Blockade of IGF2R improves muscle regeneration and ameliorates Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a debilitating fatal X-linked muscle disorder. Recent findings indicate that IGFs play a central role in skeletal muscle regeneration and development. Among IGFs, insulinlike growth factor 2 (IGF2) is a key regulator of cell growth, survival, migration and differentiation. The type 2 IGF receptor (IGF2R) modulates circulating and tissue levels of IGF2 by targeting it to lysosomes for degradation. We found that IGF2R and the store-operated Ca2+ channel CD20 share a common hydrophobic binding motif that stabilizes their association. Silencing CD20 decreased myoblast differentiation, whereas blockade of IGF2R increased proliferation and differentiation in myoblasts via the calmodulin/calcineurin/NFAT pathway. Remarkably, anti-IGF2R induced CD20 phosphorylation, leading to the activation of sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase (SERCA) and removal of intracellular Ca2+ . Interestingly, we found that IGF2R expression was increased in dystrophic skeletal muscle of human DMD patients and mdx mice. Blockade of IGF2R by neutralizing antibodies stimulated muscle regeneration, induced force recovery and normalized capillary architecture in dystrophic mdx mice representing an encouraging starting point for the development of new biological therapies for DMD.

Anti-inflammatory Effect of Somatostatin Analogue Octreotide on Rheumatoid Arthritis Synoviocytes.

Somatostatin and its analogues are known to have modulatory effects on immune response and their anti-proliferative, anti-angiogenic, and analgesic properties make them attractive candidates for a therapeutic use in immune-mediated diseases, such as rheumatoid arthritis. Here, we demonstrate the ability of the somatostatin analogue octreotide to inhibit interleukin-15 and to increase interleukin-10 production by rheumatoid arthritis fibroblast-like synovial cells maintained in a chronic inflammatory state. We also prove that the inhibitory effect of octreotide on interleukin-15 and tumor necrosis factor-α production depended on the increase in interleukin-10, since neutralizing anti-interleukin-10 antibody was able to partially reverse this inhibition. In addition, our observations suggest an octreotide control on purinergic signaling, with an inhibitory effect on purinergic P2X and P2Y receptors activation. This would have great implications, considering the roles of P2 receptors in the onset of inflammation. Data here reported extend knowledge on the biological action of octreotide and underline its multiple effects on immune response, which could make octreotide an attractive and valid support for the therapy of diseases where several inflammatory mediators are involved, such as rheumatoid arthritis, and in which the simultaneous action on different aspects can be a successful strategy.

Characterization of a Monoclonal Antibody Specific for the Growth Hormone Secretagogue Receptor.

Ghrelin is an orexigenic peptide hormone that primarily regulates growth hormone secretion, food intake, and energy homeostasis. It has been shown to also play a role in numerous higher brain functions, such as the regulation of inflammation and cell proliferation. Ghrelin is the endogenous ligand of the growth hormone secretagogue receptor (GHSR), a G-protein-coupled receptor highly expressed in brain and detectable in some peripheral tissues. The wide distribution of ghrelin receptor and the number of tissues and cell types known to respond to ghrelin suggest that a number of systems may be affected by treatment with this hormone or its analogues. In this study, we characterized a new GHSR specific monoclonal antibody recognizing specifically the ghrelin receptor. This could be a useful tool for immunoassays aimed at obtaining insights into the physiological and pathological significance of the GHSR/ghrelin system.

Optimized "in vitro" culture conditions for human rheumatoid arthritis synovial fibroblasts.

The composition of synovial fluid in rheumatoid arthritis (RA) is complex and strongly influences the microenvironment of joints and it is an inseparable element of the disease. Currently, "in vitro" studies are performed on RA cells cultured in the presence of either recombinant proinflammatory cytokines-conditioned medium or medium alone. In this study, we evaluated the use of synovial fluid, derived from RA patients, as optimal culture condition to perform "in vitro" studies on RA synovial fibroblasts. We observed that synovial fluid is more effective in inducing cell proliferation with respect to TNF-alpha or culture medium alone. Spontaneous apoptosis in fibroblasts was also decreased in response to synovial fluid. The expression of proinflammatory cytokines in the presence of synovial fluid was significantly elevated with respect to cells cultured with TNF-alpha or medium, and the overall morphology of cells was also modified. In addition, modulation of intracellular calcium dynamics elicited in response to synovial fluid or TNF-alpha exposure is different and suggests a role for the purinergic signalling in the modulation of the effects. These results emphasize the importance of using RA synovial fluid in "in vitro" studies involving RA cells, in order to reproduce faithfully the physiopathological environmental characteristic of RA joints.

A soluble biocompatible guanidine-containing polyamidoamine as promoter of primary brain cell adhesion and in vitro cell culturing.

This paper reports on a novel application of an amphoteric water-soluble polyamidoamine named AGMA1 bearing 4-butylguanidine pendants. AGMA1 is an amphoteric, prevailingly cationic polyelectrolyte with isoelectric point of about 10. At pH 7.4 it is zwitterionic with an average of 0.55 excess positive charges per unit, notwithstanding it is highly biocompatible. In this work, it was found that AGMA1 surface-adsorbed on cell culturing coverslips exhibits excellent properties as adhesion and proliferation promoter of primary brain cells such as microglia, as well as of hippocampal neurons and astrocytes. Microglia cells cultured on AGMA1-coated coverslips substrate displayed the typical resting, ramified morphology of those cultured on poly-L-lysine and poly-L-ornithine, employed as reference substrates. Mixed cultures of primary astrocytes and neuronal cells grown on AGMA1- and poly-L-lysine coated coverslips were morphologically undistinguishable. On both substrates, neurons differentiated axon and dendrites and eventually established perfectly functional synaptic contacts. Quantitative immunocytochemical staining revealed no difference between AGMA1 and poly-L-lysine. Electrophysiological experiments allowed recording neuron spontaneous activity on AGMA1. In addition, cell cultures on both AGMA1 and PLL displayed comparable excitatory and inhibitory neurotransmission, demonstrating that the synaptic contacts formed were fully functional.

Pharmacology on microfluidics: multimodal analysis for studying cell-cell interaction.

Understanding the mechanisms of cell-cell interaction is a key unanswered question in modern pharmacology, given crosstalk defects are at the basis of many pathologies. Microfluidics represents a valuable tool for analyzing intercellular communication mediated by transmission of soluble signals, as occurring for example between neurons and glial cells in neuroinflammation, or between tumor and surrounding cells in cancer. However, the use of microfluidics for studying cell behavior still encompasses many technical and biological challenges. In this review, a state of the art of successes, potentials and limitations of microfluidics applied to key biological questions in modern pharmacology is analyzed and commented.

Mesenchymal stem cell differentiation on electrochemically modified titanium: an optimized approach for biomedical applications.

PURPOSE: To speed up the osteointegration process, surface-treated titanium has been widely used in dental and orthopedic applications. The present work describes a new silicon-based anodic spark deposition (ASD) treatment and investigates the properties of the surfaces obtained, focusing on their capability to modulate the osteogenic differentiation potential of adult mesenchymal stem cells (MSCs). METHODS: The surfaces examined were obtained from commercially pure grade 2 titanium by a single-step ASD (SUM) eventually followed by a thermal treatment in alkali solution (SUMNa), while acid-etched titanium (AE; NextMaterials s.r.l.) was selected as a control. Their morphology, elemental composition, crystallographic structure of the Ti2O layer, wettability and topography were evaluated by scanning electron microscopy, energy-dispersive X-ray spectroscopy, thin-film X-ray diffraction, contact angle measurements and laser profilometry, respectively. MSCs' response to surface properties was assessed by examining cell morphology and viability by scanning electron microscopy and Alamar Blue assay®, while their osteogenic differentiation potential was investigated by evaluating the levels of the enzyme alkaline phosphatase (ALP) and the degree of calcium accumulation by Alizarin Red-S (AR-S) staining. RESULTS: The proposed ASD treatment has allowed the obtaining of surfaces with round-shaped micrometric pores, enriched in calcium, phosphorus and silicon and significantly more wettable than controls; furthermore, the treatment has been shown to promote MSC proliferation and the degree of in vitro mineralization. CONCLUSIONS: The described ASD treatment may be an effective technique to modify the surface cues of titanium implants, aiming at enhancing the conveying of osteoprogenitor cells and their functional differentiation in bone cells.

The expression of GHS-R in primary neurons is dependent upon maturation stage and regional localization.

Ghrelin is a hormone with a crucial role in the regulation of appetite, regulation of inflammation, glucose metabolism and cell proliferation. In the brain ghrelin neurons are located in the cortex (sensorimotor area, cingular gyrus), and the fibres of ghrelin neurons in hypothalamus project directly to the dorsal vagal complex (DVC). Ghrelin binds the growth hormone secretagogue receptor (GHS-R) a G-protein-coupled receptor with a widespread tissue distribution, indeed these receptors are localized both in nonnervous, organs/tissues (i.e. adipose tissue, myocardium, adrenals, gonads, lung, liver, arteries, stomach, pancreas, thyroid, and kidney) as well as in central nervous system (CNS) and higher levels of expression in the pituitary gland and the hypothalamus and lower levels of expression in other organs, including brain. A GHS-R specific monoclonal antibody has been developed and characterized and through it we demonstrate that GHS-R is expressed in primary neurons and that its expression is dependent upon their developmental stage and shows differences according to the brain region involved, with a more pronounced expression in hippocampal rather than cortical neurons. A characterization of GHS-R within the central nervous system is of extreme importance in order to gain insights on its role in the modulation of neurodegenerative events such as Alzheimer's disease.

A simple method to generate adipose stem cell-derived neurons for screening purposes.

Strategies involved in mesenchymal stem cell (MSC) differentiation toward neuronal cells for screening purposes are characterized by quality and quantity issues. Differentiated cells are often scarce with respect to starting undifferentiated population, and the differentiation process is usually quite long, with high risk of contamination and low yield efficiency. Here, we describe a novel simple method to induce direct differentiation of MSCs into neuronal cells, without neurosphere formation. Differentiated cells are characterized by clear morphological changes, expression of neuronal specific markers, showing functional response to depolarizing stimuli and electrophysiological properties similar to those of developing neurons. The method described here represents a valuable tool for future strategies aimed at personalized screening of therapeutic agents in vitro.

Overflow microfluidic networks: application to the biochemical analysis of brain cell interactions in complex neuroinflammatory scenarios.

Neuroinflammation plays a central role in neurodegenerative diseases and involves a large number of interactions between different brain cell types. Unraveling the complexity of cell-cell interaction in neuroinflammation is crucial for both clarifying the molecular mechanisms involved and increasing efficacy in drug development. Here, we provide a versatile analytical method for specifically addressing cell-to-cell communication, using primary brain cells, a microfluidic device, and a multiparametric readout approach. Different cell types are plated in separate chambers of a microfluidic network so that culturing conditions can be independently controlled and single cell types can be selectively primed with different stimuli. When chambers are microfluidically connected, the specific contribution of each cell type can be finely monitored by analyzing morphology, vitality, calcium dynamics, and electrophysiology parameters. We exemplify this approach by examining the role of astrocytes derived from two different brain regions (cortex and hippocampus) on neuronal viability in two types of neuroinflammatory insults, namely, metabolic stress and exposure to amyloid ß fibrils, and demonstrate regional differences in glial control of neuronal physiopathology. In particular, we show that during metabolic stress, cortical but not hippocampal astrocytes play a neuroprotective role; also, in an exacerbated inflammatory scenario consisting in the exposure to Aß + IL-1ß, hippocampal but not cortical astrocytes play a detrimental role on neurons. Aside from bringing novel insights into the glial role in neuroinflammation, the method presented here represents a promising tool for addressing a wide range of biological and biochemical phenomena, characterized by a complex interaction of multiple cell types.

Expression of CD20 reveals a new store-operated calcium entry modulator in skeletal muscle.

Among the scarce available data about the biological role of the membrane protein CD20, there is some evidence that this protein functions as a store-operated Ca(2+) channel and/or regulates transmembrane Ca(2+) trafficking. Recent findings indicate that store-operated Ca(2+) entry (SOCE) plays a central role in skeletal muscle function and development, but there remain a number of unresolved issues relating to SOCE modulation in this tissue. Here we describe CD20 expression in skeletal muscle, verifying its membrane localization in myoblasts and adult muscle fibers. Additionally, we show that inhibition of CD20 through antibody binding or gene silencing resulted in specific impairment of SOCE in C2C12 myoblasts. Our results provide novel insights into the CD20 expression pattern, and suggest that functional CD20 is required for SOCE to consistently occur in C2C12 myoblasts. These findings may contribute to future identification of mechanisms and molecules involved in the fine regulation of store-operated Ca(2+) entry in skeletal muscle.

Microglial microvesicle secretion and intercellular signaling.

Microvesicles (MVs) are released from almost all cell brain types into the microenvironment and are emerging as a novel way of cell-to-cell communication. This review focuses on MVs discharged by microglial cells, the brain resident myeloid cells, which comprise ∼10-12% of brain population. We summarize first evidence indicating that MV shedding is a process activated by the ATP receptor P2X(7) and that shed MVs represent a secretory pathway for the inflammatory cytokine IL-ß. We then discuss subsequent findings which clarify how IL-1 ß can be locally processed and released from MVs into the extracellular environment. In addition, we describe the current understanding about the mechanism of P2X(7)-dependent MV formation and membrane abscission, which, by involving sphingomyelinase activity and ceramide formation, may share similarities with exosome biogenesis. Finally we report our recent results which show that microglia-derived MVs can stimulate neuronal activity and participate to the propagation of inflammatory signals, and suggest new areas for future investigation.

Hydrogel for cell housing in the brain and in the spinal cord.

PURPOSE: Neurons in the adult mammalian central nervous system do not proliferate or renew themselves and consequently strong interest in cell replacement therapies to repair brain and spinal cord damages has emerged in the last decade. METHODS: An injectable resorbable hydrogel with a controlled nanostructure, specifically designed for neural cell housing, was developed together with a new protocol for building three-dimensional (3D) biohybrid cell/hydrogel constructs: cells are housed within the polymeric matrix which is directly built with a specific cell culture media. This matrix was tested with standard glial populations, primary astrocytes and mesenchymal stem cells. RESULTS: Physico-chemical characterization of the hydrogel matrix confirmed a 2- week (+ 2 days) stability before massive degradation; mean mesh size of about 5 nm and thixotropic behavior with transition yield stress at 60+5 Pa. Cell survival within the hydrogel resulted in about 55 ± 5% (minimum value) survivals, data also confirmed by optical assessments. Cell viability also remained high after extraction from the gel, indicating survival to inclusion latency period. CONCLUSIONS: Since the intimate structure of the gel mimics extracellular matrix cells as would be expected to be found in an in vivo context, this polymeric formulation is a promising base for building 3D constructs for neural cell housing, in which cells are embedded and kept alive directly from the time of polycondensation.

Overflow microfluidic networks for open and closed cell cultures on chip.

Microfluidics have a huge potential in biomedical research, in particular for studying interactions among cell populations that are involved in complex diseases. Here, we present "overflow" microfluidic networks (oMFNs) for depositing, culturing, and studying cell populations, which are plated in a few microliters of cell suspensions in one or several open cell chambers inside the chip and subsequently cultured for several days in vitro (DIV). After the cells have developed their phenotype, the oMFN is closed with a lid bearing microfluidic connections. The salient features of the chips are (1) overflow zones around the cell chambers for drawing excess liquid by capillarity from the chamber during sealing the oMFN with the lid, (2) flow paths from peripheral pumps to cell chambers and between cell chambers for interactive flow control, (3) transparent cell chambers coated with cell adhesion molecules, and (4) the possibility to remove the lid for staining and visualizing the cells after, for example, fixation. Here, we use a two-chamber oMFN to show the activation of purinergic receptors in microglia grown in one chamber, upon release of adenosine triphosphate (ATP) from astrocytes that are grown in another chamber and challenged with glutamate. These data validate oMFNs as being particularly relevant for studying primary cells and dissecting the specific intercellular pathways involved in neurodegenerative and neuroinflammatory brain diseases.

A microfluidic device for depositing and addressing two cell populations with intercellular population communication capability.

We present a method for depositing cells in the microchambers of a sealed microfluidic device and establishing flow across the chambers independently and serially. The device comprises a transparent poly(dimethylsiloxane) (PDMS) microfluidic network (MFN) having 2 cell chambers with a volume of 0.49 microL, 6 microchannels for servicing the chambers, and 1 microchannel linking both chambers. The MFN is sealed with a Si chip having 6 vias and ports that can be left open or connected to high-precision pumps. Liquids are drawn through each chamber in parallel or sequentially at flow rates from 0.1 to 10 microL min(-1). Plugs of liquid as small as 0.5 microL can be passed in one chamber within 5 s to 5 min. Plugs of liquid can also be introduced into a chamber for residence times of up to 30 min. By injecting different liquids into 3 ports, 3 adjacent laminar streams of liquid can be drawn inside one chamber with lateral concentration gradients between the streams ranging from 20 to 500 microm. The flexibility of this device for depositing cells and exposing them to liquids in parallel or serially is illustrated by depositing two types of cells, murine N9 microglia and human SH-S5Y5 neuroblastoma. Microfluidic communication between the chambers is illustrated by stimulating N9 microglia using ATP to induce these cells to release plasma membrane vesicles. The vesicles are drawn through the second chamber containing neuroblastoma and collected in a port of the device for off-chip analysis using confocal fluorescence microscopy. Cells in the MFN can also be fixed using a solution of formaldehyde for further analysis after disassembly of the MFN and Si lid. This microfluidic device offers a simple, flexible, and powerful method for depositing two cell populations in separate chambers and may help investigating pathways between the cells populations.

Controlled deposition of cells in sealed microfluidics using flow velocity boundaries.

We present a method for depositing cells in a sealed microfluidic device. The device consists of a poly(dimethylsiloxane) (PDMS) microfluidic network (MFN) sealed with a Si chip. The Si chip has vias and ports that are connected to high-precision motorized pumps. The surfaces of the PDMS MFN are homogeneously coated with fibronectin cell adhesion molecules (CAMs). Flow velocity boundaries are created between vicinal microfluidic structures to prevent or permit deposition of cells in specific regions of the MFN. In narrow flow paths, cells experience a wall shear stress from the fast-moving liquid that overcomes the initial adhesion of the cells with CAMs. Conversely, cells can adhere to CAMs in larger flow paths such as cell chambers inside which the velocity of the liquid and the shear stress are reduced. Interactively changing pumping rates makes the critical velocity (the velocity at which cells deposit in the chamber but not elsewhere) easy to find. The transparent PDMS MFN allows both real-time visualization of the deposition process and cellular assays. We illustrate this method using N9 mouse microglia cells. In one experiment, approximately 75 microglia are deposited per min in a approximately 0.5 microL chamber. The deposited cells remain viable, as assessed from staining and biofunctional assays. This method is simple, reliable, fast, and flexible, and therefore is an attractive technique for depositing cells in microfluidic systems for numerous applications.

The surface-exposed chaperone, Hsp60, is an agonist of the microglial TREM2 receptor.

Triggering receptor expressed in myeloid (TREM) cells 2, a receptor expressed by myeloid cells, osteoclasts and microglia, is known to play a protective role in bones and brain. Mutations of the receptor (or of its coupling protein, DAP12) sustain in fact a genetic disease affecting the two organs, the polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy (PLOSL or Nasu-Hakola disease). So far, specific agonist(s) of TREM2 have not been identified and its (their) transduction mechanisms are largely unknown. Heat shock protein 60 (Hsp60) is a mitochondrial chaperone that can also be harboured at the cell surface. By using constructs including the extracellular domain of TREM2 and the Fc domain of IgGs we have identified Hsp60 as the only TREM2-binding protein exposed at the surface of neuroblastoma N2A cells and astrocytes, and lacking in U373 astrocytoma. Treatment with Hsp60 was found to stimulate the best known TREM2-dependent process, phagocytosis, however, only in the microglial N9 cells rich in the receptor. Upon TREM2 down-regulation, the Hsp60-induced stimulation of N9 phagocytosis was greatly attenuated. Hsp60 is also released by many cell types, segregated within exosomes or shedding vesicles which might then undergo dissolution. However, the affinity of its binding (K(d) = 3.8 microM) might be too low for the soluble chaperone released from the vesicles to the extracellular space to induce a significant activation of TREM2. It might in contrast be appropriate for the binding of TREM2 to Hsp60 exposed at the surface of cells closely interacting with microglia. The ensuing stimulation of phagocytosis could play protective effects on the brain.

Different properties of P2X(7) receptor in hippocampal and cortical astrocytes.

P2X(7) receptor is a ligand-gated ion channel, which can induce the opening of large membrane pores. Here, we provide evidence that the receptor induces pore formation in astrocytes cultured from cortex, but not from the hippocampus. Furthermore, P2X(7) receptor activation promptly induces p38 mitogen-activated protein kinase (MAPK) phosphorylation in cortical but not in hippocampal astrocytes. Given the role of p38 MAPK activation in pore opening, these data suggest that defective coupling of the receptor to the enzyme could occur in hippocampal cultures. The different capabilities of the receptor to open membrane pores cause relevant functional consequences. Upon pore formation, caspase-1 is activated and pro-IL1-beta is cleaved and released extracellularly. The receptor stimulation does not result in interleukin-1beta secretion from hippocampal astrocytes, although the pro-cytokine is present in the cytosol of lipopolysaccharide-primed cultures. These results open the possibility that activation of P2X(7) receptors differently influences the neuroinflammatory processes in distinct brain regions.

Acid sphingomyelinase activity triggers microparticle release from glial cells.

We have earlier shown that microglia, the immune cells of the CNS, release microparticles from cell plasma membrane after ATP stimulation. These vesicles contain and release IL-1beta, a crucial cytokine in CNS inflammatory events. In this study, we show that microparticles are also released by astrocytes and we get insights into the mechanism of their shedding. We show that, on activation of the ATP receptor P2X7, microparticle shedding is associated with rapid activation of acid sphingomyelinase, which moves to plasma membrane outer leaflet. ATP-induced shedding and IL-1beta release are markedly reduced by the inhibition of acid sphingomyelinase, and completely blocked in glial cultures from acid sphingomyelinase knockout mice. We also show that p38 MAPK cascade is relevant for the whole process, as specific kinase inhibitors strongly reduce acid sphingomyelinase activation, microparticle shedding and IL-1beta release. Our results represent the first demonstration that activation of acid sphingomyelinase is necessary and sufficient for microparticle release from glial cells and define key molecular effectors of microparticle formation and IL-1beta release, thus, opening new strategies for the treatment of neuroinflammatory diseases.

A role for P2X7 in microglial proliferation.

Microglia, glial cells with an immunocompetent role in the CNS, react to stimuli from the surrounding environment with alterations of their phenotypic response. Amongst other activating signals, the endotoxin lipopolysaccharide (LPS) is widely used as a tool to mimic bacterial infection in the CNS. LPS-activated microglia undergo dramatic changes in cell morphology/activity; in particular, they stop proliferating and differentiate from resting to effector cells. Activated microglia also show modifications of purinoreceptor signalling with a significant decrease in P2X(7) expression. In this study, we demonstrate that the down-regulation of the P2X(7) receptor in activated microglia may play an important role in the antiproliferative effect of LPS. Indeed, chronic blockade of the P2X(7) receptor by antagonists (oxidized ATP, KN62 and Brilliant Blue G), or treatment with the ATP-hydrolase apyrase, severely decreases microglial proliferation, down-regulation of P2X(7) receptor expression by small RNA interference (siRNA) decreases cell proliferation, and the proliferation of P2X(7)-deficient N9 clones and primary microglia, in which P2X(7) expression is down-regulated by siRNA, is unaffected by either LPS or P2X(7) antagonists. Furthermore, flow cytometric analysis indicates that exposure to oxidized ATP or treatment with LPS reversibly decreases cell cycle progression, without increasing the percentage of apoptotic cells. Overall, our data show that the P2X(7) receptor plays an important role in controlling microglial proliferation by supporting cell cycle progression.