Background & AimsThe concept of enteric glia as regulators of intestinal homeostasis is slowly gaining acceptance as a central concept in neurogastroenterology. Yet how glia contribute to intestinal disease is still poorly understood. Purines generated during inflammation drive enteric neuron death by activating neuronal P2X7 purine receptors (P2X7R); triggering adenosine triphosphate (ATP) release via neuronal pannexin-1 channels that subsequently recruits intracellular calcium ([Ca2+]i) in surrounding enteric glia. We tested the hypothesis that the activation of enteric glia contributes to neuron death during inflammation.MethodsWe studied neuroinflammation in vivo using the 2,4-dinitrobenzene sulfonic acid model of colitis and in situ using whole-mount preparations of human and mouse intestine. Transgenic mice with a targeted deletion of glial connexin-43 (Cx43) [GFAP::CreERT2+/−/Cx43f/f] were used to specifically disrupt glial signaling pathways. Mice deficient in inducible nitric oxide (NO) synthase (iNOS−/−) were used to study NO production. Protein expression and oxidative stress were measured using immunohistochemistry and in situ Ca2+ and NO imaging were used to monitor glial [Ca2+]i and [NO]i.ResultsPurinergic activation of enteric glia drove [Ca2+]i responses and enteric neuron death through a Cx43-dependent mechanism. Neurotoxic Cx43 activity, driven by NO production from glial iNOS, was required for neuron death. Glial Cx43 opening liberated ATP and Cx43-dependent ATP release was potentiated by NO.ConclusionsOur results show that the activation of glial cells in the context of neuroinflammation kills enteric neurons. Mediators of inflammation that include ATP and NO activate neurotoxic pathways that converge on glial Cx43 hemichannels. The glial response to inflammatory mediators might contribute to the development of motility disorders.

Cardiovascular disease is a leading cause of death worldwide and accounts for greater than 17.3 million deaths per year, with an estimated increase in incidence to 23.6 million by 2030 1. Cardiovascular death represents 31% of all global deaths 2 - with stroke, heart attack, and ruptured aneurysms predominantly contributing to these high mortality rates. A key risk factor for cardiovascular disease is hypertension. Although treatment or reduction in hypertension can prevent the onset of cardiovascular events, existing therapies are only partially effective. A key pathological hallmark of hypertension is increased peripheral vascular resistance due to structural and functional changes in large (conductive) and small (resistance) arteries. In this review, we discuss the clinical implications of vascular remodeling, compare the differences between vascular smooth muscle cell (VSMC) remodeling in conductive and resistance arteries, discuss the genetic factors associated with VSMC function in hypertensive patients, and provide a prospective assessment of current and future research and pharmacological targets for the treatment of hypertension.

• The KKO-inhibition ELISA and DT40-luciferase tests are novel laboratory assays for HIT.• They showed better discrimination than 2 commercial immunoassays and may improve the specificity and feasibility of HIT laboratory testing.Laboratory testing for heparin-induced thrombocytopenia (HIT) has important shortcomings. Immunoassays fail to discriminate platelet-activating from nonpathogenic antibodies. Specific functional assays are impracticable due to the need for platelets and radioisotope. We describe 2 assays that may overcome these limitations. The KKOinhibition test (KKO-I) measures the effect of plasma on binding of the HIT-like monoclonal antibody KKO to platelet factor 4 (PF4)/heparin. DT40-luciferase (DT40-luc) is a functional test comprised of a B-cell line expressing FcgRIIa coupled to a luciferase reporter. We compared these assays to polyspecific and immunoglobulin (Ig)G-specific PF4/heparin enzyme-linked immunosorbent assays (ELISAs) in samples from 58 patients with suspected HIT and circulating anti-PF4/heparin antibodies. HIT was defined as a 4Ts score ‡ 4 and positive 14 C-serotonin release assay. HIT-positive plasma demonstrated greater mean inhibition of KKO binding than HIT-negative plasma (78.9% vs 26.0%; P < .0001) and induced greater luciferase activity (3.14-fold basal vs 0.96-fold basal; P < .0001). The area under the receiver-operating characteristic curve was greater for KKO-I (0.93) than for the polyspecific (0.82; P 5 .020) and IgG-specific ELISA (0.76; P 5 .0044) and for DT40-luc (0.89) than for the IgG-specific ELISA (P 5 .046). KKO-I and DT40-luc showed better discrimination than 2 commercially available immunoassays, are simple to perform, and hold promise for improving the specificity and feasibility of HIT laboratory testing. (Blood. 2013;121(18):3727-3732) IntroductionHeparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by platelet-, monocyte-, and endothelial cell-activating antibodies that preferentially recognize ultra-large complexes of platelet factor 4 (PF4) and heparin.1,2 Laboratory testing plays a key role in the diagnosis of HIT but is associated with important shortcomings. 3 Immunoassays such as the PF4/heparin enzyme-linked immunosorbent assay (ELISA) frequently yield false-positive results due to their inability to discriminate cell-activating and potentially pathogenic antibodies from their nonpathogenic counterparts. Functional tests such as the 14 C-serotonin release assay (SRA) are more specific but are unfeasible for most clinical laboratories due to the requirement for radioisotope and fresh platelets from reactive donors. 3KKO is a murine monoclonal anti-PF4/heparin immunoglobulin (Ig)G that induces a HIT-like thrombotic thrombocytopenic disorder in a mouse model. RTO, an isotype-matched anti-PF4 antibody not dependent on heparin for comparable binding in an ELISA, does not activate platelets in vitro or cause thrombocytopenia in vivo. 4 Binding of KKO (but not RTO) to immobilized PF4/heparin is inhibited by human HIT plasma but not...

Hydrophobic and aggregation-prone, membrane proteins often prove too insoluble for conventional in vitro biochemical studies. To engineer soluble variants of human caveolin-1, a phage-displayed library of caveolin variants targeted the hydrophobic intra-membrane domain with substitutions to charged residues. Anti-selections for insolubility removed hydrophobic variants, and positive selections for binding to the known caveolin ligand HIV gp41 isolated functional, folded variants. Assays with several caveolin binding partners demonstrated the successful folding and functionality by a solubilized, full-length caveolin variant selected from the library. This caveolin variant allowed assay of the direct interaction between caveolin and cavin, an experiment requiring purified proteins. Clustered along one face of a putative helix, the solubilizing mutations suggest a structural model for the intra-membrane domain of caveolin. The approach provides a potentially general method for solubilization and engineering of membraneassociated proteins by phage display.Membrane-associated proteins contribute essential functions to the cell, including energy generation, communication, transportation, and sensing. The hydrophobic regions of membrane proteins force them into the lipophilic environment of membranes, and such proteins typically aggregate upon removal from the membrane (1). The later property makes structural and biophysical studies of membrane-associated proteins extremely challenging. Thus, in contrast to the thousands of soluble protein structures deposited in the Protein Data Bank, only about two hundred membrane protein structures have been solved to date (2). The solubilization of functional membrane proteins through mutations to the transmembrane domain remains relatively unexplored as a solution to this challenge.Largely conserved amongst higher eukaryotes, human caveolin-1 is one of three members of the caveolin family (3,4). Most frequently expressed in adipocytes and endothelial cells, caveolin-1 (hereafter referred to as caveolin) plays key roles in signal transduction and the initiation of endocytosis (5). Although involved in a variety of diseases including Alzheimer's disease, muscular dystrophy, cancer, diabetes, obesity, and asthma (6), the structure of the 178-residue, membrane-associated caveolin remains undetermined.Caveolin, associated with detergent insoluble lipid rafts, is a monotopic membrane protein (6). Thus, the region between residues 102 and 134 inserts into one leaflet of the plasma NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript membrane, and N-and C-termini remain in the cytoplasm. Oligomerized caveolin in complex with additional proteins causes invaginations in the plasma membrane, termed caveolae (7). Signaling molecules bind to caveolin, and cluster in high concentrations at caveolae (8). The caveolin scaffolding domain (CSD), comprised of caveolin residues 81-101 binds to and inhibits endothelial nitric oxide synthase (eNOS) and protein kinase A (...

Enteric glia play an important neuroprotective role in the enteric nervous system (ENS) by producing neuroprotective compounds such as the antioxidant reduced glutathione (GSH). The specific cellular pathways that regulate glial production of GSH and how these pathways are altered during, or contribute to, neuroinflammation in situ and in vivo are not fully understood. We investigated this issue using immunohistochemistry to localize GSH synthesis enzymes within the myenteric plexus and tested how the inhibition of GSH synthesis with the selective inhibitor l-buthionine sulfoximine impacts neuronal survival and inflammation. Both enteric glia and neurons express the cellular machinery necessary for GSH synthesis. Furthermore, glial GSH synthesis is necessary for neuronal survival in isolated preparations of myenteric plexus. In vivo depletion of GSH does not induce colitis but alters myenteric plexus neuronal phenotype and survival. Importantly, global depletion of glutathione is protective against some macroscopic and microscopic measures of colonic inflammation. Together, our data highlight the heterogeneous roles of GSH in the myenteric plexus of the ENS and during gastrointestinal inflammation. NEW & NOTEWORTHY Our results show that both enteric glia and neurons express the cellular machinery necessary for glutathione (GSH) synthesis and that glial GSH synthesis is necessary for neuronal survival in isolated enteric nervous system (ENS) preparations. In vivo depletion of GSH with the selective inhibitor l-buthionine sulfoximine is not sufficient to induce inflammation but does alter neuronal neurochemical composition and survival. Together, our data highlight novel heterogeneous roles for GSH in the ENS and during gastrointestinal inflammation.