Enhanced Inflammation and Accelerated Wound Closure Following Tetraphorbol Ester Application or Full-Thickness Wounding in Mice Lacking Hyaluronan Synthases Has1 and Has3

Abstract

Hyaluronan (HA) is an abundant matrix molecule whose functions in the skin remain to be fully defined. To explore the roles of HA in cutaneous injury responses, double-knockout mice (abbreviated as Has1/3 null) that lack two HA synthase enzymes (Has1 and Has3) but still express functional Has2, were used in two types of experiments: (i) application of 12-O-tetradecanoylphorbol-13-acetate (TPA), and (ii) full-thickness wounding of the skin. Uninjured Has1/3 null mice were phenotypically normal. However, after TPA, the accumulation of HA that normally occurs in wildtype epidermis was blunted in Has1/3 null epidermis. In excisional wound healing experiments, wound closure was significantly faster in Has1/3 null than in wildtype mice. Coincident with this abnormal wound healing, a marked decrease in epidermal and dermal HA and a marked increase in neutrophil efflux from cutaneous blood vessels were observed in Has1/3 null skin relative to wildtype skin. Has1/3 null wounds displayed an earlier onset of myofibroblast differentiation. In summary, selective loss of Has1 and Has3 leads to a pro-inflammatory milieu that favors recruitment of neutrophils and other inflammation-related changes in the dermis.

Hyaluronan Synthase 2 Protects Skin Fibroblasts against Apoptosis Induced by Environmental Stress

“…However, a role for HA in regulating fibroblast viability and susceptibility to apoptosis has not been reported. Previous work from our laboratory showed that in mice lacking Has1 and Has3, healing skin wounds contain more fibroblasts and myofibroblasts at 5 days after excisional wounding relative to WT wounds (28). This suggests that a change in the level of HAS enzyme expression and HA synthesis in vivo leads to a change in the equilibrium number of fibroblasts, either through an increase in proliferation, a decrease in apoptosis, or both.…”

“…C57BL/6J WT mice were obtained from JAX Laboratories (Bar Harbor, ME). Mice deficient in Has1 and Has3 (Has1/3 null) were generated as described previously (28). Pups were euthanized in ice, and the entire trunk skin was removed and incubated overnight in 0.25% trypsin without EDTA, followed by mechanical separation of epidermis from dermis.…”

“…The HA and CS content of skin fibroblast cultures (in cell layers and in conditioned media) was measured as described previously (28). Briefly, after proteolytic digestion and ethanol precipitation/ purification steps, HA in the samples was digested into disaccharides using hyaluronidase from S. dysgalactiae (2.5 milliunits/l; 100741-1A, Seikagaku America Inc.), and CS in the samples was digested into disaccharides using chondroitinase ABC (25 milliunits/l; 100330-1A, Seikagaku America) and then labeled with 2-aminoacridone (Invitrogen).…”

Hyaluronan Deficiency Due toHas3Knock-Out Causes Altered Neuronal Activity and Seizures via Reduction in Brain Extracellular Space

“…Mouse Has1- (Mack et al, 2012) and Has3-null (Bai et al, 2005) alleles were obtained from Dr John McDonald (Mayo Clinic, Scottsdale, AZ) as Has1 Ϫ/Ϫ ;Has3 Ϫ/Ϫ compound mutant mice, from which Has1 Ϫ/Ϫ and Has3 Ϫ/Ϫ mice were segregated for this study. As reported previously (Bai et al, 2005;Mack et al, 2012), Has1 Ϫ/Ϫ , Has3 Ϫ/Ϫ , and Has1 Ϫ/Ϫ ;Has3 Ϫ/Ϫ mice exhibited no apparent developmental abnormalities and had normal life spans.…”

“…Mouse Has1- (Mack et al, 2012) and Has3-null (Bai et al, 2005) alleles were obtained from Dr John McDonald (Mayo Clinic, Scottsdale, AZ) as Has1 Ϫ/Ϫ ;Has3 Ϫ/Ϫ compound mutant mice, from which Has1 Ϫ/Ϫ and Has3 Ϫ/Ϫ mice were segregated for this study. As reported previously (Bai et al, 2005;Mack et al, 2012), Has1 Ϫ/Ϫ , Has3 Ϫ/Ϫ , and Has1 Ϫ/Ϫ ;Has3 Ϫ/Ϫ mice exhibited no apparent developmental abnormalities and had normal life spans. Because Has2 Ϫ/Ϫ mice are embryonic lethal (Camenisch et al, 2000), brain-targeted conditional Has2 knockout mice (Has2 CKO ) were generated using the conditional Has2-null allele (Matsumoto et al, 2009) and the nestin-Cre transgene (Tronche et al, 1999).…”