LAM/TSC cell migration to uterus in an experimental model of lymphangioleiomyomatosis. Regulation by anti-epidermal growth factor receptor antibody and rapamycin

Lymphangioleiomyomatosis (LAM) is a rare lung disease affecting almost exclusively women, characterized by the invasion and abnormal proliferation of smooth muscle-like cells in pulmonary parenchyma and axial lymphatics. LAM cells bear mutations in Tuberous Sclerosis Complex (TSC) genes. It has been hypothesized that uterus might be the primary site of origin and one of the most frequent metastatic or disseminated site of LAM cells. We developed a mouse model to study the migratory and invasive properties of human LAM/TSC cells to the uterus. We also examined the action of rapamycin and anti-Epidermal Growth Factor Receptor (EGFR) antibody. LAM/TSC cells were endonasally administered to 3 week old immunodeficient female nude mice. 5 months later mice were divided in 4 groups: control, LAM/TSC cell-administered mice, LAM/TSC cell-administered mice treated with rapamycin, and LAM/TSC cell-administered mice treated with anti-EGFR antibody. Drugs were administered for one months. Uteri were analysed for the presence of human LAM/TSC cells by COX IV antibody, lymphangiogenesis by LYVE 1 expression and angiogenesis by counting blood vessels. LAM/TSC cells migrated to the uterus without causing any morphological lesion. Interestingly, LAM/TSC cells increased the number of blood vessels while did not cause any alteration in lymphatics vessels. Anti-EGFR antibody and rapamycin reduced the number of human LAM/TSC and counteracted the proliferation of blood vessels in uteri. Although both drugs did not change the expression of LYVE 1, localization of lymphatics was mainly in the perimetrium after drug treatment. Our data describe the strong invasive capability of human LAM/TSC cells which migrated to the uterus. LAM/ TSC cells presence is accompanied by increased angiogenesis. Anti-EGFR antibody and rapamycin were effective in reducing the LAM/TSC cell number and blood vessel proliferation

Degradation of insulin-like growth factor-I receptor occurs via ubiquitin-proteasome pathway in human lung cancer cells

Insulin-like growth factor-I receptor (IGF-IR) is often overexpressed in malignant tumors, and is involved in the establishment and maintenance of malignant phenotypes. Tyrosine kinase receptor endocytosis is commonly triggered by ligand binding and occurs via clathrin-coatedvescicles that transfer the receptor to the lysosome system for degradation. Our study aims at the evaluation of the mechanisms involved in IGF-IR downregulation in neoplastic (Npl) and non-neoplastic (non-Npl) cells. Exposure to insulin-like growth factor-I (IGF-I) of human lung adenocarcinoma cell lines (A549 and H1299) triggers IGF-IR ubiquitination and internalization processes that require energy and are preceded by the phosphorylation of receptor tyrosines. Differently from other plasma membrane substrates of the ubiquitin system, IGF-IR is degraded mostly by the proteasome in these tumor cell lines. The degradation is inhibited by lactacystin and unaffected by lysosomal inhibitors such as bafilomycin A1 and NH(4)Cl. IGF-IR is processed in a similar manner also in fresh specimens of human lung tumors, while it requires active lysosomal functions in non-Npl human lung tissues. These results suggest that the degradation routes of ubiquitinated IGF-IR diverge in normal and Npl cells, and further support the involvement of IGF-IR signaling in cancer. Such a different route for IGF-IR processing might take place sometime during development, since both proteasome and lysosome pathways are active in fetal lung human fibroblasts, IMR90 cells

Anti-EGFR antibody reduces lung nodules by inhibition of EGFR-pathway in a model of lymphangioleiomyomatosis

EGFR belongs to the HER/Erb family of tyrosine kinase receptors and its activation in cancer cells has been linked with increased proliferation, angiogenesis, and metastasis. Lymphangioleiomyomatosis (LAM) is a rare, low-grade neoplasm that occurs sporadically or in association with tuberous sclerosis complex (TSC), a genetic, multisystem disorder characterized by hamartomas in several organs. From chylous of a LAM/TSC patient, we previously isolated smooth muscle-like LAM/TSC cells which proliferation depends on EGF and monoclonal antibodies anti-EGFR reduced proliferation and caused cell death. We demonstrated that the dependence from EGF was caused by the absence of tuberin. To study the role of EGFR pathway in vivo, we developed a mouse model by administration of LAM/TSC cells to female nude mice. LAM/TSC cells caused pulmonary airspace enlargement and, after 30 weeks, nodule formation which express EGFR. Anti-EGFR antibody decreased the number and dimension of lung nodules likely for the inhibition of Erk and S6 signaling, reversed the pulmonary alterations and reduced lymphatic and blood vessels. Moreover, in pulmonary nodules anti-EGFR antibody reduced the positivity to estrogen and progesterone receptors which enhance survival of LAM cells and Snail expression. These results suggest that the inhibition of EGFR signalling have a potential in treatment of LAM/TSC lung alterations

EPO-releasing neural precursor cells promote axonal regeneration and recovery of function in spinal cord traumatic injury

BACKGROUND: Spinal cord injury (SCI) is a debilitating condition characterized by a complex of neurological dysfunctions ranging from loss of sensation to partial or complete limb paralysis. Recently, we reported that intravenous administration of neural precursors physiologically releasing erythropoietin (namely Er-NPCs) enhances functional recovery in animals following contusive spinal cord injury through the counteraction of secondary degeneration. Er-NPCs reached and accumulated at the lesion edges, where they survived throughout the prolonged period of observation and differentiated mostly into cholinergic neuron-like cells. OBJECTIVE: The aim of this study was to investigate the potential reparative and regenerative properties of Er-NPCs in a mouse experimental model of traumatic spinal cord injury. METHODS AND RESULTS: We report that Er-NPCs favoured the preservation of axonal myelin and strongly promoted the regrowth across the lesion site of monoaminergic and chatecolaminergic fibers that reached the distal portions of the injured cord. The use of an anterograde tracer transported by the regenerating axons allowed us to assess the extent of such a process. We show that axonal fluoro-ruby labelling was practically absent in saline-treated mice, while it resulted very significant in Er-NPCs transplanted animals. CONCLUSION: Our study shows that Er-NPCs promoted recovery of function after spinal cord injury, and that this is accompanied by preservation of myelination and strong re-innervation of the distal cord. Thus, regenerated axons may have contributed to the enhanced recovery of function after SCI

A New Selective PPARγ Modulator Inhibits Triglycerides Accumulation during Murine Adipocytes’ and Human Adipose-Derived Mesenchymal Stem Cells Differentiation

Understanding the molecular basis of adipogenesis is vital to identify new therapeutic targets to improve anti-obesity drugs. The adipogenic process could be a new target in the management of this disease. Our aim was to evaluate the effect of GMG-43AC, a selective peroxisome proliferator-activated receptor \u3b3 (PPAR\u3b3) modulator, during adipose differentiation of murine pre-adipocytes and human Adipose Derived Stem Cells (hADSCs). We differentiated 3T3-L1 cells and primary hADSCs in the presence of various doses of GMG-43AC and evaluated the differentiation efficiency measuring lipid accumulation, the expression of specific differentiation markers and the quantification of accumulated triglycerides. The treatment with GMG-43AC is not toxic as shown by cell viability assessments after the treatments. Our findings demonstrate the inhibition of lipid accumulation and the significant decrease in the expression of adipocyte-specific genes, such as PPAR\u3b3, FABP-4, and leptin. This effect was long lasting, as the removal of GMG-43AC from culture medium did not allow the restoration of adipogenic process. The above actions were confirmed in hADSCs exposed to adipogenic stimuli. Together, these results indicate that GMG-43AC efficiently inhibits adipocytes differentiation in murine and human cells, suggesting its possible function in the reversal of adipogenesis and modulation of lipolysis

Therapeutic effect of neural progenitor cells expanded in the 3D nano-engineered Nichoid substrate in a Parkinson’s disease preclinical model

3D microscaffoldsare becoming more and more relevant in regenerative medicine, as they lead to the creation of a structure similar to a physiologicalniche. An example is the nano-engineered Nichoid, a 3D structure in which the cells are able to proliferate. In this work,we investigated the proliferation and stemness properties of Er-NPCswhen grown inside the Nichoid, and their potential therapeutic application in the treatment of Parkinson\u2019s Disease.3D microscaffolds are becoming more and more relevant in regenerative medicine, as they lead to the creation of a structure similar to a physiological niche. An example is the nano-engineered Nichoid, a 3D structure in which the cells are able to proliferate. In this work, we investigated the proliferation and stemness properties of Er-NPCs when grown inside the Nichoid, and their potential therapeutic application in the treatment of Parkinson\u2019s Disease

Site effects of the Roio basin, L’Aquila

During the microzonation studies of the April 6th, 2009 L’Aquila earthquake, we observed local seismic amplifications in the Roio area—a plane separated from L’Aquila city center by mount Luco. Six portable, digital instruments were deployed across the plain from 15 April to mid-May 2009. This array recorded 152 aftershocks. We analyzed the ground motion from these events to determine relative site amplification within the plain and on surrounding ridges. Horizontal over vertical spectral ratio on noise data (HVSRN),aftershock recordings (HVEQ) and standard spectral ratio (SSR) showed amplifications at 1.3 and 4.0Hz on quaternary deposits. Seismic amplifications in the frequency range of 4 and 6Hz were also observed on a carbonate ridge of Colle di Roio, on the northwestern border of the plateau. A small amplification was noticed near the top of mount Luco, another rocky site. Large discrepancies in the amplification levels between methods have been observed for these sites, but the HVSRN, HVEQ and SSR gave similar results at the stations located in the Roio plain. On the rocky sites, the SSR was more reliable than the HVSRN at estimating the transfer function of the site, even if the resonance frequency seemed to be well detected by the latter method.Published809–8234.1. Metodologie sismologiche per l'ingegneria sismicaJCR Journalreserve

Counteracting neuroinflammation in experimental Parkinson's disease favors recovery of function : effects of Er-NPCs administration

Background Parkinson\u2019s disease (PD) is the second most common neurodegenerative disease, presenting with midbrain dopaminergic neurons degeneration. A number of studies suggest that microglial activation may have a role in PD. It has emerged that inflammation-derived oxidative stress and cytokine-dependent toxicity may contribute to nigrostriatal pathway degeneration and exacerbate the progression of the disease in patients with idiopathic PD. Cell therapies have long been considered a feasible regenerative approach to compensate for the loss of specific cell populations such as the one that occurs in PD. We recently demonstrated that erythropoietin-releasing neural precursors cells (Er-NPCs) administered to MPTP-intoxicated animals survive after transplantation in the recipient\u2019s damaged brain, differentiate, and rescue degenerating striatal dopaminergic neurons. Here, we aimed to investigate the potential anti-inflammatory actions of Er-NPCs infused in an MPTP experimental model of PD. Methods The degeneration of dopaminergic neurons was caused by MPTP administration in C57BL/6 male mice. 2.5\u2009 7\u2009105 GFP-labeled Er-NPCs were administered by stereotaxic injection unilaterally in the left striatum. Functional recovery was assessed by two independent behavioral tests. Neuroinflammation was investigated measuring the mRNAs levels of pro-inflammatory and anti-inflammatory cytokines, and immunohistochemistry studies were performed to evaluate markers of inflammation and the potential rescue of tyrosine hydroxylase (TH) projections in the striatum of recipient mice. Results Er-NPC administration promoted a rapid anti-inflammatory effect that was already evident 24\u2009h after transplant with a decrease of pro-inflammatory and increase of anti-inflammatory cytokines mRNA expression levels. This effect was maintained until the end of the observational period, 2\u2009weeks post-transplant. Here, we show that Er-NPCs transplant reduces macrophage infiltration, directly counteracting the M1-like pro-inflammatory response of murine-activated microglia, which corresponds to the decrease of CD68 and CD86 markers, and induces M2-like pro-regeneration traits, as indicated by the increase of CD206 and IL-10 expression. Moreover, we also show that this activity is mediated by Er-NPCs-derived erythropoietin (EPO) since the co-injection of cells with anti-EPO antibodies neutralizes the anti-inflammatory effect of the Er-NPCs treatment. Conclusion This study shows the anti-inflammatory actions exerted by Er-NPCs, and we suggest that these cells may represent good candidates for cellular therapy to counteract neuroinflammation in neurodegenerative disorders

Erythropoietin as a Neuroprotective Molecule: An Overview of Its Therapeutic Potential in Neurodegenerative Diseases

Erythropoietin (EPO) is a cytokine mainly induced in hypoxia conditions. Its major production site is the kidney. EPO primarily acts on the erythroid progenitor cells in the bone marrow. More and more studies are highlighting its secondary functions, with a crucial focus on its role in the central nervous system. Here, EPO may interact with up to four distinct isoforms of its receptor (erythropoietin receptor [EPOR]), activating different signaling cascades with roles in neuroprotection and neurogenesis. Indeed, the EPO/EPOR axis has been widely studied in the neurodegenerative diseases field. Its potential therapeutic effects have been evaluated in multiple disorders, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, spinal cord injury, as well as brain ischemia, hypoxia, and hyperoxia. EPO is showing great promise by counteracting secondary neuroinflammatory processes, reactive oxygen species imbalance, and cell death in these diseases. Multiple studies have been performed both in vitro and in vivo, characterizing the mechanisms through which EPO exerts its neurotrophic action. In some cases, clinical trials involving EPO have been performed, highlighting its therapeutic potential. Together, all these works indicate the potential beneficial effects of EPO

Methylprednisolone neutralizes the beneficial effects of erythropoietin in experimental spinal cord injury

Inflammation plays a major pathological role in spinal cord injury (SCI). Although antiinflammatory treatment using the glucocorticoid methyprednisolone sodium succinate (MPSS) improved outcomes in several multicenter clinical trials, additional clinical experience suggests that MPSS is only modestly beneficial in SCI and poses a risk for serious complications. Recent work has shown that erythropoietin (EPO) moderates CNS tissue injury, in part by reducing inflammation, limiting neuronal apoptosis, and restoring vascular autoregulation. We determined whether EPO and MPSS act synergistically in SCI. Using a rat model of contusive SCI, we compared the effects of EPO [500-5,000 units/kg of body weight (kg-bw)] with MPSS (30 mg/kg-bw) for proinflammatory cytokine production, histological damage, and motor function at 1 month after a compression injury. Although high-dose EPO and MPSS suppressed proinflarnmatory cytokines within the injured spinal cord, only EPO was associated with reduced microglial infiltration, attenuated scar formation, and sustained neurological improvement. Unexpectedly, coadministration of MPSS antagonized the protective effects of EPO, even though the EPO receptor was up-regulated normally after injury. These data illustrate that the suppression of proinflammatory cytokines alone does not necessarily prevent secondary injury and suggest that glucocorticoids should not be coadministered in clinical trials evaluating the use of EPO for treatment of SCI