title: Is molecular size a discriminating factor in hyaluronan interaction with human cells?
abstract: Nowadays there is a great interest in investigating the effect of particular hyaluronan fragments in the biomedical field and in cosmeceutical applications. Literature has reported that very low molecular weight HA (Mw<5kDa) has an inflammatory effect, whilst HA ranging from 15 to 250 has shown controversial effects. This work aims to give better elucidation on the correlation between the different sized HA fragments and their biological functions. In this respect, a simple and effective degradation strategy is used to obtain several HA fragments. Also, an hydrodynamic and structural characterization was performed in order to obtain samples suitable to evaluate cellular response. In particular an in vitro scratch test in time lapse experiments was used to study the effect of HA fragments, ranging from 1800 to 6kDa on wound dermal reparation based on human keratinocytes. All high and low Mw HA used in this study allowed for faster wound closure compared to the un-treated cells, except for 6kDa that, on the contrary, prevented repair. In addition, TGF-β 1, TNFα and IL-6, representative biomarkers of the inflammation phase occurring in wound healing process, were quantified by RT-PCR. A general up-regulation trend of these biomarkers was found with the HA molecular weight reduction. LHA6kDa was the only treatment that induced a major inflammatory response (over 30 fold increase respect to control) confirming the recent literature outcomes. IL-6 protein level evaluated through ELISA assay corroborated the previous results. Furthermore, activation of key HA receptors, such as CD44, RHAMM, TLR4, with respect to hyaluronan size, was evaluated, at transcriptional level showing selective recognition by HA 1800, 1400, 500 for CD44, whilst the lower Mw fragments activated TLR-4 moderately at 50 and 15kDa. An increase to "alarm" level was found for 6kDa fragments. Immunofluorescence staining confirmed this data. The present research work demonstrated that the diverse pharma grade hyaluronan fragments could modulate cellular processes differently. From 1800kDa down to 50kDa, CD44 was the recognized receptor and pro-inflammatory biomarkers were only slightly up-regulated during wound healing in the presence of HA. Finally our outcomes showed that the lower the fragment size the higher the concern for inflammatory cytokines up-regulation; repair process impairment was highlighted only for 6kDa chains.
authors.affiliation: Second University of Naples, Via L. De Crecchio, 7 80138 Napoli, Italy. Electronic address: antonella.dagostino@unina2.it.
authors.forename: Antonella
authors.initials: A
authors.lastname: D'Agostino
authors.affiliation: Second University of Naples, Via L. De Crecchio, 7 80138 Napoli, Italy. Electronic address: antonietta.stellavato@unina2.it.
authors.forename: Antonietta
authors.initials: A
authors.lastname: Stellavato
authors.affiliation: Second University of Naples, Via L. De Crecchio, 7 80138 Napoli, Italy. Electronic address: luisanacorsuto@hotmail.it.
authors.forename: Luisana
authors.initials: L
authors.lastname: Corsuto
authors.affiliation: Second University of Naples, Via L. De Crecchio, 7 80138 Napoli, Italy. Electronic address: paola.diana0@libero.it.
authors.forename: Paola
authors.initials: P
authors.lastname: Diana
authors.affiliation: Second University of Naples, Via L. De Crecchio, 7 80138 Napoli, Italy. Electronic address: rosanna.filosa@unina2.it.
authors.forename: Rosanna
authors.initials: R
authors.lastname: Filosa
authors.affiliation: Second University of Naples, Via L. De Crecchio, 7 80138 Napoli, Italy. Electronic address: annalisa.lagatta@unina2.it.
authors.forename: Annalisa
authors.initials: A
authors.lastname: La Gatta
authors.affiliation: Second University of Naples, Via L. De Crecchio, 7 80138 Napoli, Italy. Electronic address: mario.derosa@unina2.it.
authors.forename: Mario
authors.initials: M
authors.lastname: De Rosa
authors.affiliation: Second University of Naples, Via L. De Crecchio, 7 80138 Napoli, Italy. Electronic address: chiara.schiraldi@unina2.it.
authors.forename: Chiara
authors.initials: C
authors.lastname: Schiraldi
mesh_terms: D016207:Cytokines / Q000378:metabolism; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D006820:Hyaluronic Acid / Q000737:chemistry*; D007249:Inflammation; D015603:Keratinocytes / Q000187:drug effects*; D008970:Molecular Weight
chemical_list: D016207:Cytokines; D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid
keywords: Hyaluronic; Inflammatory biomarker; Molecular size; Receptors