Tunable Microfibers Suppress Fibrotic Encapsulation via Inhibition of TGFβ Signaling

Fibrotic encapsulation limits the efficacy and lifetime of implantable biomedical devices. Microtopography has shown promise in the regulation of myofibroblast differentiation, a key driver of fibrotic encapsulation. However, existing studies have not systematically isolated the requisite geometric parameters for suppression of myofibroblast differentiation via microtopography, and there has not been in vivo validation of this technology to date. To address these issues, a novel lamination method was developed to afford more control over topography dimensions. Specifically, in this study we focus on fiber length and its effect on myofibroblast differentiation. Fibroblasts cultured on films with microfibers exceeding 16 μm in length lost the characteristic morphology associated with myofibroblast differentiation, while shorter microfibers of 6 μm length failed to produce this phenotype. This increase in length corresponded to a 50% decrease in fiber stiffness, which acts as a mechanical cue to influence myofibroblast differentiation. Longer microfiber films suppressed expression of myofibroblast specific genes (αSMA, Col1α2, and Col3α1) and TGFβ signaling components (TGFβ1 ligand, TGFβ receptor II, and Smad3). 16 μm long microfiber films implanted subcutaneously in a mouse wound-healing model generated a substantially thinner fibrotic capsule and less deposition of collagen in the wound bed. Together, these results identify a critical feature length threshold for microscale topography-mediated repression of fibrotic encapsulation. This study also demonstrates a simple and powerful strategy to improve surface biocompatibility and reduce fibrotic encapsulation around implanted materials

A Critical Role for Myosin IIB in Dendritic Spine Morphology and Synaptic Function

Dendritic spines show rapid motility and plastic morphology, which may mediate information storage in the brain. It is presently believed that polymerization/depolymerization of actin is the primary determinant of spine motility and morphogenesis. Here, we show that myosin IIB, a molecular motor that binds and contracts actin filaments, is essential for normal spine morphology and dynamics and represents a distinct biophysical pathway to control spine size and shape. Myosin IIB is enriched in the postsynaptic density (PSD) of neurons. Pharmacologic or genetic inhibition of myosin IIB alters protrusive motility of spines, destabilizes their classical mushroom-head morphology, and impairs excitatory synaptic transmission. Thus, the structure and function of spines is regulated by an actin-based motor in addition to the polymerization state of actin

Rule of Myosin two and Rap2 in synaptic structure and function

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2008.Includes bibliographical references.Synapses, the connections between neurons, exhibit both structural and functional plasticity, and these changes could underlie learning and memory. Two synaptic phenomena that have been studied extensively are Hebbian plasticity and changes in dendritic spine morphology. Recent proteomics studies have uncovered many proteins that reside in the synapse and could play critical roles in these processes. Among these are the molecular motor myosin II and the Ras family GTPase Rap2. Myosin II can move and contract actin filaments in non-neuronal cells, and it represents a novel way to alter spine structure, which is classically thought to occur through actin polymerization and depolymerization. Rap GTPases are the closest relatives to Ras, which is well established as a positive regulator of spines and synaptic transmission. In vitro evidence indicates that Raps could act antagonistically to Ras in neurons, inhibiting spine growth and synaptic strength. To study myosin II's role in dendritic spine morphology and synaptic function, we inhibited myosin II function either pharmacologically or genetically in dissociated hippocampal neurons. Knockdown of myosin II by RNA interference resulted in loss of mature mushroom-shaped spines, and an increase in thin, filopodia-like structures. Treatment with blebbistatin, a chemical inhibitor of myosin II, phenocopied this result. Live imaging revealed that mature spines unravel into filopodia within tens of minutes of myosin II inhibition by blebbistatin. Furthermore, blebbistatin treatment led to decreases in levels of the glutamatergic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), as well as impairment of synaptic transmission.(cont.) To study Rap2 neuronal function in vivo, we created transgenic mice that expressed constitutively active Rap2 (Rap2V12) in postnatal forebrain. Consistent with an inhibitory role for Rap at synapses, Rap2V12 mice exhibited reduced levels of phospho-ERK (pERK) and a reduction in spine density and length. Behaviorally these mice were hyperactive and showed impairments in spatial learning. In addition, Rap2V12 mice showed normal acquisition of fear memories, but were defective in the extinction of contextual fear. Fear extinction has been associated with several psychiatric disorders, including posttraumatic stress disorder (PTSD), and Rap2V12 mice might offer a potential therapeutic model for such diseases.by Jubin Wonsun Ryu.Ph.D

Constitutively Active Rap2 Transgenic Mice Display Fewer Dendritic Spines, Reduced Extracellular Signal-Regulated Kinase Signaling, Enhanced Long-Term Depression, and Impaired Spatial Learning and Fear Extinction

Within the Ras superfamily of GTPases, Rap1 and Rap2 are the closest homologs to Ras. In non-neural cells, Rap signaling can antagonize Ras signaling. In neurons, Rap also seems to oppose Ras in terms of synaptic function. Whereas Ras is critical for long-term potentiation (LTP), Rap1 has been shown to be required for long-term depression (LTD), and Rap2 has been implicated in depotentiation. Moreover, active Rap1 and Rap2 cause loss of surface AMPA receptors and reduced miniature EPSC amplitude and frequency in cultured neurons. The role of Rap signaling in vivo, however, remains poorly understood. To study the function of Rap2 in the brain and in behavior, we created transgenic mice expressing either constitutively active (Rap2V12) or dominant-negative (Rap2N17) mutants of Rap2 in postnatal forebrain. Multiple lines of Rap2N17 mice showed only weak expression of the transgenic protein, and no phenotype was observed. Rap2V12 mice displayed fewer and shorter dendritic spines in CA1 hippocampal neurons, and enhanced LTD at CA3–CA1 synapses. Behaviorally, Rap2V12 mice showed impaired spatial learning and defective extinction of contextual fear, which correlated with reduced basal phosphorylation of extracellular signal-regulated kinase (ERK) and blunted activation of ERK during fear extinction training. Our data support the idea that Rap2 opposes Ras–ERK signaling in the brain, thereby inhibiting dendritic spine development/maintenance, promoting synaptic depression rather than LTP, and impairing learning. The findings also implicate Rap2 signaling in fear extinction mechanisms, which are thought to be aberrant in anxiety disorders and posttraumatic stress disorder

Specific Trans-Synaptic Interaction with Inhibitory Interneuronal Neurexin Underlies Differential Ability of Neuroligins to Induce Functional Inhibitory Synapses

Synaptic transmission depends on the matching and alignment of presynaptically released transmitters and postsynaptic neurotransmitter receptors. Neuroligin (NL) and Neurexin (Nrxn) proteins are trans-synaptic adhesion molecules that are important in validation and maturation of specific synapses. NL isoforms NL1 and NL2 have specific functional roles in excitatory and inhibitory synapses, respectively, but the molecular basis behind this distinction is still unclear. We show here that the extracellular domain of NL2 confers its unique ability to enhance inhibitory synaptic function when overexpressed in rat hippocampal pyramidal neurons, whereas NL1 normally only promotes excitatory synapses. This specificity is conferred by presynaptic Nrxn isoforms, as NL1 can also induce functional inhibitory synapse connections when the presynaptic interneurons ectopically express an Nrxn isoform that binds to NL1. Our results indicate that trans-synaptic interaction with differentially expressed presynaptic Nrxns underlies the distinct functions of NL1 and NL2, and is sufficient to induce functional inhibitory synapse formation.University of Massachusetts (System) {Start-up Funds)Whitehall Foundatio

Nanotopography Facilitates <i>in Vivo</i> Transdermal Delivery of High Molecular Weight Therapeutics through an Integrin-Dependent Mechanism

Transdermal delivery of therapeutics is restricted by narrow limitations on size and hydrophobicity. Nanotopography has been shown to significantly enhance high molecular weight paracellular transport <i>in vitro</i>. Herein, we demonstrate for the first time that nanotopography applied to microneedles significantly enhances transdermal delivery of etanercept, a 150 kD therapeutic, in both rats and rabbits. We further show that this effect is mediated by remodeling of the tight junction proteins initiated via integrin binding to the nanotopography, followed by phosphorylation of myosin light chain (MLC) and activation of the actomyosin complex, which in turn increase paracellular permeability