A multilayered scaffold for regeneration of smooth muscle and connective tissue layers

Tissue regeneration often requires recruitment of different cell types and rebuilding of two or more tissue layers to restore function. Here, we describe the creation of a novel multilayered scaffold with distinct fiber organizations—aligned to unaligned and dense to porous—to template common architectures found in adjacent tissue layers. Electrospun scaffolds were fabricated using a biodegradable, tyrosine-derived terpolymer, yielding densely-packed, aligned fibers that transition into randomly-oriented fibers of increasing diameter and porosity. We demonstrate that differently-oriented scaffold fibers direct cell and extracellular matrix (ECM) organization, and that scaffold fibers and ECM protein networks are maintained after decellularization. Smooth muscle and connective tissue layers are frequently adjacent in vivo; we show that within a single scaffold, the architecture supports alignment of contractile smooth muscle cells and deposition by fibroblasts of a meshwork of ECM fibrils. We rolled a flat scaffold into a tubular construct and, after culture, showed cell viability, orientation, and tissue-specific protein expression in the tube were similar to the flat-sheet scaffold. This scaffold design not only has translational potential for reparation of flat and tubular tissue layers but can also be customized for alternative applications by introducing two or more cell types in different combinations

Augmenting the antinociceptive effects of nicotinic acetylcholine receptor activity through lynx1 modulation.

Neuronal nicotinic acetylcholine receptors (nAChRs) of the cholinergic system have been linked to antinociception, and therefore could be an alternative target for pain alleviation. nAChR activity has been shown to be regulated by the nicotinic modulator, lynx1, which forms stable complexes with nAChRs and has a negative allosteric action on their function. The objective in this study was to investigate the contribution of lynx1 to nicotine-mediated antinociception. Lynx1 contribution was investigated by mRNA expression analysis and electrophysiological responses to nicotine in the dorsal raphe nucleus (DRN), a part of the pain signaling pathway. In vivo antinociception was investigated in a test of nociception, the hot-plate analgesia assay with behavioral pharmacology. Lynx1/α4β2 nAChR interactions were investigated using molecular dynamics computational modeling. Nicotine evoked responses in serotonergic and GABAergic neurons in the DRN are augmented in slices lacking lynx1 (lynx1KO). The antinociceptive effect of nicotine and epibatidine is enhanced in lynx1KO mice and blocked by mecamylamine and DHβE. Computer simulations predict preferential binding affinity of lynx1 to the α:α interface that exists in the stoichiometry of the low sensitivity (α4)3(β2)2 nAChRs. Taken together, these data point to a role of lynx1 in mediating pain signaling in the DRN through preferential affinity to the low sensitivity α4β2 nAChRs. This study suggests that lynx1 is a possible alternative avenue for nociceptive modulation outside of opioid-based strategies

Overall architecture of lynx1 at α4:α4 nAChR interface.

<p>Molecular dynamic simulations of lynx1 binding to α4 nAChR subunits. The cell membrane is represented as a dashed line.</p

Mediation of <i>lynx1</i> through α4β2 nAChRs.

<p>(A) Antinociceptive responses in wt and <i>lynx1</i>KO mice after I.P. injection of the non-selective α4β2* nAChR agonist, epibatidine (5 μg·kg^-1) (n = 24 wt, 21 KO, p = 0.029, Student’s T-test). Mice were tested on the hot-plate 15 minutes after injection. Epibatidine-mediated antinociception is augmented in <i>lynx1</i>KO mice compared to wt mice. Data presented as mean ± SEM time. *P<0.05 compared to wt controls. wt: wild type, KO: <i>lynx1</i> knockout. (B) Antinociceptive responses in wt and <i>lynx1</i>KO mice after I.P. injection of the α4β2 nAChRs inhibitor dihydro-β-erythroidine hydrobromide (DHβE) (3.0 mg·kg^-1) and nicotine (0.5 mg·kg^-1) (nicotine treated <i>lynx1</i>KO mice (n = 8) vs. nicotine+DHβE treated <i>lynx1</i>KO mice (n = 6) using the hot-plate assay. Mice were injected with DHβE 25 minutes and nicotine 15 minutes prior to hot-plate testing. Injections of DHβE blocks the antinociceptive effect of nicotine in <i>lynx1</i>KO mice. Data indicates that lynx1 operates through the α4β2 nAChR to modulate antinociception. Data presented as mean ± SEM time. wt: wild type, KO: <i>lynx1</i> knockout. (C) Schematic of lynx1 binding to the LS stoichiometry of α4β2 nAChRs preferentially over the HS stoichiometry. α4β2 nAChR pentamers shown in the high sensitivity (HS) and low sensitivity (LS) stoichiometry, made up of (α4)₂(β2)₃ vs. (α4)₂(β2)₃ nAChRs respectively. In our model, lynx1 preferentially binds and stabilizes the LS stoichiometry.</p

<i>lynx1</i> does not influence nicotine-mediated locomotor performance or body temperature.

<p>(A) Effect of nicotine on locomotion in wt and <i>lynx1</i>KO mice after I.P. injections of nicotine concentrations 0.5 mg·kg^-1 (n = 7 wt, 8 KO), 1.0mg·kg^-1 (n = 6 wt, 6 KO), 1.5mg·kg^-1 (6 wt, 6 KO). Locomotion were examined by scoring leg movements (seconds) in the time period 15–20 minutes post injection. Injection of nicotine induce the same amount of hypolocomotion in both genotypes. Each data point presented as mean ± SEM. wt: wild type, KO: <i>lynx1</i> knockout. (B)The locomotor performance after nicotine injection (0.5 mg·kg^-1) was binned into 5 minute time windows and showed no significant effect of genotype at any time window. (C) Effect of nicotine on body temperature in wt and <i>lynx1</i>KO mice after I.P. injections of either saline (n = 11 wt, 11 KO), or nicotine concentrations 1.0 mg·kg^-1 (n = 9 wt, n = 11 KO) and 2.5 mg·kg^-1 (n = 7 wt, 7 KO). Each bar presented as mean ± SEM.</p

Establishing <i>lynx1</i> expression in brain regions associated with nociception.

<p>(A) Evidence of <i>lynx1</i> mRNA expression in the dorsal raphe nucleus via RT-PCR using <i>lynx1</i>-specific primers (expected band size of 62 bp). Expression of TPH2 (expected band size of 147 bp) validates that the isolation is in the correct region of interest. (B) Schematic of the brainstem at the level of the DRN, as a coronal plane of section (C) Expression of lynx1 protein (green) in the dorsal raphe nucleus, using anti-lynx1 pAb immunofluorescence staining [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199643#pone.0199643.ref039" target="_blank">39</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199643#pone.0199643.ref064" target="_blank">64</a>]and donkey anti-rabbit Cy2 secondary antibody, imaged at 4x magnification. (D and E) Side by side labeling of lynx1 (anti-lynx1 mAb, Alexa red), and TPH2 (anti-TPH2 pAb, Cy2, green), 10x magnification, scale bar = 200 μm. (F) Dual labeling immunofluorescence staining using anti-lynx1 mAb (red) and anti-TPH2 pAb (green), merge (yellow), 20x magnification, scale bar = 100 μm. (G) Dual labeling immunofluorescence staining using anti-lynx1 mAb (red) and anti-TPH2 pAb (green), merge (yellow), 40x magnification, scale bar = 50 μm.</p

Structural comparison between α4:α4/lynx1 and β2:β2/lynx1 complex model.

<p>(A) α4:α4/lynx1 complex model. (B) β2:β2/lynx1 complex model (C) Structural details of boxed in A. (D) Structural details of boxed in B.</p

The effect of nicotine on antinociception assessed on a hot-plate assay.

<p>(A) Antinociceptive responses in wt and <i>lynx1</i>KO mice after I.P. injections of saline (n = 8 wt, 8 KO. p = 0.899, two-way ANOVA, cohen’s D 0.13) or nicotine concentrations of 0.5 mg·kg^-1 (n = 8 wt, 18 KO. p = 0.122, two-way ANOVA, cohen’s D 1.36), 1.0mg·kg^-1 (n = 8 wt, 14 KO. p = 0.032, two-way ANOVA, cohen’s D 1.09) and 1.5mg·kg^-1 (8 wt, 8 KO. p = 0.657, two-way ANOVA, cohen’s D 0.13) using the hot-plate assay. ED₅₀ was 1.05 mg·kg^-1 for wt and 0.44 mg·kg^-1 for the lynx1KO group.</p

MicroRNA 4423 is a primate-specific regulator of airway epithelial cell differentiation and lung carcinogenesis.

Smoking is a significant risk factor for lung cancer, the leading cause of cancer-related deaths worldwide. Although microRNAs are regulators of many airway gene-expression changes induced by smoking, their role in modulating changes associated with lung cancer in these cells remains unknown. Here, we use next-generation sequencing of small RNAs in the airway to identify microRNA 4423 (miR-4423) as a primate-specific microRNA associated with lung cancer and expressed primarily in mucociliary epithelium. The endogenous expression of miR-4423 increases as bronchial epithelial cells undergo differentiation into mucociliary epithelium in vitro, and its overexpression during this process causes an increase in the number of ciliated cells. Furthermore, expression of miR-4423 is reduced in most lung tumors and in cytologically normal epithelium of the mainstem bronchus of smokers with lung cancer. In addition, ectopic expression of miR-4423 in a subset of lung cancer cell lines reduces their anchorage-independent growth and significantly decreases the size of the tumors formed in a mouse xenograft model. Consistent with these phenotypes, overexpression of miR-4423 induces a differentiated-like pattern of airway epithelium gene expression and reverses the expression of many genes that are altered in lung cancer. Together, our results indicate that miR-4423 is a regulator of airway epithelium differentiation and that the abrogation of its function contributes to lung carcinogenesis

MicroRNA 4423 is a primate-specific regulator of airway epithelial cell differentiation and lung carcinogenesis.

Smoking is a significant risk factor for lung cancer, the leading cause of cancer-related deaths worldwide. Although microRNAs are regulators of many airway gene-expression changes induced by smoking, their role in modulating changes associated with lung cancer in these cells remains unknown. Here, we use next-generation sequencing of small RNAs in the airway to identify microRNA 4423 (miR-4423) as a primate-specific microRNA associated with lung cancer and expressed primarily in mucociliary epithelium. The endogenous expression of miR-4423 increases as bronchial epithelial cells undergo differentiation into mucociliary epithelium in vitro, and its overexpression during this process causes an increase in the number of ciliated cells. Furthermore, expression of miR-4423 is reduced in most lung tumors and in cytologically normal epithelium of the mainstem bronchus of smokers with lung cancer. In addition, ectopic expression of miR-4423 in a subset of lung cancer cell lines reduces their anchorage-independent growth and significantly decreases the size of the tumors formed in a mouse xenograft model. Consistent with these phenotypes, overexpression of miR-4423 induces a differentiated-like pattern of airway epithelium gene expression and reverses the expression of many genes that are altered in lung cancer. Together, our results indicate that miR-4423 is a regulator of airway epithelium differentiation and that the abrogation of its function contributes to lung carcinogenesis