Search

Cell migration/adhesion assays on HuR KD cells and quality controls on the their xenotransplants.

Epithelial-to-mesenchymal transition (EMT) is a core process underlying cell movement during embryonic development and morphogenesis. Cancer cells hijack this developmental program to execute a multi-step cascade, leading to tumorigenesis and metastasis. CD133 (PROM1), a marker of cancer stem cells, has been shown to facilitate EMT in various cancers, but the regulatory networks controlling CD133 gene expression and function in cancer remain incompletely delineated. In this study, we show that a ribonucleoprotein complex including the long noncoding RNA MALAT1 and the RNA-binding protein HuR (ELAVL1) binds the CD133 promoter region to regulate its expression. In luminal nonmetastatic MCF-7 breast cancer cells, HuR silencing was sufficient to upregulate N-cadherin (CDH2) and CD133 along with a migratory and mesenchymal-like phenotype. Furthermore, we found that in the basal-like metastatic cell line MDA-MB-231 and primary triple-negative breast cancer tumor cells, the repressor complex was absent from the CD133-regulatory region, but was present in the MCF-7 and primary ER+ tumor cells. The absence of the complex from basal-like cells was attributed to diminished expression of MALAT1, which, when overexpressed, dampened CD133 levels. In conclusion, our findings suggest that the failure of a repressive complex to form or stabilize in breast cancer promotes CD133 upregulation and an EMT-like program, providing new mechanistic insights underlying the control of prometastatic processes. Cancer Res; 76(9); 2626–36. ©2016 AACR.

Rescue of phenotype by CD133 silencing in HuR KD cells and assessment of HuR binding to CD133 mRNA.

Quality controls on HuR and MALAT1 silencing

Evaluation of CHART protein fraction.

HuR expression ablation does not induce anoikis.

Clinical characteristics of the tumor specimens form which primary cancer stem cells were isolated.

Primers used in PCR reactions. Gene silencing and over-expression tools.

legends for supplemental figures

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.

Epithelial-to-mesenchymal transition (EMT) is a core process underlying cell movement during embryonic development and morphogenesis. Cancer cells hijack this developmental program to execute a multi-step cascade, leading to tumorigenesis and metastasis. CD133 (PROM1), a marker of cancer stem cells, has been shown to facilitate EMT in various cancers, but the regulatory networks controlling CD133 gene expression and function in cancer remain incompletely delineated. In this study, we show that a ribonucleoprotein complex including the long noncoding RNA MALAT1 and the RNA-binding protein HuR (ELAVL1) binds the CD133 promoter region to regulate its expression. In luminal nonmetastatic MCF-7 breast cancer cells, HuR silencing was sufficient to upregulate N-cadherin (CDH2) and CD133 along with a migratory and mesenchymal-like phenotype. Furthermore, we found that in the basal-like metastatic cell line MDA-MB-231 and primary triple-negative breast cancer tumor cells, the repressor complex was absent from the CD133-regulatory region, but was present in the MCF-7 and primary ER+ tumor cells. The absence of the complex from basal-like cells was attributed to diminished expression of MALAT1, which, when overexpressed, dampened CD133 levels. In conclusion, our findings suggest that the failure of a repressive complex to form or stabilize in breast cancer promotes CD133 upregulation and an EMT-like program, providing new mechanistic insights underlying the control of prometastatic processes. Cancer Res; 76(9); 2626–36. ©2016 AACR.

The adipose tissue is a source of inflammatory proteins, such as TNF, IL-6, and CXCL8. Most of their production occurs in macrophages that act as scavengers of dying adipocytes. The application of an orbital mechanical force for 6–10 min at 97 g to the adipose tissue, lipoaspirated and treated according to Coleman procedures, abolishes the expression of TNF-α and stimulates the expression of the anti-inflammatory protein TNF-stimulated gene-6 (TSG-6). This protein had protective and anti-inflammatory effects when applied to animal models of rheumatic diseases. We examined biopsy, lipoaspirate, and mechanically activated fat and observed that in addition to the increased TSG-6, Sox2, Nanog, and Oct4 were also strongly augmented by mechanical activation, suggesting an effect on stromal cell stemness. Human adipose tissue-derived mesenchymal stem cells (hADSCs), produced from activated fat, grow and differentiate normally with proper cell surface markers and chromosomal integrity, but their anti-inflammatory action is far superior compared to those mesenchymal stem cells (MSCs) obtained from lipoaspirate. The expression and release of inflammatory cytokines from THP-1 cells was totally abolished in mechanically activated adipose tissue-derived hADSCs. In conclusion, we report that the orbital shaking of adipose tissue enhances its anti-inflammatory properties, and derived MSCs maintain such enhanced activity.

The subcutaneous adipose tissue provides a clear advantage over other mesenchymal stem cell sources due to the ease with which it can be accessed, as well as the ease of isolating the residing stem cells. Human adipose-derived stem cells (hADSCs), localized in the stromal–vascular portion, can be isolated ex vivo using a combination of washing steps and enzymatic digestion. In this study, we report that microfragmented human lipoaspirated adipose tissue is a better stem cell source compared to normal lipoaspirated tissue. The structural composition of microfragments is comparable to the original tissue. Differently, however, this procedure activates the expression of antigens, such as β-tubulin III. The hADSCs derived from microfragmented lipoaspirate tissue were systematically characterized for growth features, phenotype, and multipotent differentiation potential. They fulfill the definition of mesenchymal stem cells, although with a higher neural phenotype profile. These cells also express genes that constitute the core circuitry of self-renewal such as OCT4, SOX2, and NANOG, and neurogenic lineage genes such as NEUROD1, PAX6, and SOX3. Such findings suggest further studies by evaluating Microfrag-AT hADSC action in animal models of neurodegenerative conditions.

Tuberous Sclerosis Complex (TSC) is characterized by the presence of benign tumors in the brain, kidneys, heart, eyes, lungs, and skin. The typical brain lesions are cortical tubers, subependimal nodules and subependymal giant-cell astrocytomas. The occurrence of malignant astrocytomas such as glioblastoma is rare. We report on a child with a clinical diagnosis of TSC and a rapidly evolving glioblastoma multiforme. Genetic analysis identified a de novo mutation in TSC2. Molecular characterization of the tumor was performed and discussed, as well as a review of the literature where cases of TSC and glioblastoma multiforme are described. Although the co-occurrence of TSC and glioblastoma multiforme seems to be rare, this possible association should be kept in mind, and proper clinical and radiological follow up should be recommended in these patients.

The only available treatment of traumatic spinal cord injury (TSCI) is high-dose methylprednisolone (MP) administered acutely after injury. However, as the efficacy of MP is controversial, we assessed the superiority of erythropoietin (EPO) versus MP in improving clinical outcome of acute TSCI. Patients aged 18 to 65 years after C5-T12 injury, and grade A or B of the ASIA Impairment Scale (AIS), admitted within 8 h, hemodynamically stable, were randomized to MP according to the NASCIS III protocol or EPO iv (500 UI/kg, repeated at 24 and 48 h). Patients were assessed by an investigator blind to treatment assignment at baseline and at day 3, 7, 14, 30, 60 and 90. Primary end point: number of responders (reduction of at least one AIS grade). Secondary end points: treatment safety and the effects of drugs on a number of disability measures. Frequentistic and post hoc Bayesian analyses were performed. Eight patients were randomized to MP and 11 to EPO. Three patients (27.3 %) on EPO and no patients on MP reached the primary end point (p = 0.17). No significant differences were found for the other disability measures. No adverse events or serious adverse events were reported in both groups. The Bayesian analysis detected a 91.8 % chance of achieving higher success rates on the primary end point with EPO in the intention-to-treat population with a 95 % chance the difference between EPO and MP falling in the range (-0.10, 0.51) and a median value of 0.2. The results of Bayesian analysis favored the experimental treatment.

The complex architecture of adult brain derives from tightly regulated migration and differentiation of precursor cells generated during embryonic neurogenesis. Changes at transcriptional level of genes that regulate migration and differentiation may lead to neurodevelopmental disorders. Androgen receptor (AR) is a transcription factor that is already expressed during early embryonic days. However, AR role in the regulation of gene expression at early embryonic stage is yet to be determinate. Long non-coding RNA (lncRNA) Sox2 overlapping transcript (Sox2OT) plays a crucial role in gene expression control during development but its transcriptional regulation is still to be clearly defined. Here, using Bicalutamide in order to pharmacologically inactivated AR, we investigated whether AR participates in the regulation of the transcription of the lncRNASox2OTat early embryonic stage. We identified a new DNA binding region upstream of Sox2 locus containing three androgen response elements (ARE), and found that AR binds such a sequence in embryonic neural stem cells and in mouse embryonic brain. Our data suggest that through this binding, AR can promote the RNA polymerase II dependent transcription of Sox2OT. Our findings also suggest that AR participates in embryonic neurogenesis through transcriptional control of the long non-coding RNA Sox2OT.

Spinal cord injury presents a significant therapeutic challenge since the treatments available are mostly vain. The use of stem cells to treat this condition represents a promising new therapeutic strategy; therefore, a variety of stem cell treatments have been recently examined in animal models of CNS trauma. In this work, we analyzed the effects of third trimester amniotic fluid cells in a mouse model of spinal cord injury. Among the different cultures used for transplantation, some were able to induce a significant improvement in motor recovery (cultures #3.5, #3.6 and #7.30), evaluated by means of open field free locomotion. All effective cell cultures expressed the surface marker nerve/glial antigen 2, ortholog of the human chondroitin sulfate proteoglycan 4, which is present on several types of immature progenitor cells. The improved motor functional recovery was correlated with higher myelin preservation in the ventral horn white matter and an increased vascularization in the peri-lesion area. Real-Time PCR analysis showed higher expression levels of vascular endothelial growth factor and hypoxia-inducible factor-1α mRNA two days after cells transplantation compared to PBS-treated animals, indicating that an angiogenic pathway might have been activated by these cells, possibly through the production of hepatocyte growth factor. This cytokine appears to be produced mostly in filtering organs, such as the lung, of the transplanted animals and is likely released in the blood suggesting an endocrine role of hepatocyte growth factor in targeting the injury site.

Tuberous sclerosis complex (TSC) is a multi-systemic syndrome caused by mutations in TSC1 or TSC2 gene. In TSC2-null cells, Rheb, a member of the Ras family of GTPases, is constitutively activated. Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase and block the synthesis of isoprenoid lipids with inhibition of Rheb farnesylation and RhoA geranylgeranylation. The effects of rosuvastatin on the function of human TSC2 −/− and TSC2 -/meth α -actin smooth muscle (ASM) cells have been investigated. The TSC2 −/− and TSC2 -/meth ASM cells, previously isolated in our laboratory from the renal angiomyolipoma of two TSC patients, do not express tuberin and bear loss of heterozigosity caused by a double hit on TSC2 and methylation of TSC2 promoter, respectively. Exposure to rosuvastatin affected TSC2 -/meth ASM cell growth and promoted tuberin expression by acting as a demethylating agent. This occurred without changes in interleukin release. Rosuvastatin also reduced RhoA activation in TSC2 -/meth ASM cells, and it required coadministration with the specific mTOR (mammalian target of rapamycin) inhibitor rapamycin to be effective in TSC2 −/− ASM cells. Rapamycin enhanced rosuvastatin effect in inhibiting cell proliferation in TSC2 −/− and TSC2 -/meth ASM cells. Rosuvastatin alone did not alter phosphorylation of S6 and extracellular signal-regulated kinase (ERK), and at the higher concentration, rosuvastatin and rapamycin slightly decreased ERK phosphorylation. These results suggest that rosuvastatin may potentially represent a treatment adjunct to the therapy with mTOR inhibitors now in clinical development for TSC. In particular, rosuvastatin appears useful when the disease is originated by epigenetic defects.

The advent of therapies for lymphangioleiomyiomatosis (LAM) has made early diagnosis important in women with tuberous sclerosis complex (TSC), although the lifelong cumulative radiation exposure caused by CT imaging screening should not be underestimated. In 200 TSC outpatients of San Paolo Hospital (Milan, Italy) we retrospectively investigated 1) the role of pulmonary function tests (PFTs) for screening purpose, 2) the association between LAM and other manifestations of TSC (e.g. demography, extrapulmonary manifestations, genetic mutations, etc.), and 3) the features of patients with multifocal micronodular pneumocyte hyperplasia (MMPH). Eighty-six adult women underwent computed tomography for LAM diagnosis; pulmonary involvement was found in 66 patients (77%, 49% LAM with or without MMPH, and 28% MMPH alone). LAM patients were older, with a higher rate of pneumothorax, presented more frequently renal and hepatic angiomyolipomas, and tended to have a TSC2 mutation profile. PFTs, assessed in 64% of women unaffected by cognitive impairments, revealed a lower lung diffusion capacity in LAM patients. In multivariate analysis age, but not PFTs, resulted independently associated with LAM diagnosis. Patients with MMPH alone did not show specific clinical, functional of genetic features. A mild respiratory impairment was most common in LAM-TSC patients; PFTs, even if indicated to assess pulmonary function impairment, are feasible in a limited number of patients, and are not significantly useful as a screening tool in women with TSC.

Since 2005, sex determining region y-box 2 (SOX2) has drawn the attention of the scientific community for being one of the key transcription factors responsible for pluripotency induction in somatic stem cells. Our research investigated the turnover regulation of SOX2 mRNA in human adipose-derived stem cells, considered one of the most valuable sources of somatic stem cells in regenerative medicine. Mitoxantrone is a drug that acts on nucleic acids primarily used to treat certain types of cancer and was recently shown to ameliorate the outcome of autoimmune diseases such as multiple sclerosis. In addition, mitoxantrone has been shown to inhibit the binding of human antigen R (HuR) RNA-binding protein to tumor necrosis factor-α mRNA. Our results show that HuR binds to the 3'-untranslated region of SOX2 mRNA together with the RNA-induced silencing complex miR145. The HuR binding works by stabilizing the interaction between the 3'-untranslated region and the RNA-induced silencing complex. Cell exposure to mitoxantrone leads to HuR detachment and the subsequent prolongation of the SOX2 mRNA half-life. The prolonged SOX2 half-life allows improvement of the spheroid-forming capability of the adipose-derived stem cells. The silencing of HuR confirmed the above observations and illustrates how the RNA-binding protein HuR may be a required molecule for regulation of SOX2 mRNA decay.

Pulmonary lymphangioleiomyomatosis (LAM) is a rare lung disease caused by mutations in the tumor suppressor genes encoding Tuberous Sclerosis Complex (TSC) 1 and TSC2. The protein product of the TSC2 gene is a well-known suppressor of the mTOR pathway. Emerging evidence suggests that the pituitary hormone prolactin (Prl) has both endocrine and paracrine modes of action. Here, we have investigated components of the Prl system in models for LAM. In a TSC2 (+/-) mouse sarcoma cell line, down-regulation of TSC2 using siRNA resulted in increased levels of the Prl receptor. In human LAM cells, the Prl receptor is detectable by immunohistochemistry, and the expression of Prl in these cells stimulates STAT3 and Erk phosphorylation, as well as proliferation. A high affinity Prl receptor antagonist consisting of Prl with four amino acid substitutions reduced phosphorylation of STAT3 and Erk. Antagonist treatment further reduced the proliferative and invasive properties of LAM cells. In histological sections from LAM patients, Prl receptor immuno reactivity was observed. We conclude that the Prl receptor is expressed in LAM, and that loss of TSC2 increases Prl receptor levels. It is proposed that Prl exerts growth-stimulatory effects on LAM cells, and that antagonizing the Prl receptor can block such effects.

Lymphangioleiomyomatosis (LAM) is an interstitial lung disease characterized by invasion and proliferation of abnormal smooth muscle (ASM) cells in lung parenchyma and axial lymphatics. LAM cells bear mutations in tuberous sclerosis ( TSC ) genes. TSC2 −/− ASM cells, derived from a human renal angiomyolipoma, require epidermal growth factor (EGF) for proliferation. Blockade of EGF receptors (EGFR) causes cell death. TSC2 −/− ASM cells, previously labeled with PKH26-GL dye, were endonasally administered to 5-week-old immunodeficient female nude mice, and 4 or 26 weeks later anti-EGFR antibody or rapamycin was administered twice a week for 4 consecutive weeks. TSC2 −/− ASM cells infiltrated lymph nodes and alveolar lung walls, causing progressive destruction of parenchyma. Parenchymal destruction was efficiently reversed by anti-EGFR treatment and partially by rapamycin treatment. Following TSC2 −/− ASM cell administration, lymphangiogenesis increased in lungs as indicated by more diffuse LYVE1 expression and high murine VEGF levels. Anti-EGFR antibody and rapamycin blocked the increase in lymphatic vessels. This study shows that TSC2 −/− ASM cells can migrate and invade lungs and lymph nodes, and anti-EGFR antibody is more effective than rapamycin in promoting lung repair and reducing lymphangiogenesis. The development of a model to study metastasis by TSC cells will also help to explain how they invade different tissues and metastasize to the lung.

This study was aimed at the isolation of neural precursor cells (NPCs) capable of resisting to a prolonged ischemic insult as this may occur at the site of traumatic and ischemic CNS injuries. Adult mice were anesthetized and then killed by cervical dislocation. The cadavers were maintained at room temperature or at 4 °C for different time periods. Post mortem neural precursors (PM-NPCs) were isolated, grown in vitro and their differentiation capability was investigated by evaluating the expression of different neuronal markers. PM-NPCs differentiate mostly in neurons, show activation of hypoxia-inducible factor-1 and MAPK, and express both erythropoietin (EPO) and its receptor (EPO-R). The exposure of PM-NPCs to neutralizing antibodies to EPO or EPO-R dramatically reduced the extent of neuronal differentiation to about 11% of total PM-NPCs. The functionality of mTOR and MAPK is also required for the expression of the neuronal phenotype by PM-NPCs. These results suggest that PM-NPCs can be isolated from animal cadaver even several hours after death and their self-renewable capability is comparable to normal neural precursors. Differently, their ability to achieve a neural phenotype is superior to that of NPCs, and this is mediated by the activation of hypoxia-induced factor 1 and EPO signaling. PM-NPCs may represent good candidates for transplantation studies in animal models of neurodegenerative diseases.

Erythropoietin (EPO), originally identified for its critical function in regulating production and survival of erythrocytes, is a member of the type 1 cytokine superfamily. Recent studies have shown that EPO has cytoprotective effects in a wide variety of cells and tissues. Here is presented the analysis of EPO effects on spinal cord injury (SCI), considering both animal experiments concerning to mechanisms of neurodegeneration in SCI and EPO as a neuroprotective agent, and some evidences coming from ongoing clinical trials. The evidences underling that EPO could be a promising therapeutic agent in a variety of neurological insults, including trauma, are mounting. In particular, it is highlighted that administration of EPO or other recently generated EPO analogues such as asialo-EPO and carbamylated-EPO demonstrate interesting preclinical and clinical characteristics, rendering the evaluation of these tissue-protective agents imperative in human clinical trials. Moreover the demonstration of rhEPO and its analogues’ broad neuroprotective effects in animal models of cord lesion and in human trial like stroke, should encourage scientists and clinicians to design clinical trials assessing the efficacy of these pharmacological compounds on SCI.

Spinal cord injury (SCI) triggers a complex ischemic and inflammatory reaction, involving activation of neurotransmitter systems, in particular glutamate, culminating in cell death. We hypothesized that SCI might lead to alteration in the RNA editing of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors that govern critical determinants of neuronal survival. To this end, we examined the molecular changes set in motion by SCI that affect the channel properties of AMPA receptors. SCI strongly reduced the level of AMPA receptor R/G editing, involving not only the site of the lesion but also adjacent areas of the cord spared by the lesion. The effects, with changes for some subunits and loci, were observed as long as 30 days after lesioning and may correlate with a partial decrease in enzymatic activity of adenosine deaminase acting on RNA 2 (ADAR2), as deduced from the analysis of ADAR2 self-editing. The reduced editing at the R/G site of glutamate receptor subunits (GluRs) is likely to reduce post-synaptic excitatory responses to glutamate, thus limiting the progression of cell death; however, prolonged suppression of GluR function in later stages may hinder synaptic plasticity. These observations provide the first direct evidence of the potential contribution of RNA editing to excitatory neural injury and recovery after SCI.

The cells comprising pulmonary lymphangioleiomyomatosis (LAM) and renal angiomyolipomas (AMLs) are heterogeneous, with variable mixtures of cells exhibiting differentiation towards smooth muscle, fat, and vessels. Cells grown from LAM and AMLs have likewise tended to be heterogeneous. The discovery that LAM and AMLs contain cells with mutations in the TSC1 or TSC2 genes is allowing investigators to discriminate between “two-hit” cells and neighboring cells, providing insights into disease pathogenesis. In rare cases, it has been possible to derive cells from human tumors, including AMLs and TSC skin tumors that are highly enriched for TSC2-/- cells. Cells derived from an Eker rat uterine leiomyoma (ELT3 cells) are Tsc2-null and these have been used in a rodent cell models for LAM. Further improvements in the ability to reliably grow well-characterized TSC2-/- cells from human tumors are critical to developing in vitro and in vivo model systems for studies of LAM pathogenesis and treatment.

Tuberous sclerosis complex (TSC) is an autosomal-dominant disease that is caused by mutations in either the TSC1 or TSC2 gene. Smooth muscle-like cells (ASMs) were isolated from an angiomyolipoma of a patient with TSC. These cells lacked tuberin, were labeled by both HMB45 and CD44v6 antibodies, and had constitutive S6 phosphorylation. The cells bear a germline TSC2 intron 8-exon 9 junction mutation, but DNA analysis and polymerase chain reaction amplification failed to demonstrate loss of heterozygosity. Testing for an epigenetic alteration, we detected methylation of the TSC2 promoter. Its biological relevance was confirmed by tuberin expression and a reduction in HMB45 labeling and S6 constitutive phosphorylation after exposure to the chromatin-remodeling agents, trichostatin A and 5-azacytidine. These cells were named TSC2(-/meth) ASMs. Their proliferation required epidermal growth factor in the medium as previously described for TSC2(-/-) ASMs. Blockade of epidermal growth factor with monoclonal antibodies caused the death of TSC2(-/meth) ASMs. In addition, rapamycin effectively blocked the proliferation of these cells. Our data show for the first time that methylation of the TSC2 promoter might cause a complete loss of tuberin in TSC2 cells, and that the pathogenesis of angiomyolipomas might also originate from epigenetic defects in smooth muscle cells. Additionally, the effect of chromatin-remodeling agents in these cells suggests a further avenue for the treatment of TSC as well as lymphangioleiomyomatosis.

Spinal cord injury (SCI) is a devastating event which causes dramatic changes in the everyday life of the patient. We have found that acute SCI reduced BDNF expression selectively in the hippocampus of lesioned rats, a decrease which persists at least 1 week, thus identifying the modulation of the neurotrophin biosynthesis as an important mechanism underlying brain vulnerability to SCI. These data are the first to show that SCI alters hippocampal BDNF expression and identify the neurotrophin as a potential target through which SCI changes brain functions, a notion that might prove useful in understanding the mechanisms underlying brain vulnerability to SCI.

Using a standardized rat model of contusive spinal cord injury (SCI; [Gorio A, Gokmen N, Erbayraktar S, Yilmaz O, Madaschi L, Cichetti C, Di Giulio AM, Vardar E, Cerami A, Brines M (2002) Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma. Proc Natl Acad Sci U S A 99:9450-9455]), we previously showed that the administration of recombinant human erythropoietin (rhEPO) improves both tissue sparing and locomotory outcome. In the present study, to better understand rhEPO-mediated effects on chronic astrocyte response to SCI in rat, we have used immunocytochemical methods combined with confocal and electron microscopy to investigate, 1 month after injury, the effects of a single rhEPO administration on the expression of a) aquaporin 4 (AQP4), the main astrocytic water channel implicated in edema development and resolution, and two molecules (dystrophin and syntrophin) involved in its membrane anchoring; b) glial fibrillary acidic protein (GFAP) and vimentin as markers of astrogliosis; c) chondroitin sulfate proteoglycans of the extracellular matrix which are upregulated after SCI and can inhibit axonal regeneration and influence neuronal and glial properties. Our results show that rhEPO administration after SCI modifies astrocytic response to injury by increasing AQP4 immunoreactivity in the spinal cord, but not in the brain, without apparent modifications of dystrophin and syntrophin distribution. Attenuation of astrogliosis, demonstrated by the semiquantitative analysis of GFAP labeling, was associated with a reduction of phosphacan/RPTP zeta/beta, whereas the levels of lecticans remained unchanged. Finally, the relative volume of a microvessel fraction was significantly increased, indicating a pro-angiogenetic or a vasodilatory effect of rhEPO. These changes were consistently associated with remarkable reduction of lesion size and with improvement in tissue preservation and locomotor recovery, confirming previous observations and underscoring the potentiality of rhEPO for the therapeutic management of SCI.

Although effective and safe, carotid endarterectomy (CEA) implies a reduced blood flow to the brain and likely an ischemia/reperfusion event. The high rate of uneventful outcomes associated with CEA suggests the activation of brain endogenous protection mechanisms aimed at limiting the possible ischemia/reperfusion damage. This study aims at assessing whether CEA triggers protective mechanisms such as brain release of erythropoietin and nitric oxide. CEA was performed in 12 patients; blood samples were withdrawn simultaneously from the surgically exposed ipsilateral jugular and leg veins before, during (2 and 40 min) and after clamp removal (2 min). Plasma antioxidant capacity, carbonylated proteins, erythropoietin, nitrates and nitrites (NOx) were determined. No changes in intraoperative EEG, peripheral and transcranial blood oxygen saturation were detectable, and no patients showed any neurologic sign after the intervention. Antioxidant capacity and protein carbonylation in plasma were unaffected. Differently, erythropoietin, VEGF, TNF-α and NOx increased during clamping in the jugular blood (2 and 40 min), while no changes were observed in the peripheral circulation. These results show that blood erythropoietin, VEGF, TNF-α, and NOx increased in the brain during uncomplicated CEA. This may represent an endogenous self-activated neuroprotective mechanism aimed at the prevention of ischemia/reperfusion damage.

Spinal cord injury (SCI) is a debilitating clinical condition, characterized by a complex of neurological dysfunctions. Neural stem cells from the subventricular zone of the forebrain have been considered a potential tool for cell replacement therapies. We recently isolated a subclass of neural progenitors from the cadaver of mouse donors. These cells, named postmortem neural precursor cells (PM-NPCs), express both erythropoietin (EPO) and its receptor. Their EPO-dependent differentiation abilities produce a significantly higher percentage of neurons than regular NSCs. The cholinergic yield is also higher. The aim of the present study was to evaluate the potential repair properties of PM-NPCs in a mouse model of traumatic SCI. Labeled PM-NPCs were administered intravenously; then the functional recovery and the fate of transplanted cells were studied. Animals transplanted with PM-NPCs showed a remarkable improved recovery of hindlimb function that was evaluated up to 90 days after lesion. This was accompanied by reduced myelin loss, counteraction of the invasion of the lesion site by the inflammatory cells, and an attenuation of secondary degeneration. PM-NPCs migrate mostly at the injury site, where they survive at a significantly higher extent than classical NSCs. These cells accumulate at the edges of the lesion, where a reach neuropile is formed by MAP2- and β-tubulin III-positive transplanted cells that are also mostly labeled by anti-ChAT antibodies.

Tuberous Sclerosis Complex (TSC) is characterized by the presence of benign tumors in the brain, kidneys, heart, eyes, lungs, and skin. The typical brain lesions are cortical tubers, subependimal nodules and subependymal giant-cell astrocytomas. The occurrence of malignant astrocytomas such as glioblastoma is rare. We report on a child with a clinical diagnosis of TSC and a rapidly evolving glioblastoma multiforme. Genetic analysis identified a de novo mutation in TSC2. Molecular characterization of the tumor was performed and discussed, as well as a review of the literature where cases of TSC and glioblastoma multiforme are described. Although the co-occurrence of TSC and glioblastoma multiforme seems to be rare, this possible association should be kept in mind, and proper clinical and radiological follow up should be recommended in these patients.

EGFR belongs to the HER/ErbB 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 whose proliferation depends on EGF and monoclonal anti-EGFR antibodies reduced proliferation and caused cell death. We demonstrated that the dependency from EGF was caused by the absence of tuberin. To study the role of EGFR pathwayin 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 has a potential in treatment of LAM/TSC lung alterations.

Primary biliary cirrhosis (PBC) is a chronic liver disease characterized by severe intraepatic cholestasis. Pruritus often occurs during the course of the illness. We designed a study aimed at assessing whether pruritus is associated with dysfunction of signal transduction. Seventeen female patients affected by PBC were enrolled into the study and divided in two groups according to severity of liver disease. Leukocytes were isolated from peripheral blood and Gi, Go and Gs protein expressions were evaluated by western blotting, while G protein function was assessed by measuring cyclic adenosine phosphate formation. The expression of all types of G proteins was increased in leukocytes of PBC patients. The basal adenylate activity was significally higher than control in patients with less severe liver disease, while it was lower than normal in those with severe liver disease. Incubation of patient leukocytes with guanosine triphosphate-gamma-S and Gs protein activators failed to enhance cAMP production, while N-formyl-met-leu-phe was more effective in reducing cAMP production. The expression of all G proteins was non-selectively increased in PBC leukocytes, while adenylate cyclase activity was significantly modified. However, the observed changes in G proteins expression and in adenylate cyclase activity are not related to the presence of pruritus.