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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 affects cellular recruitment and edema formation.Animals were topically treated either with vehicle (control group) or HYAL 16 U daily. Paraffin-wound sections were stained with HE to evaluate the inflammatory infiltrate response by image analysis. (A) The sections were photographed at 400x. The ImageJ software was used to count the inflammatory cells in wound tissue specimens at day 2, 7, 14 and 21 post wounding in at least ten random optic fields per group. (B) Histogram of a quantitative analysis of inflammatory infiltrate counted. (C) Tissue neutrophil accumulation determined by MPO levels in wound biopsies. (D) The total protein content was measured according to Coomassie assay. Values represent mean ± 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-g002: HYAL affects cellular recruitment and edema formation.Animals were topically treated either with vehicle (control group) or HYAL 16 U daily. Paraffin-wound sections were stained with HE to evaluate the inflammatory infiltrate response by image analysis. (A) The sections were photographed at 400x. The ImageJ software was used to count the inflammatory cells in wound tissue specimens at day 2, 7, 14 and 21 post wounding in at least ten random optic fields per group. (B) Histogram of a quantitative analysis of inflammatory infiltrate counted. (C) Tissue neutrophil accumulation determined by MPO levels in wound biopsies. (D) The total protein content was measured according to Coomassie assay. Values represent mean ± SEM (n = 8 wounds/group), *P<0.05, **P<0.01, ***P<0.001 compared to control group by one-way-ANOVA.

Mentions: Histological analysis from the skin revealed significantly increased cellularity in the wounds of 16 U HYAL treated rats at days 2 and 7 compared to controls (Figure 2A, B). The density of cells 14 and 21 days post wounding were similar between treated and control groups and gradually decreased to physiological levels at 21st day. To evaluate whether neutrophils may have accumulated or have been activated, the activity of myeloperoxidase (MPO) was studied. As expected, MPO activity was very low in the intact skin (day zero) (Figure 2C). However, at day 2 post-wounding, MPO levels markedly increased in the animals treated with 16 U HYAL. At day 7, MPO activity in treated rats declined to similar levels as in the controls and both gradually declined thereafter to normal concentration at day 14 as observed in the day zero. To study if the decrease in MPO activity may have an influence on the edema we analyzed the total protein content in homogenate skin biopsies. We observed that HYAL significantly reduced the edema formation evidenced by lower amount of protein at day 2 and at day 7 after excision wounds being daily treated with 16 U HYAL and compared to vehicle-control group (Figure 2D). After 14 and 21 days no difference was observed between experimental groups, and the amount of protein declined to similar concentrations detected in unwound control tissue.


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 affects cellular recruitment and edema formation.Animals were topically treated either with vehicle (control group) or HYAL 16 U daily. Paraffin-wound sections were stained with HE to evaluate the inflammatory infiltrate response by image analysis. (A) The sections were photographed at 400x. The ImageJ software was used to count the inflammatory cells in wound tissue specimens at day 2, 7, 14 and 21 post wounding in at least ten random optic fields per group. (B) Histogram of a quantitative analysis of inflammatory infiltrate counted. (C) Tissue neutrophil accumulation determined by MPO levels in wound biopsies. (D) The total protein content was measured according to Coomassie assay. Values represent mean ± 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

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

pone-0112297-g002: HYAL affects cellular recruitment and edema formation.Animals were topically treated either with vehicle (control group) or HYAL 16 U daily. Paraffin-wound sections were stained with HE to evaluate the inflammatory infiltrate response by image analysis. (A) The sections were photographed at 400x. The ImageJ software was used to count the inflammatory cells in wound tissue specimens at day 2, 7, 14 and 21 post wounding in at least ten random optic fields per group. (B) Histogram of a quantitative analysis of inflammatory infiltrate counted. (C) Tissue neutrophil accumulation determined by MPO levels in wound biopsies. (D) The total protein content was measured according to Coomassie assay. Values represent mean ± SEM (n = 8 wounds/group), *P<0.05, **P<0.01, ***P<0.001 compared to control group by one-way-ANOVA.
Mentions: Histological analysis from the skin revealed significantly increased cellularity in the wounds of 16 U HYAL treated rats at days 2 and 7 compared to controls (Figure 2A, B). The density of cells 14 and 21 days post wounding were similar between treated and control groups and gradually decreased to physiological levels at 21st day. To evaluate whether neutrophils may have accumulated or have been activated, the activity of myeloperoxidase (MPO) was studied. As expected, MPO activity was very low in the intact skin (day zero) (Figure 2C). However, at day 2 post-wounding, MPO levels markedly increased in the animals treated with 16 U HYAL. At day 7, MPO activity in treated rats declined to similar levels as in the controls and both gradually declined thereafter to normal concentration at day 14 as observed in the day zero. To study if the decrease in MPO activity may have an influence on the edema we analyzed the total protein content in homogenate skin biopsies. We observed that HYAL significantly reduced the edema formation evidenced by lower amount of protein at day 2 and at day 7 after excision wounds being daily treated with 16 U HYAL and compared to vehicle-control group (Figure 2D). After 14 and 21 days no difference was observed between experimental groups, and the amount of protein declined to similar concentrations detected in unwound control tissue.

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