Abstract
IntroductionHyaluronic acid (hyaluronan; HA) is an essential component of the extracellular matrix (ECM) of the skin. The HA-degrading enzyme hyaluronidase (HYAL) is critically involved in the HA-metabolism. Yet, only little information is available regarding the skin’s HA–HYAL interactions on the molecular and cellular levels.ObjectiveTo analyze the dose- and time-dependent molecular and cellular effects of HYAL on structural cells and the HA-metabolism in the skin.Materials and methodsChip-based, genome-wide expression analyses (Affymetrix® GeneChip PrimeView™ Human Gene Expression Array), quantitative real-time PCR analyses, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (DAB), and in vitro wound healing assays were performed to assess dose-dependent and time-kinetic effects of HA and HYAL (bovine hyaluronidase, Hylase “Dessau”) on normal human dermal fibroblasts (NHDF), primary human keratinocytes in vitro and human skin samples ex vivo.ResultsGenome-wide expression analyses revealed an upregulation of HA synthases (HAS) up to 1.8-fold change in HA- and HYAL-treated NHDF. HA and HYAL significantly accelerated wound closure in an in vitro model for cutaneous wound healing. HYAL induced HAS1 and HAS2 mRNA gene expression in NHDF. Interestingly, low concentrations of HYAL (0.015 U/ml) resulted in a significantly higher induction of HAS compared to moderate (0.15 and 1.5 U/ml) and high concentrations (15 U/ml) of HYAL. This observation corresponded to increased concentrations of HA measured by ELISA in conditioned supernatants of HYAL-treated NHDF with the highest concentrations observed for 0.015 U/ml of HYAL. Finally, immunohistochemical analysis of human skin samples incubated with HYAL for up to 48 h ex vivo demonstrated that low concentrations of HYAL (0.015 U/ml) led to a pronounced accumulation of HA, whereas high concentrations of HYAL (15 U/ml) reduced dermal HA-levels.ConclusionHYAL is a bioactive enzyme that exerts multiple effects on the HA-metabolism as well as on the structural cells of the skin. Our results indicate that HYAL promotes wound healing and exerts a dose-dependent induction of HA-synthesis in structural cells of the skin. Herein, interestingly the most significant induction of HAS and HA were observed for the lowest concentration of HYAL.
Highlights
Hyaluronic acid is an essential component of the extracellular matrix (ECM) of the skin
Genome-wide expression analyses revealed an upregulation of HA synthases (HAS) up to 1.8-fold change in HA- and HYAL-treated normal human dermal fibroblasts (NHDF)
Low concentrations of HYAL (0.015 U/ml) resulted in a significantly higher induction of HAS compared to moderate (0.15 and 1.5 U/ml) and high concentrations (15 U/ml) of HYAL. This observation corresponded to increased concentrations of HA measured by enzyme-linked immunosorbent assay (ELISA) in conditioned supernatants of HYAL-treated NHDF with the highest concentrations observed for 0.015 U/ ml of HYAL
Summary
Hyaluronic acid (hyaluronan; HA) is an essential component of the extracellular matrix (ECM) of the skin. The extracellular matrix (ECM) of the skin is a complex network of macromolecules, and plays an important role in the regulation of numerous cellular mechanisms such as proliferation, adhesion, migration, and gene regulation. A functionally and quantitatively important component of dermal ECM is hyaluronic acid (hyaluronan; HA) [26]. Half of all HA in the body is contained within skin tissue. The different HAS isoforms produce HA which differs primarily in the polymer size. The half-life of HA is organ-dependent and is approximately 24 h in the skin. The degradation of HA is mediated via free chemical radicals and different hyaluronidases (HYAL1 and HYAL2) first into smaller fragments of different sizes, which are further degraded [51]
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