Search

Background: Cell-based therapies for multiple sclerosis (MS), including those employing autologous bone marrow-derived mesenchymal stromal cells (MSC) are being examined in clinical trials. However, recent studies have identified abnormalities in the MS bone marrow microenvironment. Objective: We aimed to compare the secretome of MSC isolated from control subjects (C-MSC) and people with MS (MS-MSC) and explore the functional relevance of findings. Methods: We employed high throughput proteomic analysis, enzyme-linked immunosorbent assays and immunoblotting, as well as in vitro assays of enzyme activity and neuroprotection. Results: We demonstrated that, in progressive MS, the MSC secretome has lower levels of mitochondrial fumarate hydratase (mFH). Exogenous mFH restores the in vitro neuroprotective potential of MS-MSC. Furthermore, MS-MSC expresses reduced levels of fumarate hydratase (FH) with downstream reduction in expression of master regulators of oxidative stress. Conclusions: Our findings are further evidence of dysregulation of the bone marrow microenvironment in progressive MS with respect to anti-oxidative capacity and immunoregulatory potential. Given the clinical utility of the fumaric acid ester dimethyl fumarate in relapsing–remitting MS, our findings have potential implication for understanding MS pathophysiology and personalised therapeutic intervention.

The overall survival for patients with primary glioblastoma is very poor. Glioblastoma contains a subpopulation of glioma stem cells (GSC) that are responsible for tumour initiation, treatment resistance and recurrence. PPARα is a transcription factor involved in the control of lipid, carbohydrate and amino acid metabolism. We have recently shown that PPARα gene and protein expression is increased in glioblastoma and has independent clinical prognostic significance in multivariate analyses. In this work, we report that PPARα is overexpressed in GSC compared to foetal neural stem cells. To investigate the role of PPARα in GSC, we knocked down its expression using lentiviral transduction with short hairpin RNA (shRNA). Transduced GSC were tagged with luciferase and stereotactically xenografted into the striatum of NOD‐SCID mice. Bioluminescent and magnetic resonance imaging showed that knockdown (KD) of PPARα reduced the tumourigenicity of GSC in vivo. PPARα‐expressing control GSC xenografts formed invasive histological phenocopies of human glioblastoma, whereas PPARα KD GSC xenografts failed to establish viable intracranial tumours. PPARα KD GSC showed significantly reduced proliferative capacity and clonogenic potential in vitro with an increase in cellular senescence. In addition, PPARα KD resulted in significant downregulation of the stem cell factors c‐Myc, nestin and SOX2. This was accompanied by downregulation of the PPARα‐target genes and key regulators of fatty acid oxygenation ACOX1 and CPT1A, with no compensatory increase in glycolytic flux. These data establish the aberrant overexpression of PPARα in GSC and demonstrate that this expression functions as an important regulator of tumourigenesis, linking self‐renewal and the malignant phenotype in this aggressive cancer stem cell subpopulation. We conclude that targeting GSC PPARα expression may be a therapeutically beneficial strategy with translational potential as an adjuvant treatment. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Objective Friedreich's ataxia is an incurable inherited neurological disease caused by frataxin deficiency. Here, we report the neuroreparative effects of myeloablative allogeneic bone marrow transplantation in a humanized murine model of the disease. Methods Mice received a transplant of fluorescently tagged sex-mismatched bone marrow cells expressing wild-type frataxin and were assessed at monthly intervals using a range of behavioral motor performance tests. At 6 months post-transplant, mice were euthanized for protein and histological analysis. In an attempt to augment numbers of bone marrow-derived cells integrating within the nervous system and improve therapeutic efficacy, a subgroup of transplanted mice also received monthly subcutaneous infusions of the cytokines granulocyte-colony stimulating factor and stem cell factor. Results Transplantation caused improvements in several indicators of motor coordination and locomotor activity. Elevations in frataxin levels and antioxidant defenses were detected. Abrogation of disease pathology throughout the nervous system was apparent, together with extensive integration of bone marrow-derived cells in areas of nervous tissue injury that contributed genetic material to mature neurons, satellite-like cells, and myelinating Schwann cells by processes including cell fusion. Elevations in circulating bone marrow-derived cell numbers were detected after cytokine administration and were associated with increased frequencies of Purkinje cell fusion and bone marrow-derived dorsal root ganglion satellite-like cells. Further improvements in motor coordination and activity were evident. Interpretation Our data provide proof of concept of gene replacement therapy, via allogeneic bone marrow transplantation, that reverses neurological features of Friedreich's ataxia with the potential for rapid clinical translation. Ann Neurol 2018;83:779-793.

Background: Autologous bone-marrow-derived cells are currently employed in clinical studies of cell-based therapy in multiple sclerosis (MS) although the bone marrow microenvironment and marrow-derived cells isolated from patients with MS have not been extensively characterised. Objectives: To examine the bone marrow microenvironment and assess the proliferative potential of multipotent mesenchymal stromal cells (MSCs) in progressive MS. Methods: Comparative phenotypic analysis of bone marrow and marrow-derived MSCs isolated from patients with progressive MS and control subjects was undertaken. Results: In MS marrow, there was an interstitial infiltrate of inflammatory cells with lymphoid (predominantly T-cell) nodules although total cellularity was reduced. Controlling for age, MSCs isolated from patients with MS had reduced in vitro expansion potential as determined by population doubling time, colony-forming unit assay, and expression of β-galactosidase. MS MSCs expressed reduced levels of Stro-1 and displayed accelerated shortening of telomere terminal restriction fragments (TRF) in vitro. Conclusion: Our results are consistent with reduced proliferative capacity and ex vivo premature ageing of bone-marrow-derived cells, particularly MSCs, in MS. They have significant implication for MSC-based therapies for MS and suggest that accelerated cellular ageing and senescence may contribute to the pathophysiology of progressive MS.

© 2016 John Wiley & Sons Ltd Aims: PPARα agonists are in current clinical use as hypolipidaemic agents and show significant antineoplastic effects in human glioblastoma models. To date however, the expression of PPARα in large-scale glioblastoma datasets has not been examined. We aimed to investigate the expression of the transcription factor PPARα in primary glioblastoma, the relationship between PPARα expression and patients’ clinicopathological features and other molecular markers associated with gliomagenesis. Methods and results: With protein immunoblotting techniques and reverse transcription quantitative real-time PCR, PPARα was found to be significantly overexpressed in glioblastoma compared with control brain tissue (P = 0.032 and P = 0.005). PPARA gene expression was found to be enriched in the classical glioblastoma subtype within The Cancer Genome Atlas (TCGA) dataset. Although not associated with overall survival when assessed by immunohistochemistry, cross-validation with the TCGA dataset and multivariate analyses identified PPARA gene expression as an independent prognostic marker for overall survival (P = 0.042). Finally, hierarchical clustering revealed novel, significant associations between high PPARA expression and a putative set of glioblastoma molecular mediators including EMX2, AQP4, and NTRK2. Conclusions: PPARα is overexpressed in primary glioblastoma and high PPARA expression functions as an independent prognostic marker in the glioblastoma TCGA dataset. Further studies are required to explore genetic associations with high PPARA expression and to analyse the predictive role of PPARα expression in glioblastoma models in response to PPARα agonists.

Objectives: Friedreich's ataxia is a devastating neurological disease currently lacking any proven treatment. We studied the neuro-protective effects of the cytokines granulocyte-colony stimulating factor and stem cell factor in a humanised murine model of Friedreich's ataxia. Methods: Mice received monthly subcutaneous infusions of cytokines while also being assessed at monthly time points using an extensive range of behavioural motor performance tests. After 6 months of treatment, neurophysiological evaluation of both sensory and motor nerve conduction was performed. Subsequently, mice were sacrificed for mRNA, protein and histological analysis of the dorsal root ganglion, spinal cord and cerebellum. Results: Cytokine administration resulted in significant reversal of biochemical, neuropathological, neurophysiological and behavioural deficits associated with Friedreich's ataxia. Both granulocyte-colony stimulating factor and stem cell factor had pronounced effects on frataxin levels (the primary molecular defect in the pathogenesis of the disease), and on regulators of frataxin expression. Sustained improvements in motor coordination and locomotor activity were seen, even after onset of neurological symptoms. Treatment also restored the duration of sensory nerve compound potentials. Improvements in peripheral nerve conduction positively correlated with cytokine-induced increases in frataxin expression, providing a link between increases in frataxin and neurophysiological function. Abrogation of disease-related pathology was also evident, with reductions in inflammation/gliosis and increased neural stem cell numbers in areas of tissue injury. Interpretation: These experiments show that cytokines already clinically used in other conditions offer the prospect of a novel, rapidly translatable, disease-modifying and neuroprotective treatment for Friedreich's ataxia. This article is protected by copyright. All rights reserved.

The potential of autologous cell-based therapies including those using multipotent mesenchymal stromal cells (MSCs) is being investigated for multiple sclerosis (MS) and other neurological conditions. However, the phenotype of MSC in neurological diseases has not been fully characterized. We have previously shown that MSC isolated from patients with progressive MS (MS-MSC) have reduced expansion potential, premature senescence, and reduced neuroprotective potential in vitro. In view of the role of antioxidants in ageing and neuroprotection, we examined the antioxidant capacity of MS-MSC demonstrating that MS-MSC secretion of antioxidants superoxide dismutase 1 (SOD1) and glutathione S-transferase P (GSTP) is reduced and correlates negatively with the duration of progressive phase of MS. We confirmed reduced expression of SOD1 and GSTP by MS-MSC along with reduced activity of SOD and GST and, to examine the antioxidant capacity of MS-MSC under conditions of nitrosative stress, we established an in vitro cell survival assay using nitric oxide-induced cell death. MS-MSC displayed differential susceptibility to nitrosative stress with accelerated senescence and greater decline in expression of SOD1 and GSTP in keeping with reduced expression of master regulators of antioxidant responses nuclear factor erythroid 2-related factor 2 and peroxisome proliferator-activated receptor gamma coactivator 1-α. Our results are compatible with dysregulation of antioxidant responses in MS-MSC and have significant implications for development of autologous MSC-based therapies for MS, optimization of which may require that these functional deficits are reversed. Furthermore, improved understanding of the underlying mechanisms may yield novel insights into MS pathophysiology and biomarker identification.

Background The preschool years offer a unique window of opportunity to instill healthy life-style behaviors and promote cardiovascular health. Objectives This study sought to evaluate the effect of a 3-year multidimensional school-based intervention to improve life-style–related behaviors. Methods We performed a cluster-randomized controlled intervention trial involving 24 public schools in Madrid, Spain, that were assigned to either the SI! Program intervention or the usual curriculum and followed for 3 years. The SI! Program aimed to instill and develop healthy behaviors in relation to diet, physical activity, and understanding how the human body and heart work. The primary outcome was change in the overall knowledge, attitudes, and habits (KAH) score (range 0 to 80). The intervention’s effect on adiposity markers was also evaluated. Results A total of 2,062 children from 3 to 5 years of age were randomized. After 3 years of follow-up, the overall KAH score was 4.9% higher in children in the intervention group compared with the control group (21.7 vs. 16.4; p Conclusions The SI! Program is an effective strategy for instilling healthy habits among preschoolers, translating into a beneficial effect on adiposity, with maximal effect when started at the earliest age and maintained over 3 years. Wider adoption may have a meaningful effect on cardiovascular health promotion. (Evaluation of the Program SI! for Preschool Education: A School-Based Randomized Controlled Trial [Preschool_PSI!]; NCT01579708 )

Introduction and objectives Program SI! is a multi-level, school-based intervention for the promotion of cardiovascular health from early childhood. The aim of this paper is to characterize the prevalence of obesity and high blood pressure in the preschoolers enrolled in the study, and to compare various criteria for classifying obesity. Methods The study was a cluster-randomized controlled intervention trial including 24 state schools in Madrid (Spain). Weight, height, triceps and subscapular skinfold thicknesses, waist circumference, and systolic and diastolic blood pressure were measured in 2011 children (1009 boys and 1002 girls) aged 3 to 5 years (3.7 [0.9]). Body mass index and blood pressure were classified by corresponding task force criteria. Obesity was studied by 6 different criteria. Associations of body mass index, body weight, body fat, and waist circumference on blood pressure were examined, and the risk of high blood pressure in relation to tertiles of body mass index was calculated. Results The prevalence of obesity according to the International Obesity Task Force varied from 2% at age 3 to 8% at age 5, and the overall prevalence of high blood pressure (≥ 90th percentile) was 20%. Sex- and age-specific criteria for obesity showed better agreement with the reference than a single generalized cutoff. The risk of high blood pressure was higher for the highest tertile of body mass index distribution. Conclusions The highest prevalence of obesity and high blood pressure was found among older children. The classification of obesity in children was more accurate using sex- and age-specific cutoffs.

Resumen Introduccion y objetivos El Programa SI! es una intervencion escolar de promocion de salud cardiovascular en la infancia. El objetivo de este articulo es caracterizar la prevalencia de obesidad y presion arterial alta entre los preescolares del estudio del Programa SI! y comparar distintos criterios de clasificacion de obesidad. Metodos El diseno es una intervencion controlada y aleatorizada por grupos en 24 colegios publicos de Madrid. Se midio peso, talla, pliegues tricipital y subescapular, circunferencia de la cintura y presion arterial en 2.011 preescolares (1.009 ninos y 1.002 ninas) de 3 a 5 (3,7 ± 0,9) anos. El indice de masa corporal y la presion arterial se clasificaron siguiendo criterios internacionales. Se estudiaron seis criterios diferentes de obesidad, los efectos del indice de masa corporal, peso, porcentaje de grasa y circunferencia de la cintura en la presion arterial y el riesgo de presion arterial alta por terciles de indice de masa corporal. Resultados La prevalencia de obesidad oscilo del 2% (ninos de 3 anos) al 8% (ninos de 5) y la de presion arterial alta fue del 20%. Se ha encontrado mejor concordancia con la referencia internacional para los criterios de obesidad especificos para sexo y edad que con puntos de corte unicos. El riesgo de presion arterial alta aumento en cada tercil de indice de masa corporal. Conclusiones Los ninos mayores mostraron la mayor prevalencia de obesidad y presion arterial alta. La clasificacion de obesidad fue mas precisa utilizando criterios especificos para sexo y edad.

Axonal injury is often characterized by axonal transport defects and abnormal accumulation of intra-axonal components. Nitric oxide (NO) has a key role in mediating inflammatory axonopathy in many neurodegenerative diseases, but little is known about how nitrosative/oxidative stress affects axonal transport or whether reductions in kinesin superfamily protein (KIF) expression correlate with axon pathology. KIFs are molecular motors that have a key role in axonal and dendritic transport, and impairment of these mechanisms has been associated with a number of neurological disorders. This study shows that rat cortical neurons exposed to NO display both a time-dependent decrease in KIF gene/protein expression and neurofilament phosphorylation in addition to a reduction in axonal length and neuronal survival. Because mesenchymal stem cells (MSCs) represent a promising therapeutic candidate for neuronal/axonal repair, this study analyzes the capacity of MSCs to protect neurons and axonal transport mechanisms from NO damage. Results show that coculture of MSCs with NO-exposed neurons results in the preservation of KIF expression, axonal length, and neuronal survival. Altogether, these results suggest a potential mechanism involved in the disruption of axonal transport and abnormal accumulation of proteins in axons during nitrosative insult. We hypothesize that impaired axonal transport contributes, per se, to progression of injury and provide further evidence of the therapeutic potential of MSCs for neurodegenerative disorders. © 2015 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

AimsHistopathological tissue samples are being increasingly used as sources of nucleic acids in molecular pathology translational research. This study investigated the suitability of glioblastoma and control central nervous system (CNS) formalin-fixed paraffin embedded (FFPE) tissue-derived RNA for gene expression analyses.MethodsTotal RNA was extracted from control (temporal lobe resection tissue) and glioblastoma FFPE tissue samples. RNA purity (260/280 ratios) was determined and RNA integrity number (RIN) analysis was performed. RNA was subsequently used for RT-qPCR for two reference genes,18SandGAPDH.ResultsReference gene expression was equivalent between control and glioblastoma tissue when using RNA extracted from FFPE tissue, which has key implications for biological normalisation for CNS gene expression studies. There was a significant difference between the mean RIN values of control and glioblastoma FFPE tissue. There was no significant correlation between 260/280 or RIN values versus total RNA yield. The age of the tissue blocks did not influence RNA yield, fragmentation or purity. There was no significant correlation between RIN or 260/280 ratios and mean qPCR cycle threshold for either reference gene.ConclusionsThis study showed that routinely available CNS FFPE tissue is suitable for RNA extraction and downstream gene expression studies, even after 60 months of storage. Substantial RNA fragmentation associated with glioblastoma and control FFPE tissue blocks did not preclude downstream RT-qPCR gene expression analyses. Cross validation with both archival and prospectively collated FFPE specimens is required to further demonstrate that CNS tissue blocks can be used in novel translational molecular biomarker studies.

Background Clinical trials using ex vivo expansion of autologous mesenchymal stromal cells (MSCs) are in progress for several neurological diseases including multiple sclerosis (MS). Given that environment alters MSC function, we examined whether in vitro expansion, increasing donor age and progressive MS affect the neuroprotective properties of the MSC secretome. Methods Comparative analyses of neuronal survival in the presence of MSC-conditioned medium (MSCcm) isolated from control subjects (C-MSCcm) and those with MS (MS-MSCcm) were performed following (1) trophic factor withdrawal and (2) nitric oxide–induced neurotoxicity. Results Reduced neuronal survival following trophic factor withdrawal was seen in association with increasing expansion of MSCs in vitro and MSC donor age. Controlling for these factors, there was an independent, negative effect of progressive MS. In nitric oxide neurotoxicity, MSCcm-mediated neuroprotection was reduced when C-MSCcm was isolated from higher-passage MSCs and was negatively associated with increasing MSC passage number and donor age. Furthermore, the neuroprotective effect of MSCcm was lost when MSCs were isolated from patients with MS. Discussion Our findings have significant implications for MSC-based therapy in neurodegenerative conditions, particularly for autologous MSC therapy in MS. Impaired neuroprotection mediated by the MSC secretome in progressive MS may reflect reduced reparative potential of autologous MSC-based therapy in MS and it is likely that the causes must be addressed before the full potential of MSC-based therapy is realized. Additionally, we anticipate that understanding the mechanisms responsible will contribute new insights into MS pathogenesis and may also be of wider relevance to other neurodegenerative conditions.

AimsUnderstanding the causes of axonal pathology remains a key goal in the pursuit of new therapies to target disease progression in MS. Anterograde axonal transport of many proteins vital for axonal viability is mediated by the motor protein KIF5A, which has been linked to several neurological diseases. This study aimed to investigate the expression of KIF5A protein and its associated cargoes: amyloid precursor protein (APP) and neurofilament (NF) in post-mortem MS and control white matter and to determine if KIF5A expression is influenced by the presence of MS risk single nucleotide polymorphisms (SNPs) identified in the region of the KIF5A gene.MethodsUsing immunoblotting assays we analyzed the expression of KIF5A, APP and NF phospho-isoforms in 23 MS cases and 12 controls.ResultsWe found a significant reduction in KIF5A and associated cargoes in MS white matter and an inverse correlation between KIF5A and APP/NF protein levels. Furthermore, homozygous carriers of MS risk gene SNPs show significantly lower levels of KIF5A protein compared to MS patients with no copies of the risk SNPs.ConclusionsWe conclude that reduced expression of axonal motor KIF5A may have important implications in determining axonal transport deficits and ongoing neurodegeneration in MS.

Background and objectives Cellular therapies using Mesenchymal Stem Cells (MSCs) represent a promising approach for the treatment of degenerative diseases, in particular for mesengenic tissue regeneration. However, before the approval of clinical trials in humans, in vitro studies must be performed aimed at investigating MSCs' biology and the mechanisms regulating their proliferation and differentiation abilities. Besides studies on human MSCs (hMSCs), MSCs derived from rodents have been the most used cellular type for in vitro studies. Nevertheless, the transfer of the results obtained using animal MSCs to hMSCs has been hindered by the limited knowledge regarding the similarities existing between cells of different origins. Aim of this paper is to highlight similarities and differences and to clarify the sometimes reported different results obtained using these cells. Methods and results We compare the differentiation ability into mesengenic lineages of rat and human MSCs cultured in their standard conditions. Our results describe in which way the source from which MSCs are derived affects their differentiation potential, depending on the mesengenic lineage considered. For osteogenic and chondrogenic lineages, the main difference between human and rat MSCs is represented by differentiation time, while for adipogenesis hMSCs have a greater differentiation potential. Conclusions These results on the one hand suggest to carefully evaluate the transfer of results obtained with animal MSCs, on the other hand they offer a clue to better apply MSCs into clinical practice.

Background: Peroxisomes are organelles in eukaryotic cells with multiple functions including the detoxification of reactive oxygen species, plasmalogen synthesis and β-oxidation of fatty acids. Recent evidence has implicated peroxisomal dysfunction in models of multiple sclerosis (MS) disease progression. Objectives: Our aims were to determine whether there are changes in peroxisomes in MS grey matter (GM) compared to control GM. Methods: We analysed cases of MS and control GM immunocytochemically to assess peroxisomal membrane protein (PMP70) and neuronal proteins. We examined the expression of ABCD3 (the gene that encodes PMP70) in MS and control GM. Analyses of very long chain fatty acid (VLCFA) levels in GM were performed. Results: PMP70 immunolabelling of neuronal somata was significantly lower in MS GM compared to control. Calibration of ABCD3 gene expression with reference to glyceraldehyde 3-phsophate dehydrogenase (GAPDH) revealed overall decreases in expression in MS compared to controls. Mean PMP70 counts in involved MS GM negatively correlated to disease duration. Elevations in C26:0 (hexacosanoic acid) were found in MS GM. Conclusions: Collectively, these observations provide evidence that there is an overall reduction in peroxisomal gene expression and peroxisomal proteins in GM neurons in MS. Changes in peroxisomal function may contribute to neuronal dysfunction and degeneration in MS.

Objectives Friedreich's ataxia is a devastating neurological disease currently lacking any proven treatment. We studied the neuroprotective effects of the cytokines, granulocyte‐colony stimulating factor (G‐CSF) and stem cell factor (SCF) in a humanized murine model of Friedreich's ataxia. Methods Mice received monthly subcutaneous infusions of cytokines while also being assessed at monthly time points using an extensive range of behavioral motor performance tests. After 6 months of treatment, neurophysiological evaluation of both sensory and motor nerve conduction was performed. Subsequently, mice were sacrificed for messenger RNA, protein, and histological analysis of the dorsal root ganglia, spinal cord, and cerebellum. Results Cytokine administration resulted in significant reversal of biochemical, neuropathological, neurophysiological, and behavioural deficits associated with Friedreich's ataxia. Both G‐CSF and SCF had pronounced effects on frataxin levels (the primary molecular defect in the pathogenesis of the disease) and a regulators of frataxin expression. Sustained improvements in motor coordination and locomotor activity were observed, even after onset of neurological symptoms. Treatment also restored the duration of sensory nerve compound potentials. Improvements in peripheral nerve conduction positively correlated with cytokine‐induced increases in frataxin expression, providing a link between increases in frataxin and neurophysiological function. Abrogation of disease‐related pathology was also evident, with reductions in inflammation/gliosis and increased neural stem cell numbers in areas of tissue injury. Interpretation These experiments show that cytokines already clinically used in other conditions offer the prospect of a novel, rapidly translatable, disease‐modifying, and neuroprotective treatment for Friedreich's ataxia. Ann Neurol 2017;81:212–226

Purkinje cell pathology is a common finding in a range of inherited and acquired cerebellar disorders, with the degree of Purkinje cell injury dependent on the underlying aetiology. Purkinje cells have an unparalleled resistance to insult and display unique regenerative capabilities within the central nervous system. Their response to cell injury is not typical of most neurons and likely represents both degenerative, compensatory and regenerative mechanisms. Here we present a pathological study showing novel and fundamental insights into Purkinje cell injury, remodelling and repair in Friedreich’s ataxia; the most common inherited ataxia. Analysing post-mortem cerebellum tissue from patients who had Friedreich's ataxia, we provide evidence of significant injury to the Purkinje cell axonal compartment with relative preservation of both the perikaryon and its extensive dendritic arborisation. Axonal remodelling of Purkinje cells was clearly elevated in the disease. For the first time in a genetic condition, we have also shown a disease-related increase in the frequency of Purkinje cell fusion and heterokaryon formation in Friedreich's ataxia cases; with evidence that underlying levels of cerebellar inflammation influence heterokaryon formation. Our results together further demonstrate the Purkinje cell’s unique plasticity and regenerative potential. Elucidating the biological mechanisms behind these phenomena could have significant clinical implications for manipulating neuronal repair in response to neurological injury.

The spontaneous expression of neural markers by mesenchymal stem cells (MSCs) has been considered to be a demonstration of MSCs' predisposition to differentiate towards neural lineages. In view of their application in cell therapy for neurodegenerative diseases, it is very important to deepen the knowledge about this distinctive biological property of MSCs. In this study, we evaluated the expression of neuronal and glial markers in undifferentiated rat MSCs (rMSCs) at different culture passages (from early to late). rMSCs spontaneously expressed neural markers depending on culture passage, and they were coexpressed or not with the neural progenitor marker nestin. In contrast, the number of rMSCs expressing mesengenic differentiation markers was very low or even completely absent. Moreover, rMSCs at late culture passages were not senescent cells and maintained the MSC immunophenotype. However, their differentiation capabilities were altered. In conclusion, our results support the concept of MSCs as multidifferentiated cells and suggest the existence of immature and mature neurally fated rMSC subpopulations. A possible correlation between specific MSC subpopulations and specific neural lineages could optimize the use of MSCs in cell transplantation therapy for the treatment of neurological diseases. © 2012 Dana Foudah et al.

Cerebellar dysfunction is a significant contributor to disability in multiple sclerosis (MS). Both white matter (WM) and grey matter (GM) injury occurs within MS cerebellum and, within GM, demyelination, inflammatory cell infiltration and neuronal injury contribute to on-going pathology. The precise nature of cerebellar GM injury is, however, unknown. Oxidative stress pathways with ultimate lipid peroxidation and cell membrane injury occur extensively in MS and the purpose of this study was to investigate these processes in MS cerebellar GM. Post-mortem human cerebellar GM from MS and control subjects was analysed immunohistochemically, followed by semi-quantitative analysis of markers of cellular injury, lipid peroxidation and anti-oxidant enzyme expression. We have shown evidence for reduction in myelin and neuronal markers in MS GM, coupled to an increase in expression of a microglial marker. We also show that the lipid peroxidation product 4-hydroxynonenal co-localises with myelin and its levels negatively correlate to myelin basic protein levels. Furthermore, superoxide dismutase (SOD1 and 2) enzymes, localised within cerebellar neurons, are up-regulated, yet the activation of subsequent enzymes responsible for the detoxification of hydrogen peroxide, catalase and glutathione peroxidase are relatively deficient. These studies provide evidence for oxidative injury in MS cerebellar GM and further help define disease mechanisms within the MS brain.

The preschool years offer a unique window of opportunity to instill healthy life-style behaviors and promote cardiovascular health.This study sought to evaluate the effect of a 3-year multidimensional school-based intervention to improve life-style-related behaviors.We performed a cluster-randomized controlled intervention trial involving 24 public schools in Madrid, Spain, that were assigned to either the SI! Program intervention or the usual curriculum and followed for 3 years. The SI! Program aimed to instill and develop healthy behaviors in relation to diet, physical activity, and understanding how the human body and heart work. The primary outcome was change in the overall knowledge, attitudes, and habits (KAH) score (range 0 to 80). The intervention's effect on adiposity markers was also evaluated.A total of 2,062 children from 3 to 5 years of age were randomized. After 3 years of follow-up, the overall KAH score was 4.9% higher in children in the intervention group compared with the control group (21.7 vs. 16.4; p0.001). A peak effect was observed at the second year (improvement 7.1% higher than in the control group; p0.001). Physical activity was the main driver of the change in KAH at all evaluation times. Children in the intervention group for 2 years and 1 year showed greater improvement than control subjects (5.9%; p0.001 and 2.9%; p = 0.002, respectively). After 3 years, the intervention group showed a higher probability than the control group of reducing the triceps skinfold z-score by at least 0.1 (hazard ratio: 1.40, 95% confidence interval: 1.04 to 1.89; p = 0.027).The SI! Program is an effective strategy for instilling healthy habits among preschoolers, translating into a beneficial effect on adiposity, with maximal effect when started at the earliest age and maintained over 3 years. Wider adoption may have a meaningful effect on cardiovascular health promotion. (Evaluation of the Program SI! for Preschool Education: A School-Based Randomized Controlled Trial [Preschool_PSI!]; NCT01579708).

Stem cell-based therapies are increasingly emerging as hopeful therapeutic options for treating currently incurable chronic degenerative and inflammatory diseases of the central nervous system (CNS). Among the variety of different stem cell types, mesenchymal stem cells (MSCs) possess a wide range of practical features, in addition to a plethora of neuroprotective properties, which make them an attractive candidate as a potential cell therapy for a wide range of neurodegenerative disorders. Here we will discuss the suitability of MSCs for clinical use. We will also summarize the underlying mechanisms that drive their multiplicity of neuroprotective effects alongside their use in the treatment of neurodegenerative disease from the bench side to clinical trials.

The development of CVD risk factors carries a behavioral component that may be corrected at an early age, when behaviors are first formed, by effective health promotion initiatives. School-based interventions are an effective way to promote healthy behaviors, specially when they also reach out to the children’s most proximal environment: their families, teachers and the school itself. Within this framework, the first phase of the long-term Program SI! intervention targets children from 3 to 5 years of age aiming to establish appropriate lifestyle behaviors early in life. The intervention entails a comprehensive approach including four lifestyle-related components: correct dietary habits, promotion of physical activity, understanding how the human body and heart work, and emotion’s management. To evaluate the efficacy of the Program SI! to instill healthy behaviors in children aged 3-5, a cluster-randomized controlled trial in public schools in the city of Madrid (Spain) was initiated in 2011. A total 24 schools were selected on the basis of socio-demographic characteristics. These included 2062 children (3-5 years), 1949 families, and 125 teachers at baseline. Schools were randomized (1:1) to follow their usual school curriculum or to engage in the intervention. The primary outcome of this report is the 2-years differential change from baseline in scores for knowledge, attitudes and habits (KAH) in the four components of the intervention between intervention and control. Children were interviewed by trained psychologists and scored on overall and component-specific KAH questionnaires. The Test for Emotional Comprehension (TEC) was used to assess children’s ability to recognize emotions. Mixed linear models accounting for the cluster-randomized design were used to test for intervention effects. Fixed effects in each model were the corresponding baseline score, the class year, and the treatment group. Schools were handled as random effects. After 2 years, the Program SI! intervention increased children’s overall KAH score (5.6, 95%CI, 4.1-7.1, p

Preservation with University of Wisconsin (UW) solution has been implicated in coronary artery endothelial damage and loss of endothelium-dependent vasodilatation. Therefore, the objective of this study was to investigate the effect of this solution on basal nitric oxide (NO) release from porcine coronary endothelial cells (CEC). Cultures were exposed to cold (4 degrees C) storage in UW solution for 6, 8 and 12 h. Parallel cultures were incubated with control medium at 37 degrees C. After treatment, NO release was evaluated by nitrite production, a stable metabolite of NO. Activity of the constitutive endothelial nitric oxide synthase (eNOS) was measured by the conversion [3H]-l-arginine to [3H]-l-citrulline and eNOS protein expression by Western blotting. Nitrite production by control cells was augmented with increasing times of incubation, whereas no change was observed in those cultures preserved with UW solution. Activity of eNOS was significantly decreased compared to the respective control group by cold storage of cells for longer periods than 6 h. Such decrease was correlated with a diminished eNOS protein expression in CEC preserved with UW solution after 8- and 12-h storage. These results suggest that prolonged hypothermic storage of CEC with UW solution does not preserve basal NO release because of a certain loss of eNOS protein, which may contribute to the reported injury of heart transplants after long-term preservation.

1 1Cardiac fibroblasts play an important role in the pathophysiology of cardiac remodelling induced by hypertension and myocardial infarction by undergoing proliferation and depositing extracellular matrix proteins such as collagen. We have examined the effects of atrial natriuretic peptide (ANP) on proliferation and collagen synthesis by adult rat and human cardiac fibroblasts in culture. 2 2In cells from both species radioligand studies using 125I-ANP suggested that the majority of binding sites (>85%) were non-guanylyl cyclase-linked (NPR-C subtype). Nonetheless ANP (10−9 to 10−6m), in the presence of zaprinast, an inhibitor of phosphodiesterase 5 (PDE5), increased fibroblast cyclic GMP levels 3–5 fold in a concentration-dependent manner (P

The discovery in the early 1980s of atrial natriuretic peptide, a circulating peptide with natriuretic/diuretic and vasorelaxant properties was greeted with excitement since the existence of a humoral link between heart and kidney had long been predicted. A multidisciplinary investigation of atrial natriuretic peptide raised expectations of new insights into the pathogenesis of cardiovascular and renal disease and opportunities for the regulation of salt and water excretion and blood pressure. What progress has been made? Synthesis and tissue distribution Atrial natriuretic peptide mRNA has been found in many tissues but is most abundant in the atria of the heart. 1 The atrial natriuretic peptide prohormone in cardiac tissue is cleaved into two fragments, both of which enter the circulation—a C-terminal 28-aminoacid peptide (ANP 99‐126) and an N-terminal fragment (ANP 1‐98). ANP 99‐126 (hereafter known as ANP) is biologically active. Some researchers suggest that the N-terminal fragment is further processed to release three peptides (proANPs 1‐30, 31‐67, and 79‐98) which may have either renal, or vasorelaxant actions, or both. 2 Urodilatin, a peptide isolated from human urine, is ANP 95‐126; it may arise by alternative processing of the ANP prohormone in the kidney but its synthetic pathway is not yet established. 3 Two other peptides, derived from different precursor molecules encoded by separate genes, have also been isolated and exhibit structural, if not functional, homology to ANP. Together, these three peptides, ANP, brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) constitute the natriuretic-peptide family. 1

Chronic demyelination, on-going inflammation, axonal loss and grey matter neuronal injury are likely pathological processes that contribute to disease progression in multiple sclerosis (MS). Although the precise contribution of each process and their aetiological substrates is not fully known, recent evidence has implicated oxidative damage as a major cause of tissue injury in MS. The degree of tissue injury caused by oxidative molecules, such as reactive oxygen species (ROS), is balanced by endogenous anti-oxidant enzymes which detoxify ROS. Understanding endogenous mechanisms which protect the brain against oxidative injury in MS is important, since enhancing anti-oxidant responses is a major therapeutic strategy for preventing irreversible tissue injury in the disease. Our aims were to determine expression and activity levels of the hydrogen peroxide-reducing enzyme catalase in MS grey matter (GM). In MS GM, a catalase enzyme activity was elevated compared to control GM. We measured catalase protein expression by immune dot-blotting and catalase mRNA by a real-time polymerase chain reaction (RT-PCR). Protein analysis studies showed a strong positive correlation between catalase and microglial marker IBA-1 in MS GM. In addition, calibration of catalase mRNA level with reference to the microglial-specific transcript AIF-1 revealed an increase in this transcript in MS. This was reflected by the extent of HLA-DR immunolabeling in MS GM which was significantly elevated compared to control GM. Collectively, these observations provide evidence that microglial catalase activity is elevated in MS grey matter and may be an important endogenous anti-oxidant defence mechanism in MS.

BACKGROUND: Unhealthy lifestyles contribute to the development of cardiovascular risk factors, whose incidence is increasing among children and adolescents. The Program SI! is a long-term, multi-target behavioral intervention to promote healthy lifestyle habits in children through the school environment. The objective of the study is to evaluate the efficacy of this intervention in its first phase, preschoolers. METHODS: Cluster-randomized controlled trial in public schools in the city of Madrid, Spain. A total 24 schools, including 2062 children (3-5 years), 1949 families, and 125 teachers participated in the study. Schools were assigned to their usual school curriculum or to engage in an additional multi-component intervention (Program SI!). The primary outcome of this trial is 1-school year changes from baseline in scores for children's knowledge, attitudes and habits (KAH). Secondary outcomes are 1-school year changes from baseline in scores for knowledge, attitudes, and habits among parents, teachers, and the school environment. RESULTS: After 1-school year, our results indicate that the Program SI! intervention increases children's KAH scores, both overall (3.45, 95% CI, 1.84-5.05) and component-specific (Diet: 0.93, 95% CI, 0.12-1.75; Physical activity: 1.93, 95% CI, 1.17-2.69; Human body: 0.65, 95% CI, 0.07-1.24) score. CONCLUSIONS: The Program SI! is demonstrated as an effective and feasible strategy for increasing knowledge and improving lifestyle attitudes and habits among very young children. TRIAL REGISTRATION: NCT01579708, Evaluation of the Program SI! for Preschool Education: A School-Based Randomized Controlled Trial (Preschool-SI!). This work is supported by the research grant FIS-PI11/01885 (Fondo de Investigación Sanitaria del Instituto de Salud Carlos III, Madrid). Sí

Mesenchymal stem cells (MSCs) are multipotent cells that are able to differentiate into mesodermal lineages (osteogenic, adipogenic, chondrogenic), but also towards non-mesodermal derivatives (e.g. neural cells). Recent in vitro studies revealed that, in the absence of any kind of differentiation stimuli, undifferentiated MSCs express neural differentiation markers, but the literature data do not all concur. Considering their promising therapeutic potential for neurodegenerative diseases, it is very important to expand our knowledge about this particular biological property of MSCs. In this study, we confirmed the spontaneous expression of neural markers (neuronal, glial and progenitor markers) by undifferentiated human MSCs (hMSCs) and in particular, we demonstrated that the neuronal markers βIII-tubulin and NeuN are expressed by a very high percentage of hMSCs, regardless of the number of culture passages and the culture conditions. Moreover, the neuronal markers βIII-tubulin and NeuN are still expressed by hMSCs after in vitro osteogenic and adipogenic differentiation. On the other hand, chondrogenically differentiated hMSCs are negative for these markers. Our findings suggest that the expression of neuronal markers could be common to a wide range of cellular types and not exclusive for neuronal lineages. Therefore, the expression of neuronal markers alone is not sufficient to demonstrate the differentiation of MSCs towards the neuronal phenotype. Functional properties analysis is also required.

BACKGROUND: The onset of inadequate behaviors leading to the development of risk factors for chronic diseases is known to occur early in life. An effective program for health promotion should therefore focus on children and their environment, as the starting point for behavior development. The overarching objective of the Program SI! (Salud Integral - Comprehensive Health) is to intervene at the school level, to establish and develop life-lasting habits that will help preserving health during adulthood. The Program SI! comprises five consecutive subprograms according to the five stages of education in Spain, the first being in preschoolers. This study aims to evaluate the efficacy of Program SI! to establish and improve lifestyle behaviors in children (preschoolers aged 3-5 years), their parents, and teachers, and also improving the school environment. A secondary objective is to evaluate improvements in cardiovascular health-related markers (anthropometric parameters, blood pressure, and dietary and physical activity patterns) in these same children. METHODS/DESIGN: 24 public schools from the city of Madrid (Spain) were allocated through stratified randomization to intervention or control. The intervention schools follow the Program SI!, which provides didactic units, emotions cards, healthy tips, and online resources. The intervention schools integrate the Program SI! into their scholar curriculum organized in four complete weeks during each academic year during the 3 years of preschool education. Control schools follow their normal curriculum. Primary outcomes are 1-year, and 3-year changes from baseline of scores for knowledge, attitudes, and habits (KAH) of children, their parents and teachers in regards to a healthy lifestyle. Secondary outcomes are 1-year, and 3-year changes from baseline in clinical and anthropometric parameters of children. DISCUSSION: The Program SI! is a long-term health promotion program starting in 3 years old. It incorporates the traditional areas of intervention (diet and physical activity), introducing additional components such as knowledge of the human body and management of emotions to achieve a comprehensive intervention. The Program SI! is designed to be an effective, sustainable health promotion program for the adoption of healthy behaviors from early in life. TRIAL REGISTRATION: TRIAL REGISTRATION NUMBER: NCT01579708. The authors would like to thank the International SHE Foundation, intellectual owner of the Program SI!. We also thank the ALICIA Foundation, and Sesame Workshop for providing the materials needed for the intervention. This work is supported by the research grant FIS-PI11/01885 (Fondo de Investigación Sanitaria del Instituto de Salud Carlos III, Madrid). Sí

Multipotent mesenchymal stromal cells (MSCs) have neuro- and glial protective properties and can influence the differentiation and maturation of oligodendrocytes. Clinical trials employing autologous MSC transplantation for the treatment of multiple sclerosis (MS) and other neurodegenerative diseases are ongoing. Extending previous studies, we have performed detailed analysis of the phenotype of MSCs derived from patients with MS using marrow samples donated as part of the ‘ACTiMuS’ trial which is examining the efficacy of unfractionated autologous bone marrow in progressive MS (NCT01815632). In keeping with previous findings, MS MSCs have comparable mesenchymal differentiation potential to MSCs isolated from control subjects. However, donor age negatively correlates with clonogenic potential and premature senescence. Using in vitro models of neuroglial toxicity, we have demonstrated that the neuroglial protective effects of soluble factors produced by MSCs decline with expansion in vitro. Significantly, media conditioned by MSCs isolated from patients with long duration of progressive disease have reduced neuroglial protective capacity. Using tandem mass spectroscopy, we have identified over 40 factors whose secretion by MSCs is altered in progressive MS. Further work will explore their relevance to MS pathogenesis and repair with the aim of identifying new drug candidates and optimisation of cell-based therapy for MS.

Introduction. Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent cells that can differentiate into different cell lineages and have emerged as a promising tool for cell-targeted therapies and tissue engineering. Their use in a therapeutic context requires large-scale in vitro expansion, increasing the probability of genetic and epigenetic instabilities. Some evidence shows that an organized program of replicative senescence is triggered in human BM-MSCs (hBM-MSCs) on prolonged in vitro expansion that includes alterations in phenotype, differentiation potential, telomere length, proliferation rates, global gene-expression patterns, and DNA methylation profiles. Methods. In this study, we monitored the chromosomal status, the biologic behavior, and the senescence state of hBM-MSCs derived from eight healthy donors at different passages during in vitro propagation. For a more complete picture, the telomere length was also monitored in five of eight donors, whereas the genomic profile was evaluated in three of eight donors by array-comparative genomic hybridization (array-CGH). Finally, an epigenomic profile was delineated and compared between early and late passages, by pooling DNA of hBM-MSCs from four donors. Results: Our data indicate that long-term culture severely affects the characteristics of hBM-MSCs. All the observed changes (that is, enlarged morphology, decreased number of cell divisions, random loss of genomic regions, telomere shortening) might be regulated by epigenetic modifications. Gene Ontology analysis revealed that specific biologic processes of hBM-MSCs are affected by variations in DNA methylation from early to late passages. Conclusions: Because we revealed a significant decrease in DNA methylation levels in hBM-MSCs during long-term culture, it is very important to unravel how these modifications can influence the biologic features of hBM-MSCs to keep track of this organized program and also to clarify the conflicting observations on hBM-MSC malignant transformation in the literature.

The aim of the present study was to analyse the ability of phorbol 12,13-dibutyrate (PDB) to activate protein kinase C (PKC), measured by its capacity to translocate the enzyme from the cytosol to the membrane fraction, as well as to induce vasconstrictive responses in segments from branches of bovine cerebral arteries. PDB (0·1 μm) produced a marked translocation of PKC activity from the cytosolic to the membranous fraction. This drug induced concentration-dependent contractions which were slow in onset. The contraction elicited by PDB was reduced by the PKC inhibitor, staurosporine (1 and 10 Nm), but unaltered by both Ca2+-free medium containing 3 Mm EGTA and the Ca2+-channel antagonist, nifedipine (1 μm). Preincubation of segments with PDB (10 and 30 Nm) reduced the vasoconstriction elicited by 5-hydroxytryptamine (5-HT) in a concentration- and preincubation time-dependent manner. These data indicate that bovine cerebral arteries possess cytosolic and membranous PKC activities, that the vasoconstrictive responses induced by PDB were independent of extracellular Ca2+, that cytosolic C-kinase is translocated to the membrane and probably down-regulated by PDB, and that this enzyme is not involved in 5-HT responses, but is down-regulated by PDB.

The aim was to determine whether high glucose levels interfere with nitric oxide (NO) production and inducible NO synthase (iNOS) protein expression in interleukin-1beta-stimulated vascular smooth muscle cells from normotensive Wistar Kyoto and spontaneously hypertensive rats. Cells were incubated with either normal (5.5 mM) or high (22 mM) d-glucose for 72 h and with interleukin-1beta (10 ng/ml) for the last 24 h. High glucose increased nitrite levels, iNOS expression and protein kinase C activity in cells from normotensive rats and had no effect in cells from hypertensive rats. High glucose effects on nitrite production and iNOS expression was abolished by the selective inhibitor for the protein kinase C-betaII, 5,21:12,17-dimetheno-18H-dibenzo[i,o]pyrrolo[3,4-1] [1,8]diacyclohexadecine-18,20 (19H)-dione, 8-[(dimethylamino) methyl]-6,7,8,9,10,11-hexahydro-monomethanesulfonate (LY379196, 30 nM). Calphostin C (1 microM) and LY379196 (10 microM) reduced nitrite levels and iNOS expression only in cells from normotensive rats treated with both media. These results suggest that high glucose increases inducible nitric oxide synthase induction and subsequent NO production by activating the protein kinase C-betaII; this mechanism seems to be altered in hypertension.

Release of CD34+ haematopoietic stem cells (HSC) into the peripheral circulation has been demonstrated in patients receiving anti-CD49d monoclonal antibody for the treatment of multiple sclerosis (MS). It is not known whether these cells are mobilised during an acute MS relapse or by treatment with other disease-modifying drugs (DMDs). We analysed peripheral blood samples from patients with MS including those on disease modifying therapy and during acute relapses. Circulating numbers of CD34/133+ cells were determined using flow cytometry. A novel, multi-channel flow cytometric approach was developed to identify multipotent mesenchymal stromal cells (MSCs). The finding of mobilisation of CD34+ HSCs with anti-CD49d antibody infusion was replicated. Increases in circulating numbers of CD34+ HSCs were also observed in patients treated with glatiramer acetate and in MS patients experiencing an acute relapse, irrespective of ongoing treatment with DMDs. CD133+ subsets were also increased in all groups. Circulating MSCs were not detected in the peripheral blood of patients or controls. Our results suggest a role for HSC mobilisation during treatment for MS and in acute relapse. Further work will examine the role of these cells in the pathogenesis of MS and endogenous repair.

Background The aim of this study was to determine the effect of University of Wisconsin solution (UWS) incubation on bradykinin-induced vasodilation. Methods Porcine coronary arteries were incubated in Krebs-Henseleit solution (KHS) or UWS at 4°C for 20 hours. Endothelium-dependent relaxation to bradykinin and endothelium-independent relaxation to nitric oxide were tested after U46619 or KCl pre-contraction. Nitric oxide synthase activity and protein expression was determined by [ 3 H]-l-citrulline formation and western blot analysis, respectively. Results The relaxation to bradykinin (0.1 to 300 nmol/liter) after U46619 (30 to 300 nmol/liter) pre-contraction was similar with both KHS and UWS pre-incubation; however, it was reduced after KCl pre-contraction (15 to 20 mmol/liter), this reduction being greater after UWS incubation. The inhibitory effect of N G -nitro-l-arginine methylester (0.1 mmol/liter) on bradykinin-induced relaxation was lower in UWS- than KHS-incubated segments after U46619 pre-contraction, but similar after KCl pre-contraction; however, the inhibitory effect of 0.5 mmol/liter ouabain was unaffected. Tetraethylammonium (5 mmol/liter) reduced the response to bradykinin more strongly after UWS pre-incubation. UWS did not modify relaxation to nitric oxide (0.1 to 30 μmol/liter) in pre-incubated UWS or KHS segments. UWS failed to modify both total nitric oxide synthase activity and endothelial nitric oxide synthase expression. Conclusions UWS incubation decreased nitric oxide participation and increased the hyperpolarizing mechanisms produced by bradykinin.

Background . Hypothermia-induced changes in cell volume and ionic transport systems of coronary endothelial cells may play a role in the development of coronary artery disease in cardiac transplant recipients. Methods . Coronary endothelial cells were incubated in University of Wisconsin solution or culture control medium for up to 48 hours at 4°C. Parallel control cultures were incubated at 37°C. Na/K-ATPase and Na/K/Cl cotransport activities were determined as ouabain- and furosemide-sensitive 86 Rb + uptake, respectively. Cell volume changes and cell death were analyzed by a FACScan flow cytometer and the release of lactate dehydrogenase, respectively. Results . Coronary endothelial cells stored in University of Wisconsin solution up to 6 hours showed an increased Na/K-ATPase activity compared to control cells, whereas no changes were observed in Na/K/Cl cotransport activity or cell volume. Long-term preservation (24 and 48 hours) was associated with a partial loss of cell viability, as demonstrated by lactate dehydrogenase release, and dramatic alterations in ionic transport system activities. Conclusions . University of Wisconsin solution seems to prevent coronary endothelial cells Na/K/Cl cotransport activity changes during cold preservation, which could alter cell volume regulation and cause cell injury.

Objective To investigate the effect of ouabain on inducible nitric oxide synthase (iNOS) activity and expression in cytokine-stimulated vascular smooth muscle cells (VSMC) from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Methods VSMC were treated for 24 h and afterwards, nitric oxide (NO) release was determined by the production of nitrite, a stable metabolite of NO. Activity of iNOS was measured by the conversion of [ 3 H]-L-arginine to [ 3 H]-L-citrulline and iNOS protein expression by Western blotting. Results Ouabain (0.01 -1 mmol/I) further enhanced interleukin-1β (II-1β)-induced nitrite production by WKY and SHR VSMC, although a more pronounced effect was observed in SHR cells (maximum response 52.1 ± 5.2 and 71.2 ± 6.4% of II-1β effect in WKY and SHR cells, respectively). Such response on NO release was mimicked by the calcium ionophore A 23187 (0.01-1 μmol/l) and abolished by the voltage-operated calcium channels (VOCC) nifedipine (0.1 μmol/l). Expression of iNOS showed that ouabain increased the synthesis of the enzyme in WKY and SHR VSMC stimulated with II-1 β, and this effect was higher in SHR cells. The increased iNOS expression was significantly reduced by nifedipine. Conclusions Ouabain stimulation of iNOS expression and activity in II-1 β-stimulated VSMCs from WKY rats and SHR seems to be related to increased intracellular calcium influx through VOCC. The more pronounced effect observed in SHR VSMC could be explained by an altered calcium entry in the hypertensive strain.

The vascular Na+ pump maintains intracellular ionic concentration and controls membrane potential. Its inhibition by cardiac glycosides enhances the intracellular Na+ concentration. This in turn activates the Na+-Ca2+ exchange mechanism, which induces intracellular Ca2+ increase, membrane depolarization, and noradrenaline release from perivascular adrenergic nerve endings; mechanisms that promote vasoconstriction. This article reviews the relevance of the Na+ pump in vascular tone regulation and the modulation of its activity by the endothelium. The endothelium negatively modulates the vasoconstriction elicited by Na+ pump inhibition by the release of nitric oxide, according to some authors, or an unknown factor, as suggested by others. The possible existence of endogenous digitalis-like factors is also reviewed, as is the involvement of the vascular Na+ pump in some cardiovascular disorders and aging.

Age-related changes in the blood prooxidant-antioxidant state, as well as its influence on the relaxant responses to acetylcholine (ACh) were studied in the tail artery from 6-, 24- and 30-month-old Sprague-Dawley (SD) rats. Malondialdehyde (MDA) plasma levels increased 2 and 3 times in 24- and 30-month-old rats, respectively, when compared with 6-month-old rats (0.43±0.09 μM). This increase was accompanied by an induction of 6-phosphogluconate dehydrogenase (6PG-DH) and glutathione reductase (GR) activities in red blood cells from 24-month-old rats. In 30-month-old rats, a further induction of these enzymatic activities, as well as glucose-6-phosphate dehydrogenase (G6P-DH) and glutathione peroxidase (GPx) activities was observed. No differences with age were found in the concentration-response curves to ACh in isolated tail artery segments from 6- and 24-month-old rats precontracted with 0.3 μM noradrenaline (NA). However, a decrease in sensitivity to ACh-induced relaxation was observed in 30-month-old rats; EC30 values were 3.5 (1.3–8.0)×10−7 M and 18.1 (8.9–30.1)×10−7 M for 6- and 30-month-old rats, respectively. Moreover, a decrease in maximum ACh relaxation (10 μM) was found in 30-month-old rats in comparison with that obtained in 6-month-old rats (58.5±3.9% and 42.5±3.4% of previous NA contraction, respectively). Incubation of tail artery segments with MDA (0.5, 1 or 10 μM) caused a reduction of ACh-induced relaxations that was different in the three ages. Thus, the reduction of ACh-induced relaxations became significant with 0.5 μM MDA in 6-, with 1 μM MDA in 24-, and with 10 μM MDA in 30-month-old rats. In addition, MDA did not cause a shift in the concentration-response curve to ACh, but a decrease in the maximum response. Superoxide dismutase (SOD; 150 u ml−1, a superoxide anion scavenger) reversed the inhibitory effect of MDA on ACh-induced relaxations at all ages studied. We conclude that: (1) ageing produces an increase in lipid peroxidation process, as indicated by the increase in MDA plasma levels, that is accompanied by an induction of lipid peroxide detoxification enzymes; (2) the changes in prooxidant-antioxidant equilibrium with age contribute, at least partially, to the impairment of the relaxant responses evoked by ACh; and (3) the effect of MDA appears to be mediated by superoxide anion at all ages studied. British Journal of Pharmacology (1998) 123, 113–121; doi:10.1038/sj.bjp.0701595

Abstract The endothelium exerts a large influence on the underlying vascular smooth muscle, not only by the release of both contracting and relaxing factors but also by its ability to synthesize a large number of molecules that influence vascular smooth muscle growth. In addition to well-characterized growth promoters or growth inhibitors, some endothelium-derived factors, originally described as vasoactive compounds, seem to possess growth-regulatory properties. The vasoconstrictor endothelin-1 elicited a dose-dependent increase of cultured vascular smooth muscle cell DNA synthesis with a maximal effect of 57±14% over basal levels, whereas vasodilators such as prostacyclin, sodium nitroprusside, and 8-bromoguanosine 3′:5′-cyclic monophosphate reduced DNA synthesis by 19±5%, 22±2%, and 31±3%, respectively. Medium conditioned by cultured bovine aortic endothelial cells markedly stimulated both DNA synthesis and proliferation of smooth muscle cells. When medium was conditioned in the presence of the endothelin-converting enzyme inhibitor phosphoramidon, the mitogenic effect was significantly reduced, thus indicating a role for endothelin in the stimulation of smooth muscle cell growth by endothelial cells. However, when both cell types were maintained in a coculture system, a 13±2% decrease of DNA synthesis was observed in smooth muscle cultures. The addition of the nitric oxide synthase inhibitor N ω -nitro- l -arginine methyl ester, the cyclooxygenase inhibitor indomethacin, or both during the coculture period did not revert the antiproliferative effect of endothelial cells in coculture, thereby indicating it is not likely due to these unstable endothelium-derived vasorelaxant molecules.

The vascular effects of endothelin-1 (ET-1) were compared with those elicited by phorbol 12,13-dibutyrate (PDB), an activator of the protein kinase C (PKC), to analyze the involvement of this enzyme on ET-1 responses. PDB and ET-1 caused slow-developing contractions (sustained and transient, respectively), which were reduced by the PKC inhibitor, staurosporine (1 and 10 nM). Only the contractile effects evoked by ET-1 were reduced in Ca-free medium and by the Ca channel antagonist, nifedipine (1 microM), and increased by the Ca channel agonist, BAY K 8644 (10 nM). PDB (10 and 30 nM) preincubation reduced the vasoconstriction elicited by 5-hydroxytryptamine (5-HT; 0.01, 0.1 and 1 microM) in a way dependent on phorbol concentration and preincubation time, whereas ET-1 (1 nM) increased the contractile response to 5-HT (0.1 microM). Furthermore, PDB (0.1 microM) also reduced the responses elicited by ET-1 (30 microM) and vice versa. ET-1 (0.1 microM) induced transient translocation of PKC activity from the cytosol to the membrane, which was less than that produced by PDB (0.1 microM). Electrical stimulation induced [3H]noradrenaline (NA) release, which was increased by PDB (10 and 100 nM) and not affected by ET-1 (10 nM). These results indicate: (1) the responses induced by PDB and ET-1 were independent and dependent on extracellular Ca, respectively; (2) PKC is involved in NA release and 5-HT responses, but mainly in desensitization of these responses, and (3) PKC is activated by ET-1 and is implicated in vascular actions of ET-1, but other mechanisms, such as the activation of ET-1 receptors and opening of dihydropyridine-sensitive Ca channels also appear to be involved.

In vascular smooth muscle cell (VSMC) cultures from Sprague-Dawley (SD) and hypertensive transgenic rats for the mouse renin gene Ren-2 (TGR), the DNA synthesis, which was analyzed by the uptake of [3H]thymidine, was higher in TGR than SD VSMCs (2.5- to 8-fold, mean of 5.6-fold) under basal conditions. DNA synthesis was increased by fetal calf serum (10%) in SD cells more than in TGR VSMCs, and was decreased by heparin (400 micrograms/ml) and by phorbol-12,13-dibutyrate (10(-7) M) in TGR VSMCs to a higher degree than in SD cells. Neither endothelin (10(-7) M), angiotensinogen (10(-8) M), the renin inhibitor CGP 29,287 (10(-4) M), angiotensin I (10(-7) M), captopril (10(-5) M), angiotensin II (10(-7) M), nor saralasin (10(-6) M) modified DNA synthesis in either type of VSMCs. Sodium nitroprusside (10(-4) and 10(-3) M) increased DNA synthesis in both kinds of VSMCs but in TGR cultures it became toxic at 10(-3) M. 8-Bromocyclic GMP (10(-7) to 10(-5) M) reduced DNA synthesis in SD cells more than in TGR VSMCs. These results suggest that (a) cellular mechanisms of proliferation appear to be more activated in TGR VSMCs, likely involving a protein kinase C-dependent pathway but not the renin-angiotensin system, and (b) in both type of cells, sodium nitroprusside possesses proliferative properties whereas 8-bromocyclic GMP has antiproliferative properties.

Endothelial interference with ouabain effects on vascular smooth muscle was analyzed in isolated human placental arteries and veins, carotid arteries from reserpinized guinea pigs (to eliminate the adrenergic actions of the glycoside), and aorta from Wistar-Kyoto (WKY) rats that were 5 weeks, 3, 6, 12, and 18 months old. After endothelium removal, ouabain-evoked contractions were increased in human placental vessels and guinea pig carotid arteries. These effects were not mimicked by the blockade of nitric oxide, prostaglandin, or leukotriene synthesis. Bioassay experiments suggest the release of an endothelial diffusable factor(s). 86Rb+ uptake, a method to measure sodium-pump activity, was significantly reduced by the removal of endothelium. In aortas from WKY rats, ouabain caused contractions that were increased with the age of animals until 12 months. Endothelium removal increased ouabain effects in vessels from animals of 3 and 6 months but not in older ages. These results suggest (a) that there is an inhibitory endothelial modulation of ouabain-induced vasoconstriction by the release of a diffusible factor(s); (b) that this substance is not related to nitric oxide, prostaglandins, or leukotrienes; (c) that its mechanism of action could be stimulating the activity of vascular sodium pump and/or antagonizing its inhibition by ouabain; and (d) that the endothelial modulation is lost in the elderly.

Cerebellar ataxia commonly occurs in multiple sclerosis, particularly in chronic progressive disease. Previous reports have highlighted both white matter and grey matter pathological changes within the cerebellum; and demyelination and inflammatory cell infiltrates appear commonly. As Purkinje cell axons are the sole output of the cerebellar cortex, understanding pathologic processes within these cells is crucial to develop strategies to prevent their loss and thus reduce ataxia. We studied pathologic changes occurring within Purkinje cells of the cerebellum. Using immunohistochemic techniques, we found changes in neurofilament phosphorylation states within Purkinje cells, including loss of dephosphorylated neurofilament and increased phosphorylated and hyperphosphorylated neurofilament. We also found Purkinje axonal spheroids and Purkinje cell loss, both of which occurred predominantly within areas of leucocortical demyelination within the cerebellar cortex. These changes have important implications for the study of cerebellar involvement in multiple sclerosis and may help design therapies to reduce the burden of ataxia in the condition.

Erratum After publication of this study [1], we discovered that the unit of measurement used to quantify changes in axonal morphology had been incorrectly labelled. Both the methods section ‘Purkinje cell axonal morphology’ and ‘Table 2 legend’ should read:All axonal changes are expressed as either frequency per standardized unit length of the Purkinje cell layer (1 centimetre) or frequency per Purkinje cell.