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

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

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