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Hyaluronidase modulates inflammatory response and accelerates the cutaneous wound healing.

Fronza M, Caetano GF, Leite MN, Bitencourt CS, Paula-Silva FW, Andrade TA, Frade MA, Merfort I, Faccioli LH - PLoS ONE (2014)

Bottom Line: Hyaluronidases are enzymes that degrade hyaluronan an important constituent of the extracellular matrix.They have been used as a spreading agent, improving the absorption of drugs and facilitating the subcutaneous infusion of fluids.Moreover, HYAL increased gene expression of peroxisome proliferator-activated receptors (PPAR) γ and PPAR β/δ, the collagen content in the early stages of healing processes as well as angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil; Departamento de Farmácia, Universidade de Vila Velha, Vila Velha, Espirito Santo, Brazil.

ABSTRACT
Hyaluronidases are enzymes that degrade hyaluronan an important constituent of the extracellular matrix. They have been used as a spreading agent, improving the absorption of drugs and facilitating the subcutaneous infusion of fluids. Here, we investigated the influence of bovine testes hyaluronidase (HYAL) during cutaneous wound healing in in vitro and in vivo assays. We demonstrated in the wound scratch assay that HYAL increased the migration and proliferation of fibroblasts in vitro at low concentration, e.g. 0.1 U HYAL enhanced the cell number by 20%. HYAL presented faster and higher reepithelialization in in vivo full-thickness excisional wounds generated on adult Wistar rats back skin already in the early phase at 2nd day post operatory compared to vehicle-control group. Wound closured area observed in the 16 U and 32 U HYAL treated rats reached 38% and 46% compared to 19% in the controls, respectively. Histological and biochemical analyses supported the clinical observations and showed that HYAL treated wounds exhibited increased granulation tissue, diminished edema formation and regulated the inflammatory response by modulating the release of pro and anti-inflammatory cytokines, growth factor and eicosanoids mediators. Moreover, HYAL increased gene expression of peroxisome proliferator-activated receptors (PPAR) γ and PPAR β/δ, the collagen content in the early stages of healing processes as well as angiogenesis. Altogether these data revealed that HYAL accelerates wound healing processes and might be beneficial for treating wound disorders.

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HYAL modulates cytokines and induces eicosanoid generation in the skin wound biopsies.Homogenates were prepared from the wound biopsies obtained from animals at day 0, 2, 7, 14 and 21 treated with 16 U HYAL or with vehicle-control. (A) IL1-α, (B) TNF-α, (C) IL-4, (D) IL-10, (E) TGF-β, (F) PGE2, (G) LTB4 and (H) PGD2 were assayed by ELISA. Data are means ± SEM (n = 8 wounds/group), *P<0.05, **P<0.01, ***P<0.001 compared to control group by one-way-ANOVA.
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pone-0112297-g003: HYAL modulates cytokines and induces eicosanoid generation in the skin wound biopsies.Homogenates were prepared from the wound biopsies obtained from animals at day 0, 2, 7, 14 and 21 treated with 16 U HYAL or with vehicle-control. (A) IL1-α, (B) TNF-α, (C) IL-4, (D) IL-10, (E) TGF-β, (F) PGE2, (G) LTB4 and (H) PGD2 were assayed by ELISA. Data are means ± SEM (n = 8 wounds/group), *P<0.05, **P<0.01, ***P<0.001 compared to control group by one-way-ANOVA.

Mentions: Neutrophils were proven to be attracted to the wounded sites which may be induced by various cytokines. However, neutrophils themselves can also be activated releasing cytokines which have an impact on further steps in the wound healing process. Therefore, we evaluated the impact of HYAL on different cytokines. In fact, 16 U HYAL altered the release of pro- and anti-inflammatory cytokines in a time-dependent manner (Figure 3). Enhanced production of IL1-α, TNF-α, IL-4 and IL-10 at day 2 was observed compared to vehicle-control group (Figure 3A, B, C e D). Except of IL1-α all other cytokines declined at day 7 and this continuously. Only TNF-α was still slightly, but significantly increased at day 7 compared to the controls. After 14 days, there were no significant changes in the levels of the studied cytokines, while at day 21, a significant decrease in the IL1-α and TNF-α amount could be observed. TGF-β production was increased after 2 days, peaked at day 7 and then decreased after 14 and 21 days to control level in the wound biopsies (Figure 3E). Levels of IL-6, IL1-β, IFN-γ and IL-5 were not detected after HYAL treatment during the time-course of this experiment. Eicosanoids were also altered by HYAL treatment. Compared to controls, we observed a significant peak in the production of PGE2 and LTB4 after 2 days of HYAL treatment (Figure 3F, G). Subsequently, both lipid mediators decreased to baseline levels, after 14 days for LTB4 and 21 days for PGE2 post-wounding, respectively. Low concentrations of PGD2 where observed in the first days of HYAL treatment, however a significant increase in the production of PGD2 was observed at day 7 (Figure 3H).


Hyaluronidase modulates inflammatory response and accelerates the cutaneous wound healing.

Fronza M, Caetano GF, Leite MN, Bitencourt CS, Paula-Silva FW, Andrade TA, Frade MA, Merfort I, Faccioli LH - PLoS ONE (2014)

HYAL modulates cytokines and induces eicosanoid generation in the skin wound biopsies.Homogenates were prepared from the wound biopsies obtained from animals at day 0, 2, 7, 14 and 21 treated with 16 U HYAL or with vehicle-control. (A) IL1-α, (B) TNF-α, (C) IL-4, (D) IL-10, (E) TGF-β, (F) PGE2, (G) LTB4 and (H) PGD2 were assayed by ELISA. Data are means ± SEM (n = 8 wounds/group), *P<0.05, **P<0.01, ***P<0.001 compared to control group by one-way-ANOVA.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230982&req=5

pone-0112297-g003: HYAL modulates cytokines and induces eicosanoid generation in the skin wound biopsies.Homogenates were prepared from the wound biopsies obtained from animals at day 0, 2, 7, 14 and 21 treated with 16 U HYAL or with vehicle-control. (A) IL1-α, (B) TNF-α, (C) IL-4, (D) IL-10, (E) TGF-β, (F) PGE2, (G) LTB4 and (H) PGD2 were assayed by ELISA. Data are means ± SEM (n = 8 wounds/group), *P<0.05, **P<0.01, ***P<0.001 compared to control group by one-way-ANOVA.
Mentions: Neutrophils were proven to be attracted to the wounded sites which may be induced by various cytokines. However, neutrophils themselves can also be activated releasing cytokines which have an impact on further steps in the wound healing process. Therefore, we evaluated the impact of HYAL on different cytokines. In fact, 16 U HYAL altered the release of pro- and anti-inflammatory cytokines in a time-dependent manner (Figure 3). Enhanced production of IL1-α, TNF-α, IL-4 and IL-10 at day 2 was observed compared to vehicle-control group (Figure 3A, B, C e D). Except of IL1-α all other cytokines declined at day 7 and this continuously. Only TNF-α was still slightly, but significantly increased at day 7 compared to the controls. After 14 days, there were no significant changes in the levels of the studied cytokines, while at day 21, a significant decrease in the IL1-α and TNF-α amount could be observed. TGF-β production was increased after 2 days, peaked at day 7 and then decreased after 14 and 21 days to control level in the wound biopsies (Figure 3E). Levels of IL-6, IL1-β, IFN-γ and IL-5 were not detected after HYAL treatment during the time-course of this experiment. Eicosanoids were also altered by HYAL treatment. Compared to controls, we observed a significant peak in the production of PGE2 and LTB4 after 2 days of HYAL treatment (Figure 3F, G). Subsequently, both lipid mediators decreased to baseline levels, after 14 days for LTB4 and 21 days for PGE2 post-wounding, respectively. Low concentrations of PGD2 where observed in the first days of HYAL treatment, however a significant increase in the production of PGD2 was observed at day 7 (Figure 3H).

Bottom Line: Hyaluronidases are enzymes that degrade hyaluronan an important constituent of the extracellular matrix.They have been used as a spreading agent, improving the absorption of drugs and facilitating the subcutaneous infusion of fluids.Moreover, HYAL increased gene expression of peroxisome proliferator-activated receptors (PPAR) γ and PPAR β/δ, the collagen content in the early stages of healing processes as well as angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil; Departamento de Farmácia, Universidade de Vila Velha, Vila Velha, Espirito Santo, Brazil.

ABSTRACT
Hyaluronidases are enzymes that degrade hyaluronan an important constituent of the extracellular matrix. They have been used as a spreading agent, improving the absorption of drugs and facilitating the subcutaneous infusion of fluids. Here, we investigated the influence of bovine testes hyaluronidase (HYAL) during cutaneous wound healing in in vitro and in vivo assays. We demonstrated in the wound scratch assay that HYAL increased the migration and proliferation of fibroblasts in vitro at low concentration, e.g. 0.1 U HYAL enhanced the cell number by 20%. HYAL presented faster and higher reepithelialization in in vivo full-thickness excisional wounds generated on adult Wistar rats back skin already in the early phase at 2nd day post operatory compared to vehicle-control group. Wound closured area observed in the 16 U and 32 U HYAL treated rats reached 38% and 46% compared to 19% in the controls, respectively. Histological and biochemical analyses supported the clinical observations and showed that HYAL treated wounds exhibited increased granulation tissue, diminished edema formation and regulated the inflammatory response by modulating the release of pro and anti-inflammatory cytokines, growth factor and eicosanoids mediators. Moreover, HYAL increased gene expression of peroxisome proliferator-activated receptors (PPAR) γ and PPAR β/δ, the collagen content in the early stages of healing processes as well as angiogenesis. Altogether these data revealed that HYAL accelerates wound healing processes and might be beneficial for treating wound disorders.

Show MeSH
Related in: MedlinePlus