The Anti-Inflammatory Effect of Lactose-Modified Hyaluronic Acid Molecules on Primary Bronchial Fibroblasts of Smokers

Abstract

The progression of smoking-related diseases is characterized by macrophage-mediated inflammation, which is responsible for an increased expression of proinflammatory cytokines and galectins, molecules that bind specifically to β-galactoside sugars. This study aimed to assess the anti-inflammatory and antioxidant effects of a broad selection of differently lactose-modified hyaluronic acids (HA) named HYLACH®, which are able to bind proinflammatory galectins. The best HYLACH ligands for Gal-3 were selected in silico and their activities were tested in vitro on primary human bronchial fibroblasts obtained from smokers and inflamed with the conditioned medium of activated U937 monocytes. Changes in cell viability, ROS generation, proinflammatory mediators, and MMP expression, at both gene and protein levels, were analyzed. The in silico results show that HYLACH with a percentage of lactosylation of 10–40% are the best ligands for Gal-3. The in vitro study revealed that HYLACH compounds with 10, 20, and 40% lactosylation (HYLACH-1-2-3) administrated to inflamed cell cultures counteracted the oxidative damage and restored gene and protein expression for IL-1β, TNF-α, IL-6, Gal-1, Gal-3, and MMP-3 to near baseline values. The evidence that HYLACH attenuated macrophage-induced inflammation, inhibited MMP expression, and exhibited antioxidative effects provide an initial step toward the development of a therapeutic treatment suitable for smoking-related diseases.

A novel biomimetic probe for galectin‐3 recognition: Chemical synthesis and structural characterization of a β‐galactose branched sodium hyaluronate

“…The synthesis has been performed to obtain compounds with a DS between 10% and 40%, which were found to provide better results in preliminary in silico studies. 8 The syntheses were performed in aqueous media, in the presence of a commercially available condensing agent (CA), DMTMM, 24 known to be highly soluble and stable in water for an extended period of time. 25 This allows the formation of an ammonium salt of DMTMM which reacts, by a substitution mechanism, with the carboxylic group of HA to form the active triazine-ester followed by a nucleophilic attack of LAC-NH 2 .…”

“…25 This allows the formation of an ammonium salt of DMTMM which reacts, by a substitution mechanism, with the carboxylic group of HA to form the active triazine-ester followed by a nucleophilic attack of LAC-NH 2 . 7 To synthesize samples with different DS values in a range considered ideal according to previous biological evaluation, 8 various reaction parameters, such as stoichiometry of the triazine condenser (DMTMM) and LAC-NH 2, and pH, were optimized.…”

“…Interaction studies were performed on saccharide ligands and OHA and HYLACH ® (OHY1 and OHY2), obtained by enzymatical hydrolysis. OHY1 and OHY2 were selected due to the similarity of molecular weight and DS with HYLACH 1 and HYLACH 3, respectively, cited by Donato et al 8 ITC results showed that the interactions between Gal-3 and saccharide ligands, OHA and OHY are all energetically favorable binding reactions (ΔG negative) and entropically driven (TΔS > 0), except for OHY2. For small ligands (LAC-NH 2 , galactose, and lactose) the stoichiometry of the interaction (N) is in the order of 1 protein:1 ligand, and the dissociation constant (K D ) is the order of µM, with values similar to data reported for lactose.…”

“…The possible increase in rigidity combined with fewer available negative charges may decrease the electrostatic interactions and increase the specificity of HYLACH ® binding. In addition, HYLACH ® (OHY1 and OHY2), but not HA, have been reported to provide anti-inflammatory properties in an in vitro inflammation model of lung fibroblasts as showed by Donato et al8 This is likely to be the consequence that although with the same affinity of HA, the specific occupation of the CRD pocket by LAC-NH 2 , triggers Gal-3 function in reduction of both gene and protein expression of fundamental profibrotic molecules as reported by Donato et al18 CDCD measurements were performed to address conformational changes of Gal-3 upon ligand binding. Specifically, the CD signal in the far-UV (200-250 nm) provides information concerning possible changes in the protein secondary structure and conformation (αhelices, β-sheet, and unstructured regions) that may be induced by interactions with other ligands.…”

“…HYLACH ® has already been reported to attenuate macrophage-induced inflammation and inhibit Gal-3 expression among other ECM proteins, while also exhibiting antioxidative effects. 8 In addition, the superiority of HYLACH in comparison to HA in reducing both gene and protein expression of fundamental profibrotic molecules has been demonstrated. 18 Two HA with different molecular weights (90 and 400 kDa) were employed.…”