-step clinical method in problems associated with skin disorders. Here we present a case of a nine-year-old girl with a congenital giant nevus treated by surgical approach, with primary co-cultures of keratinocytes, melanocytes and fibroblasts obtained from autologous skin biopsy. Generally these lesions need to be removed to avoid the risk of transformation into malignant melanoma. With this purpose we analyzed the melanocytes contained in the new skin substitute for the presence of genetic alterations correlated to increased risk for melanoma. The organotypical cultures were designed including an engineered scaffold of a non-woven mesh of hyaluronic acid (HYAFF11). This biomaterial has been previously demonstrated to be the most suitable to maintain polarity and to support the in vitro constructs. Six dermal-epidermal skin substitutes were transplanted and 14 days after surgery the re-epithelialized area was about 90%. Our results suggest that this new dermal-epidermal construct not only reduces hospitalization time and ameliorates scar retraction, but might also represent a solution for the high risk of developing a tumour derived from the original nevus.

UVB-induced activation and internalization of keratinocyte growth factor receptor

Ultraviolet irradiation of mammalian cells induces several events that include activation of growth factor receptors and triggering of signal transduction pathway. Most of the UV responses are mediated by the production of reactive oxygen species (ROS) and can be blocked by antioxidants. In this study, we analysed the effect of UVB irradiation at physiologic doses and that of the pro-oxidant agent cumene hydroperoxide (CUH) on the activation of the receptor for keratinocyte growth factor (KGF), a key mediator of epithelial growth and differentiation. Exposure to both UVB (30–150 mJ/cm2) and CUH (200 μM of NIH3T3 KGFR (KGF receptors) transfectants caused a rapid tyrosine phosphorylation and activation of KGFR similar to that induced by KGF, and internalization of the activated receptor. The KGFR expression appeared unmodified by the treatments. Ultrastructural observations of both UVB- and CUH-treated cells showed a normal morphology of the plasma membranes and intracellular organelles. The antioxidant N-acetylcysteine inhibited UVB-induced receptor phosphorylation. The generation of an intracellular oxidative stress was detected as a decrease of catalase activity and of vitamin E, and reduced glutathione levels, whereas superoxide dismutase activity was not significantly modified. A peroxidation of polyunsaturated fatty acids of cell membranes was observed after both treatments, associated with the intracellular oxidative stress. Similar biochemical events were observed on NIH3T3 untransfected control cells, suggesting that KGFR activation follows intracellular generation of ROS and is not associated with a scavenging effect. Taken together our results demonstrate that exposure to UVB and to oxidant stimuli induces a rapid intracellular production of ROS, which in turn are capable of triggering KGFR activation and internalization, similar to those induced by KGF.

Silencing of Keratinocyte Growth Factor Receptor Restores 5-Fluorouracil and Tamoxifen Efficacy on Responsive Cancer Cells

Background Keratinocyte growth factor receptor (KGFR) is a splice variant of the FGFR2 gene expressed in epithelial cells. Activation of KGFR is a key factor in the regulation of physiological processes in epithelial cells such as proliferation, differentiation and wound healing. Alterations of KGFR signaling have been linked to the pathogenesis of different epithelial tumors. It has been also hypothesized that its specific ligand, KGF, might contribute to the development of resistance to 5-fluorouracil (5-FU) in epithelial cancers and tamoxifen in estrogen-positive breast cancers. Methodology/Principal Findings Small interfering RNA was transfected into a human keratinocyte cell line (HaCaT), a breast cancer derived cell line (MCF-7) and a keratinocyte primary culture (KCs) to induce selective downregulation of KGFR expression. A strong and highly specific reduction of KGFR expression was observed at both RNA (reduction = 75.7%, P = 0.009) and protein level. KGFR silenced cells showed a reduced responsiveness to KGF treatment as assessed by measuring proliferation rate (14.2% versus 39.0% of the control cells, P<0.001) and cell migration (24.6% versus 96.4% of the control cells, P = 0.009). In mock-transfected MCF-7 cells, KGF counteracts the capacity of 5-FU to inhibit cell proliferation, whereas in KGFR silenced cells KGF weakly interferes with 5-FU antiproliferative effect (11.2% versus 28.4% of the control cells, P = 0.002). The capacity of 5-FU to induce cell death is abrogated by co-treatment with KGF, whereas in KGFR silenced cells 5-FU efficiently induces cell death even combined to KGF, as determined by evaluating cell viability. Similarly, the capacity of tamoxifen to inhibit MCF-7 and KCs proliferation is highly reduced by KGF treatment and is completely restored in KGFR silenced cells (12.3% versus 45.5% of the control cells, P<0.001). Conclusions/Significance These findings suggest that selective inhibition of the KGF/KGFR pathway may provide a useful tool to ameliorate the efficacy of the therapeutic strategies for certain epithelial tumors.

The use of cultured autologous oral epithelial cells for vaginoplasty in male-to-female transsexuals: a feasibility, safety, and advantageousness clinical pilot study.

Summary: Various vaginoplasty methods in male-to-female transsexuals have been described, but none is ideal. The use of cultured autologous oral epithelium to cover the neovagina walls is presented. Six patients were operated on. Complications encountered were one case of low-located rectovaginal fistula that was treated conservatively; one case of partial glans necrosis resulting in a scarred but sensitive clitoris; one case of large labia majora requiring reduction; and one case of short vagina requiring surgical revision with autologous cultured oral cells that was successful. All patients had adequate vagina, experienced sexual intercourse, and were satisfied with results. Autologous cultured oral epithelium transplantation is feasible, safe, and advantageous. Satisfactory neovaginas were provided; the procedure is relatively easy to perform and provides a thin, mucosa-lined neovagina with a sufficient amount of secretion.

Gene Expression Profile of Patients with Mayer-Rokitansky-Küster-Hauser Syndrome: New Insights into the Potential Role of Developmental Pathways

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is a rare disease characterized by congenital aplasia of uterus and vagina. Although many studies have investigated several candidate genes, up to now none of them seem to be responsible for the aetiology of the syndrome. In our study, we identified differences in gene expression profile of in vitro cultured vaginal tissue of MRHKS patients using whole-genome microarray analysis. A group of eight out of sixteen MRKHS patients that underwent reconstruction of neovagina with an autologous in vitro cultured vaginal tissue were subjected to microarray analysis and compared with five healthy controls. Results obtained by array were confirmed by qRT-PCR and further extended to other eight MRKHS patients. Gene profiling of MRKHS patients delineated 275 differentially expressed genes, of which 133 downregulated and 142 upregulated. We selected six deregulated genes (MUC1, HOXC8, HOXB2, HOXB5, JAG1 and DLL1) on the basis of their fold change, their differential expression in most patients and their relevant role in embryological development. All patients showed upregulation of MUC1, while HOXB2 and HOXB5 were downregulated, as well as Notch ligands JAG1 and DLL1 in the majority of them. Interestingly, HOXC8 was significantly upregulated in 47% of patients, with a differential expression only in MRKHS type I patients. Taken together, our results highlighted the dysregulation of developmental genes, thus suggesting a potential alteration of networks involved in the formation of the female reproductive tract and providing a useful clue for understanding the pathophysiology of MRKHS.

Crizotinib-induced antitumour activity in human alveolar rhabdomyosarcoma cells is not solely dependent on ALK and MET inhibition

BackgroundRhabdomyosarcoma (RMS) is the most commonly diagnosed malignant soft tissue tumour in children and adolescents. Aberrant expression of Anaplastic Lymphoma Kinase (ALK) and MET gene has been implicated in the malignant progression of RMS, especially in the alveolar subtype. This observation suggests that crizotinib (PF-02341066), a kinase inhibitor against ALK and MET, may have a therapeutic role in RMS, although its antitumour activity in this malignancy has not yet been studied.MethodsRH4 and RH30 alveolar RMS (ARMS) cell lines were treated with crizotinib and then assessed by using proliferation, viability, migration and colony formation assays. Multiple approaches, including flow cytometry, immunofluorescence, western blotting and siRNA-based knock-down, were used in order to investigate possible molecular mechanisms linked to crizotinib activity.ResultsIn vitro treatment with crizotinib inhibited ALK and MET proteins, as well as Insulin-like Growth Factor 1 Receptor (IGF1R), with a concomitant robust dephosphorylation of AKT and ERK, two downstream kinases involved in RMS cell proliferation and survival. Exposure to crizotinib impaired cell growth, and accumulation at G2/M phase was attributed to an altered expression and activation of checkpoint regulators, such as Cyclin B1 and Cdc2. Crizotinib was able to induce apoptosis and autophagy in a dose-dependent manner, as shown by caspase-3 activation/PARP proteolytic cleavage down-regulation and by LC3 activation/p62 down-regulation, respectively. The accumulation of reactive oxygen species (ROS) seemed to contribute to crizotinib effects in RH4 and RH30 cells. Moreover, crizotinib-treated RH4 and RH30 cells exhibited a decreased migratory/invasive capacity and clonogenic potential.ConclusionsThese results provide a further insight into the molecular mechanisms affected by crizotinib in ARMS cells inferring that it could be a useful therapeutic tool in ARMS cancer treatment.

TNFα modulates Fibroblast Growth Factor Receptor 2 gene expression through the pRB/E2F1 pathway: identification of a non-canonical E2F binding motif.

Interactions between epithelium and mesenchyme during wound healing are not fully understood, but Fibroblast Growth Factors (FGFs) and their receptors FGFRs are recognized as key elements. FGFR2 gene encodes for two splicing transcript variants, FGFR2-IIIb or Keratinocyte Growth Factor Receptor (KGFR) and FGFR2-IIIc, which differ for tissue localization and ligand specificity. Proinflammatory cytokines play an essential role in the regulation of epithelial-mesenchymal interactions, and have been indicated to stimulate FGFs production. Here we demonstrated that upregulation of FGFR2 mRNA and protein expression is induced by the proinflammatory cytokines Tumor Necrosis Factor-α, Interleukin-1β and Interleukin 2. Furthermore, we found that TNFα determines FGFR2 transcriptional induction through activation of pRb, mediated by Raf and/or p38 pathways, and subsequent release of the transcription factor E2F1. Experiments based on FGFR2 promoter serial deletions and site-directed mutagenesis allowed us to identify a minimal responsive element that retains the capacity to be activated by E2F1. Computational analysis indicated that this element is a non-canonical E2F responsive motif. Thus far, the molecular mechanisms of FGFR2 upregulation during wound healing or in pathological events are not known. Our data suggest that FGFR2 expression can be modulated by local recruitment of inflammatory cytokines. Furthermore, since alterations in FGFR2 expression have been linked to the pathogenesis of certain human cancers, these findings could also provide elements for diagnosis and potential targets for novel therapeutic approaches.

Characterization of Wnt/ β -catenin signaling in rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and accounts for about 5% of all malignant paediatric tumours. β-Catenin, a multifunctional nuclear transcription factor in the canonical Wnt signaling pathway, is active in myogenesis and embryonal somite patterning. Dysregulation of Wnt signaling facilitates tumour invasion and metastasis. This study characterizes Wnt/β-catenin signaling and functional activity in paediatric embryonal and alveolar RMS. Immunohistochemical assessment of paraffin-embedded tissues from 44 RMS showed β-catenin expression in 26 cases with cytoplasmic/membranous expression in 9/14 cases of alveolar RMS, and 15/30 cases of embryonal RMS, whereas nuclear expression was only seen in 2 cases of embryonal RMS. The potential functional significance of β-catenin expression was tested in four RMS cell lines, two derived from embryonal (RD and RD18) RMS and two from alveolar (Rh4 and Rh30) RMS. Western blot analysis demonstrated the expression of Wnt-associated proteins including β-catenin, glycogen synthase kinase-3β, disheveled, axin-1, naked, LRP-6 and cadherins in all cell lines. Cell fractionation and immunofluorescence studies of the cell lines (after stimulation by human recombinant Wnt3a) showed reduced phosphorylation of β-catenin, stabilization of the active cytosolic form and nuclear translocation of β-catenin. Reporter gene assay demonstrated a T-cell factor/lymphoid-enhancing factor-mediated transactivation in these cells. In response to human recombinant Wnt3a, the alveolar RMS cells showed a significant decrease in proliferation rate and induction of myogenic differentiation (myogenin, MyoD1 and myf5). These data indicate that the central regulatory components of canonical Wnt/β-catenin signaling are expressed and that this pathway is functionally active in a significant subset of RMS tumours and might represent a novel therapeutic target.