Novel Sulfated Polysaccharides Disrupt Cathelicidins, Inhibit RAGE and Reduce Cutaneous Inflammation in a Mouse Model of Rosacea

Rosacea is a common disfiguring skin disease of primarily Caucasians characterized by central erythema of the face, with telangiectatic blood vessels, papules and pustules, and can produce skin thickening, especially on the nose of men, creating rhinophyma. Rosacea can also produce dry, itchy eyes with irritation of the lids, keratitis and corneal scarring. The cause of rosacea has been proposed as over-production of the cationic cathelicidin peptide LL-37.We tested a new class of non-anticoagulant sulfated anionic polysaccharides, semi-synthetic glycosaminoglycan ethers (SAGEs) on key elements of the pathogenic pathway leading to rosacea. SAGEs were anti-inflammatory at ng/ml, including inhibition of polymorphonuclear leukocyte (PMN) proteases, P-selectin, and interaction of the receptor for advanced glycation end-products (RAGE) with four representative ligands. SAGEs bound LL-37 and inhibited interleukin-8 production induced by LL-37 in cultured human keratinocytes. When mixed with LL-37 before injection, SAGEs prevented the erythema and PMN infiltration produced by direct intradermal injection of LL-37 into mouse skin. Topical application of a 1% (w/w) SAGE emollient to overlying injected skin also reduced erythema and PMN infiltration from intradermal LL-37.Anionic polysaccharides, exemplified by SAGEs, offer potential as novel mechanism-based therapies for rosacea and by extension other LL-37-mediated and RAGE-ligand driven skin diseases

Photocrosslinkable Hyaluronan-Gelatin Hydrogels for Two-Step Bioprinting

Bioprinting by the codeposition of cells and biomaterials is constrained by the availability of printable materials. Herein we describe a novel macromonomer, a new two-step photocrosslinking strategy, and the use of a simple rapid prototyping system to print a proof-of-concept tubular construct. First, we synthesized the methacrylated ethanolamide derivative of gelatin (GE-MA). Second, partial photochemical cocrosslinking of GE-MA with methacrylated hyaluronic acid (HA-MA) gave an extrudable gel-like fluid. Third, the new HA-MA:GE-MA hydrogels were biocompatible, supporting cell attachment and proliferation of HepG2 C3A, Int-407, and NIH 3T3 cells in vitro. Moreover, hydrogels injected subcutaneously in nude mice produced no inflammatory response. Fourth, using the Fab@Home printing system, we printed a tubular tissue construct. The partially crosslinked hydrogels were extruded from a syringe into a designed base layer, and irradiated again to create a firmer structure. The computer-driven protocol was iterated to complete a cellularized tubular construct with a cell-free core and a cell-free structural halo. Cells encapsulated within this printed construct were viable in culture, and gradually remodeled the synthetic extracellular matrix environment to a naturally secreted extracellular matrix. This two-step photocrosslinkable biomaterial addresses an unmet need for printable hydrogels useful in tissue engineering

Topical SAGE reduces LL-37-induced inflammation.

<p>Balb/c mice were injected intradermally as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016658#pone-0016658-g005" target="_blank">Figure 5</a>, and treated topically with GM-1111-1 (1% w/w) in a triglyceride-based emollient. <b>A–C</b>. Gross pictures of LL-37 injected skin region: <b>A</b>, no treatment; <b>B</b>, GM-1111 treatment immediately (t = 0) after LL-37 injection; <b>C</b>, GM-1111 treatment beginning at t = 12 h after LL-37 injection. <b>D</b>. H&E-stained cross-sectional view of a LL-37 injected skin sample. <b>E</b>. H&E-stained cross-sectional view of GM-1111 treatment at t = 0 in LL-37 injected skin region. <b>F</b>. H&E-stained cross-sectional view of GM-1111 at t = 12 h treatment in LL-37 injected skin region. <b>G</b>. MPO activity measurement of LL-37 injection with different treatments. <b>H</b>. Area of erythema from LL-37 injection. *P<0.05 vs intradermal LL-37 alone; n = 6 per group.</p

Topical SAGE reduces PMN infiltration and S100 calgranulin accumulation from intradermal LL-37.

<p>LL-37, PBS and LL-37 and SAGE-injected mouse skin was stained with antibody to mouse Gr-1 to examine neutrophil infiltration, and also for S100A8 calgranulin, the major RAGE-binding calgranulin present in murine PMNs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016658#pone.0016658-Fuellen1" target="_blank">[17]</a> as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016658#s4" target="_blank">Methods</a>. <b>A.</b> Staining of normal control skin for the PMN antigen Gr-1. <b>B.</b> Staining of LL-37 injected skin for the PMN antigen Gr-1. <b>C.</b> Staining of skin injected with LL-37 plus GM-1111 (applied at t = 0) for the PMN antigen Gr-1. <b>D.</b> Staining of skin injected with LL-37 plus GM-1111 (applied at t = 12 h) for the PMN antigen Gr-1. <b>E.</b> Staining of normal control skin for the RAGE ligand S100A8. <b>F.</b> Staining of LL-37 injected skin for the RAGE ligand S100A8. <b>G.</b> Staining of skin injected with LL-37 plus GM-1111 (applied at t = 0) for the RAGE ligand S100A8. <b>H.</b> Staining of skin injected with LL-37 plus GM-1111 (applied at t = 12 h) for the RAGE ligand S100A8. Figures and insets are shown at 20x and 40x magnification, respectively.</p

Anti-Inflammatory Activities of SAGE, GM-1111, <i>In Vitro</i>¹.

<p>*The average molecular mass of SAGE is 5.5 kDa and heparin is 14 kDa.</p><p>Details of ¹cell surface binding assays, ² inhibition of human leukocyte elastase (HLE) and ³solid phase binding assays are found in Methods. Detailed graphical plots of the results from cell surface binding assays, inhibition of HLE and solid phase binding assays are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016658#pone-0016658-g003" target="_blank">Figure 3</a>.</p

SAGEs bind to vascular adhesion proteins.

<p>GM-1111 was studied to determine binding affinity for P-selectin (<b>A</b>), Mac-1 (<b>B</b>), and RAGE (<b>C</b>). Binding affinity (K_D) values were 0.0036 nM for GM-1111 binding to P-selectin, 0.175 nM for GM-1111 binding to Mac-1 and 1.69 nM for GM-1111 binding to RAGE.</p

Structure of semi-synthetic glycosaminoglycan ethers (SAGEs).

<p>SAGEs can vary in molecular size, and in extent of alkylation and sulfation. GM-1111 is a low-molecular weight SAGE with an average molecular weight of 5.5 kDa.</p

HA emollient does not reduce LL-37-induced inflammation.

<p>Experiments were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016658#pone-0016658-g007" target="_blank">Figure 7</a>, but with immediate (t = 0) application of 1% w/w HA-containing emollient (53 kDa HA) or 1% w/w GM-1111 emollient. *P<0.05 vs LL-37 alone. <b>A</b>. H&E-stained cross-sectional view of LL-37 injected skin. <b>B</b>. H&E-stained section of LL-37 injected skin treated with topical HA emollient alone. <b>C</b>. H&E-stained section of LL-37 injected skin treated with topical emollient containing 1% SAGE GM-1111. <b>D</b>. MPO activity measurement of LL-37 injection with different treatments. <b>E</b>. Area of erythema from LL-37 injection. Erythema scores after LL-37 injection were: 5.1±0.1 after LL-37 alone; 4.9±0.44 after LL-37+ HA; 2.3±0.7 after LL-37+ SAGE (P<0.05 LL-37 alone vs LL-37+ SAGE). *P<0.05 vs intradermal LL-37 alone; n = 6 per group.</p

SAGE binds LL-37 and inhibits LL-37-induced interleukin-8 (IL-8) expression <i>in vitro.</i>

<p><b>A.</b> SAGE binds LL-37. Microwell plates coated with GM-111 were incubated with LL-37 at 37°C for 2 h. Plates were washed, incubated with anti-LL-37 antibody, incubated for 1 h, washed again four times and incubated with peroxidase-conjugated secondary antibody for 1 h. A colorimetric reaction was produced by addition of TMB and quantified by absorbance at 450 nm. LL-37 binds to GM-1111 with a K_D of 0.225 nM. <b>B.</b> SAGE inhibits IL-8 production LL-37 stimulated keratinocytes. Human keratinocytes were grown to confluence and treated with 3.2 µM LL-37 or LL-37 and a 4x molar excess of GM-111101 for 6 h. Supernatants were collected and placed in a sterile 96-well plate. Production of IL-8 was determined by ELISA (R&D Systems, Minneapolis, MN) in accordance with manufacturer's instructions. Co-addition of GM-1111 significantly inhibited IL-8 release into medium. *P<0.01 as compared with negative control group without LL37; **P<0.05 as compared with positive control group without GM-111101 treatment.</p

SAGEs inhibit P-selectin, HLE, interaction of RAGE with its many ligands.

<p>Data points in each figure represent the average value ± standard deviation of quadruplicate wells for each concentration of SAGE. <b>A.</b> SAGEs inhibit P-selectin. Inhibition of P-selectin glycoprotein ligand-1 (PSGL-1) binding to P-selectin by SAGEs was studied using calcein-labeled U937 cells incubated in microwells coated with P-selectin. After 1 h, plates were washed, bound cells were lysed with Triton-X100 buffer and bound cells were quantified using an excitation of 494 nm and emission of 517 nm. GM-1111 inhibits PSGL-1 attachment to P-selectin with an IC₅₀ of 25 nM. <b>B.</b> SAGEs inhibit HLE. HLE (100 nM) was incubated with GM-1111 at 1–100 nm concentrations in 0.5 M HEPES buffer for 15 min. Following incubation, the elastase substrate, Suc-Ala-Ala-Val-<i>p</i>NA was added to the reaction mixture to the final concentration of 0.3 mM. Absorbance due to <i>p</i>-NA hydrolysis was monitored for 15 min at absorbance of 405 nm. GM-1111 inhibits HLE with an IC₅₀ of 45 nM. <b>C–E.</b> SAGEs inhibit interaction of the AGE product CML-BSA, the calgranulin S100b and the alarmin HMGB-1 with RAGE. Microwell plates coated with CML-BSA (<b>C</b>), S100b calgranulin (<b>D</b>) or HMGB-1 (<b>E</b>) were incubated with RAGE-Fc chimera with or without GM-1111 for 2 h. Plates were washed, incubated with anti-RAGE antibody, incubated for 1 h, washed again four times and incubated with horse-radish peroxidase conjugated secondary for 1 h. A colorimetric reaction was produced by addition of tetramethyl benzidine chromogen (TMB) and quantified by absorbance at 450 nm. GM-1111 inhibits interaction of RAGE with CM-BSA, S100b and HMGB-1 with IC₅₀ values of 413, 275 and 80 nM, respectively. <b>F.</b> SAGEs inhibit function of RAGE as an adhesion ligand. Inhibition of Mac-1-dependent ligation of RAGE by U937 cells was studied as in <b>A</b>, but using microwells coated with RAGE. GM-1111 inhibits Mac-1 attachment to RAGE with an IC₅₀ of 7.6 nM.</p