The use of human adipose-derived stem cells in the treatment of physiological and pathological vulvar dystrophies

“Vulvar dystrophy” is characterized by chronic alterations of vulvar trophism, occurring in both physiological (menopause) and pathological (lichen sclerosus, vulvar graft-versus-host disease) conditions. Associated symptoms are itching, burning, dyspareunia and vaginal dryness. Current treatments often do not imply a complete remission of symptoms. Adipose-Derived Stem Cells (ADSCs) injection represents a valid alternative therapy to enhance trophism and tone of dystrophic tissues. We evaluated efficacy of ADSCs-based therapy in the dystrophic areas. From February to April 2013 we enrolled 8 patients with vulvar dystrophy. A biopsy specimen was performed before and after treatment. Digital photographs were taken at baseline and during the follow-up. Pain was detected with Visual Analogue Scale and sexual function was evaluated with Female Sexual Function Index. All patients received 2 treatments in 3 months. Follow-up was at 1 week , 1 and 3 months, and 1 and 2 years. We obtained a significant vulvar trophism enhancement in all patients, who reported pain reduction and sexual function improvement. Objective exam with speculum was easy to perform after treatment. We believe ADSCs-based therapy finds its application in the treatment of vulvar dystrophies, since ADSCs could induce increased vascularization due to their angiogenic properties and tissue trophism improvement thanks to their eutrophic effect

Notch3 targeting. A novel weapon against ovarian cancer stem cells

Notch signaling is frequently activated in ovarian cancer (OC) and contributes to the proliferation and survival of cultured OC cells as well as to tumor formation and angiogenesis in xenograft models. Several studies demonstrate that Notch3 expression renders cancer cells more resistant to carboplatin, contributing to chemoresistance and poor survival of OC-bearing patients. This suggests that Notch3 can represent both a biomarker and a target for therapeutic interventions in OC patients. Although it is still unclear how chemoresistance arises, different lines of evidence support a critical role of cancer stem cells (CSCs), suggesting that CSC targeting by innovative therapeutic approaches might represent a promising tool to efficiently reduce OC recurrence. To date, CSC-directed therapies in OC tumors are mainly targeted to the inhibition of CSC-related signaling pathways, including Notch. As it is increasingly evident the involvement of Notch signaling, and in particular of Notch3, in regulating stem-like cell maintenance and expansion in several tumors, here we provide an overview of the current knowledge of Notch3 role in CSC-mediated OC chemoresistance, finally exploring the potential design of innovative Notch3 inhibition-based therapies for OC treatment, aimed at eradicating tumor through the suppression of CSCs

Improvement of mouth functional disability in systemic sclerosis patients over one year in a trial of fat transplantation versus adipose-derived stromal cells

Background. Systemic sclerosis (SSc) is a multisystem disease characterized by cutaneous and visceral fibrosis. Face and mouth changes include telangiectasia, sicca syndrome, and thinning and reduction of mouth width (microcheilia) and opening (microstomia). We applied autologous fat transplantation compared with autologous adipose-derived stromal cells (ADSCs) injection to evaluate the clinical improvement of mouth opening. Methods. From February to May 2013 ten consecutive SSc patients were enrolled from the outpatient clinic of Plastic Surgery Department of Sapienza University of Rome. Patients were divided into two groups as follows: 5 patients were treated with fat transplantation and 5 patients received infiltration of ADSCs produced by cell factory of our institution. To value mouth opening, we use the Italian version of Mouth Handicap in Systemic Sclerosis Scale (IvMHISS). Mouth opening was assessed in centimetres (Maximal Mouth Opening, MMO). In order to evaluate compliance and physician and patient satisfaction, we employed a Questionnaire of Satisfaction and the Visual Analogic Scale (VAS) performed before starting study and 1 year after the last treatment. Results and Conclusion. We noticed that both procedures obtained significant results but neither one emerged as a first-choice technique. The present clinical experimentation should be regarded as a starting point for further experimental research and clinical trials

Immunomodulatory effect of adipose-derived stem cells: the cutting edge of clinical application

Adipose-derived stem cells (ASCs) represent a promising tool for soft tissue engineering as well as for clinical treatment of inflammatory and autoimmune pathologies. The well-characterized multi-differentiation potential and self-renewal properties of ASCs are coupled with their immunomodulatory ability in providing therapeutic efficacy. Yet, their impact in immune or inflammatory disorders might rely both on cell contact-dependent mechanisms and paracrine effects, resulting in the release of various soluble factors that regulate immune cells functions. Despite the widespread use of ASCs in clinical trials addressing several pathologies, the pathophysiological mechanisms at the basis of their clinical use have been not yet fully investigated. In particular, a thorough analysis of ASC immunomodulatory potential is mandatory. Here we explore such molecular mechanisms involved in ASC immunomodulatory properties, emphasizing the relevance of the milieu composition. We review the potential clinical use of ASC secretome as a mediator for immunomodulation, with a focus on in vitro and in vivo environmental conditions affecting clinical outcome. We describe some potential strategies for optimization of ASCs immunomodulatory capacity in clinical settings, which act either on adult stem cells gene expression and local microenvironment. Finally, we discuss the limitations of both allogeneic and autologous ASC use, highlighting the issues to be fixed in order to significantly improve the efficacy of ASC-based cell therapy

Fibrinolysis protease receptors promote activation of astrocytes to express pro-inflammatory cytokines.

BACKGROUND:Astrocytes contribute to the crosstalk that generates chronic neuro-inflammation in neurological diseases; however, compared with microglia, astrocytes respond to a more limited continuum of innate immune system stimulants. Recent studies suggest that the fibrinolysis system may regulate inflammation. The goal of this study was to test whether fibrinolysis system components activate astrocytes and if so, elucidate the responsible biochemical pathway. METHODS:Primary cultures of astrocytes and microglia were prepared from neonatal mouse brains. The ability of purified fibrinolysis system proteins to elicit a pro-inflammatory response was determined by measuring expression of the mRNAs encoding tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and chemokine (C-C motif) ligand 2 (CCL2). IκBα phosphorylation also was measured. Plasminogen activation in association with cells was detected by chromogenic substrate hydrolysis. The activity of specific receptors was tested using neutralizing antibodies and reagents. RESULTS:Astrocytes expressed pro-inflammatory cytokines when treated with plasminogen but not when treated with agonists for Toll-like Receptor-4 (TLR4), TLR2, or TLR9. Microglia also expressed pro-inflammatory cytokines in response to plasminogen; however, in these cells, the response was observed only when tissue-type plasminogen activator (tPA) was added to activate plasminogen. In astrocytes, endogenously produced urokinase-type plasminogen activator (uPA) converted plasminogen into plasmin in the absence of tPA. Plasminogen activation was dependent on the plasminogen receptor, α-enolase, and the uPA receptor, uPAR. Although uPAR is capable of directly activating cell-signaling, the receptor responsible for cytokine expression and IκBα phosphorylation response to plasmin was Protease-activated Receptor-1 (PAR-1). The pathway, by which plasminogen induced astrocyte activation, was blocked by inhibiting any one of the three receptors implicated in this pathway with reagents such as εACA, α-enolase-specific antibody, uPAR-specific antibody, the uPA amino terminal fragment, or a pharmacologic PAR-1 inhibitor. CONCLUSIONS:Plasminogen may activate astrocytes for pro-inflammatory cytokine expression through the concerted action of at least three distinct fibrinolysis protease receptors. The pathway is dependent on uPA to activate plasminogen, which is expressed endogenously by astrocytes in culture but also may be provided by other cells in the astrocytic cell microenvironment in the CNS

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

Can an Investigation of a Single Gene be Effective in Differentiating Certain Features of the Bipolar Disorder Profile?

Bipolar disorder (BD) is amongst the most common heritable mental disorders, but the clarification of its genetic roots has proven to be very challenging. Many single nucleotide polymorphisms (SNPs) have been identified to be associated with BD. SNPs in the CACNA1C gene have emerged as the most significantly associated with the disease. The aim of the present study is to provide a concise description of SNP 1006737 variants identified by Real Time PCR and confirm sequencing analysis with the Sanger method in order to estimate the association with BD. The molecular method was tested on 47 Sardinian subjects of whom 23 were found to not be mutated, 1 was found to be a carrier of the homozygous A allele and 23 were found to be carriers of the heterozygous G allele. Moreover, the positive results of the preliminary application suggest that the development of the screener could be extended to the other 5 genetic variables identified as associated with BD

HCV derived from sera of HCV-infected patients induces pro-fibrotic effects in human primary fibroblasts by activating GLI2

Hepatitis C virus (HCV) infection is a leading cause of liver fibrosis, especially in developing countries. The process is characterized by the excess accumulation of ECM that may lead, over time, to hepatic cirrhosis, liver failure and also to hepatocarcinoma. The direct role of HCV in promoting fibroblasts trans-differentiation into myofibroblasts, the major fibrogenic cells, has not been fully clarified. In this study, we found that HCV derived from HCV-infected patients infected and directly induced the trans-differentiation of human primary fibroblasts into myofibroblasts, promoting fibrogenesis. This effect correlated with the activation of GLI2, one of the targets of Hedgehog signaling pathway previously reported to be involved in myofibroblast generation. Moreover, GLI2 activation by HCV correlated with a reduction of autophagy in fibroblasts, that may further promoted fibrosis. GLI2 inhibition by Gant 61 counteracted the pro-fibrotic effects and autophagy inhibition mediated by HCV, suggesting that targeting HH/GLI2 pathway might represent a promising strategy to reduce the HCV-induced fibrosis

Neuropilin 1mediates keratinocyte growth factor signaling in adipose-derived stem cells: potential involvement in adipogenesis

Adipogenesis is regulated by a complex network of molecules, including fibroblast growth factors. Keratinocyte growth factor (KGF) has been previously reported to promote proliferation on rat preadipocytes, although the expression of its specific receptor, FGFR2-IIIb/KGFR, is not actually detected in mesenchymal cells. Here, we demonstrate that human adipose-derived stem cells (ASCs) show increased expression of KGF during adipogenic differentiation, especially in the early steps. Moreover, KGF is able to induce transient activation of ERK and p38 MAPK pathways in these cells. Furthermore, KGF promotes ASC differentiation and supports the activation of differentiation pathways, such as those of PI3K/Akt and the retinoblastoma protein (Rb). Notably, we observed only a low amount of FGFR2-IIIb in ASCs, which seems not to be responsible for KGF activity. Here, we demonstrate for the first time that Neuropilin 1 (NRP1), a transmembrane glycoprotein expressed in ASCs acting as a coreceptor for some growth factors, is responsible for KGF-dependent pathway activation in these cells. Our study contributes to clarify the molecular bases of human adipogenesis, demonstrating a role of KGF in the early steps of this process, and points out a role of NRP1 as a previously unknown mediator of KGF action in ASCs

DNMT3B in vitro knocking-down is able to reverse embryonal rhabdomyosarcoma cell phenotype through inhibition of proliferation and induction of myogenic differentiation

Aberrant DNA methylation has been frequently observed in many human cancers, including rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children. To date, the expression and function of the de novo DNA methyltransferase (DNMT) 3B in RMS have not yet been investigated. Our study show for the first time a significant up-regulation of DNMT3B levels in 14 RMS tumour samples and 4 RMS cell lines in comparison to normal skeletal muscle. Transfection of RD and TE671 cells, two in vitro models of embryonal RMS (ERMS), with a synthetic DNMT3B siRNA decreased cell proliferation by arresting cell cycle at G1 phase, as demonstrated by the reduced expression of Cyclin B1, Cyclin D1 and Cyclin E2, and by the concomitant up-regulation of the checkpoint regulators p21 and p27. DNMT3B depletion also impaired RB phosphorylation status and decreased migratory capacity and clonogenic potential. Interestingly, DNMT3B knock-down was able to commit ERMS cells towards myogenic terminal differentiation, as confirmed by the acquisition of a myogenic-like phenotype and by the increased expression of the myogenic markers MYOD1, Myogenin and MyHC. Finally, inhibition of MEK/ERK signalling by U0126 resulted in a reduction of DNMT3B protein, giving evidence that DNMT3B is a down-stream molecule of this oncogenic pathway.Taken together, our data indicate that altered expression of DNMT3B plays a key role in ERMS development since its silencing is able to reverse cell cancer phenotype by rescuing myogenic program. Epigenetic therapy, by targeting the DNA methylation machinery, may represent a novel therapeutic strategy against RMS