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The redox enzyme p66Shc contributes to diabetes and ischemia-induced delay in cutaneous wound healing.

Fadini GP, Albiero M, Menegazzo L, Boscaro E, Pagnin E, Iori E, Cosma C, Lapolla A, Pengo V, Stendardo M, Agostini C, Pelicci PG, Giorgio M, Avogaro A - Diabetes (2010)

Bottom Line: Genetic deletion of p66Shc prolongs life span and protects against oxidative stress.Migration of p66Shc(-/-) dermal fibroblasts in vitro was significantly faster than WT fibroblasts under both high glucose and hypoxia. p66Shc is involved in the delayed wound-healing process in the setting of diabetes and ischemia.Thus, p66Shc may represent a potential therapeutic target against this disabling diabetes complication.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical and Experimental Medicine, University of Padova Medical School, Padova, Italy. gianpaolofadini@hotmail.com

ABSTRACT

Objective: The redox enzyme p66Shc produces hydrogen peroxide and triggers proapoptotic signals. Genetic deletion of p66Shc prolongs life span and protects against oxidative stress. In the present study, we evaluated the role of p66Shc in an animal model of diabetic wound healing.

Research design and methods: Skin wounds were created in wild-type (WT) and p66Shc(-/-) control and streptozotocin-induced diabetic mice with or without hind limb ischemia. Wounds were assessed for collagen content, thickness and vascularity of granulation tissue, apoptosis, reepithelialization, and expression of c-myc and beta-catenin. Response to hind limb ischemia was also evaluated.

Results: Diabetes delayed wound healing in WT mice with reduced granulation tissue thickness and vascularity, increased apoptosis, epithelial expression of c-myc, and nuclear localization of beta-catenin. These nonhealing features were worsened by hind limb ischemia. Diabetes induced p66Shc expression and activation; wound healing was significantly faster in p66Shc(-/-) than in WT diabetic mice, with or without hind limb ischemia, at 1 and 3 months of diabetes duration and in both SV129 and C57BL/6 genetic backgrounds. Deletion of p66Shc reversed nonhealing features, with increased collagen content and granulation tissue thickness, and reduced apoptosis and expression of c-myc and beta-catenin. p66Shc deletion improved response to hind limb ischemia in diabetic mice in terms of tissue damage, capillary density, and perfusion. Migration of p66Shc(-/-) dermal fibroblasts in vitro was significantly faster than WT fibroblasts under both high glucose and hypoxia.

Conclusions: p66Shc is involved in the delayed wound-healing process in the setting of diabetes and ischemia. Thus, p66Shc may represent a potential therapeutic target against this disabling diabetes complication.

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Characteristics of the granulation tissue in SV129 mice. Several morphological characteristics of the granulation tissue were evaluated in WT and p66Shc knockout (KO), diabetic (DM) (4-week duration), and nondiabetic (ND) mice with or without hind limb ischemia. Masson trichrome staining allowed determination of collagen area and granulation tissue thickness. Reepithelialization was quantified in histological sections by measuring the percentage of re-covered epithelial gap. Capillary density within the granulation tissue was quantified by B4 isolectin immunofluorescence. Apoptosis was assessed with the ApopTag kit in the granulation tissue and at wound edges, a critical site for active wound healing. Representative images are shown for control (nondiabetic nonischemic) and diabetic ischemic WT and KO mice. n ≥ 3 mice for each group; n ≥ 5 sections for tissue samples. *P < 0.05 in KO vs. corresponding WT group. †P < 0.05 in diabetic vs. corresponding nondiabetic group. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 2: Characteristics of the granulation tissue in SV129 mice. Several morphological characteristics of the granulation tissue were evaluated in WT and p66Shc knockout (KO), diabetic (DM) (4-week duration), and nondiabetic (ND) mice with or without hind limb ischemia. Masson trichrome staining allowed determination of collagen area and granulation tissue thickness. Reepithelialization was quantified in histological sections by measuring the percentage of re-covered epithelial gap. Capillary density within the granulation tissue was quantified by B4 isolectin immunofluorescence. Apoptosis was assessed with the ApopTag kit in the granulation tissue and at wound edges, a critical site for active wound healing. Representative images are shown for control (nondiabetic nonischemic) and diabetic ischemic WT and KO mice. n ≥ 3 mice for each group; n ≥ 5 sections for tissue samples. *P < 0.05 in KO vs. corresponding WT group. †P < 0.05 in diabetic vs. corresponding nondiabetic group. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: A well-developed and vascularized granulation tissue is essential for wound healing. We show that diabetes, especially in combination with ischemia, impairs development and maturation of the granulation tissue in WT mice. After 4 weeks of diabetes in SV129 mice, collagen-stained area (18.7 ± 4.0 vs. 36.0 ± 2.0%; P = 0.002) and thickness of the granulation tissue (105.5 ± 18.4 vs. 323.0 ± 35.0 μm2; P < 0.0001) were significantly reduced in diabetic ischemic wounds compared with control wounds (Fig. 2). Within the granulation tissue of diabetic wounds, capillary density was reduced (nonischemic 91.2 ± 12.7 vs. 159.6 ± 11.2 hpf; P = 0.002; ischemic 42.8 ± 6.6 vs. 135.7 ± 18.9 hpf; P = 0.002), while apoptosis was increased (nonischemic 89.9 ± 22.9 vs. 19.2 ± 6.0 hpf; P = 0.009) compared with nondiabetic wounds. In parallel, closure of the epithelial gap was delayed in diabetic wounds (nonischemic 11.3 ± 3.6 vs. 26.6 ± 4.6%; P = 0.015; ischemic 20.6 ± 5.9% vs. 29.4 ± 6.8%; P = 0.33). All these alterations were reversed in p66Shc−/− mice: collagen area and tissue thickness were increased in diabetic wounds, while apoptosis was reduced and epithelial re-covering was significantly improved. Interestingly, capillary density within the granulation tissue was significantly lower in nondiabetic nonischemic p66Shc−/− mice compared withWT mice (50.8 ± 3.1 vs. 159.6 ± 11.2 hpf; P < 0.0001), but this difference was abrogated in the presence of either diabetes or ischemia, and was inverted in the presence of both diabetes and ischemia (92.8 ± 18.7 vs. 42.8 ± 6.6 hpf; P = 0.002). Results were confirmed in mice with the C57BL/6 genetic background after 1 month of diabetes (supplementary Fig. III). Collectively, these data using two different mouse strains strongly indicate that deletion of p66Shc reverts the negative effect of diabetes on development and ischemia-induced vascularization of the granulation tissue.


The redox enzyme p66Shc contributes to diabetes and ischemia-induced delay in cutaneous wound healing.

Fadini GP, Albiero M, Menegazzo L, Boscaro E, Pagnin E, Iori E, Cosma C, Lapolla A, Pengo V, Stendardo M, Agostini C, Pelicci PG, Giorgio M, Avogaro A - Diabetes (2010)

Characteristics of the granulation tissue in SV129 mice. Several morphological characteristics of the granulation tissue were evaluated in WT and p66Shc knockout (KO), diabetic (DM) (4-week duration), and nondiabetic (ND) mice with or without hind limb ischemia. Masson trichrome staining allowed determination of collagen area and granulation tissue thickness. Reepithelialization was quantified in histological sections by measuring the percentage of re-covered epithelial gap. Capillary density within the granulation tissue was quantified by B4 isolectin immunofluorescence. Apoptosis was assessed with the ApopTag kit in the granulation tissue and at wound edges, a critical site for active wound healing. Representative images are shown for control (nondiabetic nonischemic) and diabetic ischemic WT and KO mice. n ≥ 3 mice for each group; n ≥ 5 sections for tissue samples. *P < 0.05 in KO vs. corresponding WT group. †P < 0.05 in diabetic vs. corresponding nondiabetic group. (A high-quality digital representation of this figure is available in the online issue.)
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Related In: Results  -  Collection

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Figure 2: Characteristics of the granulation tissue in SV129 mice. Several morphological characteristics of the granulation tissue were evaluated in WT and p66Shc knockout (KO), diabetic (DM) (4-week duration), and nondiabetic (ND) mice with or without hind limb ischemia. Masson trichrome staining allowed determination of collagen area and granulation tissue thickness. Reepithelialization was quantified in histological sections by measuring the percentage of re-covered epithelial gap. Capillary density within the granulation tissue was quantified by B4 isolectin immunofluorescence. Apoptosis was assessed with the ApopTag kit in the granulation tissue and at wound edges, a critical site for active wound healing. Representative images are shown for control (nondiabetic nonischemic) and diabetic ischemic WT and KO mice. n ≥ 3 mice for each group; n ≥ 5 sections for tissue samples. *P < 0.05 in KO vs. corresponding WT group. †P < 0.05 in diabetic vs. corresponding nondiabetic group. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: A well-developed and vascularized granulation tissue is essential for wound healing. We show that diabetes, especially in combination with ischemia, impairs development and maturation of the granulation tissue in WT mice. After 4 weeks of diabetes in SV129 mice, collagen-stained area (18.7 ± 4.0 vs. 36.0 ± 2.0%; P = 0.002) and thickness of the granulation tissue (105.5 ± 18.4 vs. 323.0 ± 35.0 μm2; P < 0.0001) were significantly reduced in diabetic ischemic wounds compared with control wounds (Fig. 2). Within the granulation tissue of diabetic wounds, capillary density was reduced (nonischemic 91.2 ± 12.7 vs. 159.6 ± 11.2 hpf; P = 0.002; ischemic 42.8 ± 6.6 vs. 135.7 ± 18.9 hpf; P = 0.002), while apoptosis was increased (nonischemic 89.9 ± 22.9 vs. 19.2 ± 6.0 hpf; P = 0.009) compared with nondiabetic wounds. In parallel, closure of the epithelial gap was delayed in diabetic wounds (nonischemic 11.3 ± 3.6 vs. 26.6 ± 4.6%; P = 0.015; ischemic 20.6 ± 5.9% vs. 29.4 ± 6.8%; P = 0.33). All these alterations were reversed in p66Shc−/− mice: collagen area and tissue thickness were increased in diabetic wounds, while apoptosis was reduced and epithelial re-covering was significantly improved. Interestingly, capillary density within the granulation tissue was significantly lower in nondiabetic nonischemic p66Shc−/− mice compared withWT mice (50.8 ± 3.1 vs. 159.6 ± 11.2 hpf; P < 0.0001), but this difference was abrogated in the presence of either diabetes or ischemia, and was inverted in the presence of both diabetes and ischemia (92.8 ± 18.7 vs. 42.8 ± 6.6 hpf; P = 0.002). Results were confirmed in mice with the C57BL/6 genetic background after 1 month of diabetes (supplementary Fig. III). Collectively, these data using two different mouse strains strongly indicate that deletion of p66Shc reverts the negative effect of diabetes on development and ischemia-induced vascularization of the granulation tissue.

Bottom Line: Genetic deletion of p66Shc prolongs life span and protects against oxidative stress.Migration of p66Shc(-/-) dermal fibroblasts in vitro was significantly faster than WT fibroblasts under both high glucose and hypoxia. p66Shc is involved in the delayed wound-healing process in the setting of diabetes and ischemia.Thus, p66Shc may represent a potential therapeutic target against this disabling diabetes complication.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical and Experimental Medicine, University of Padova Medical School, Padova, Italy. gianpaolofadini@hotmail.com

ABSTRACT

Objective: The redox enzyme p66Shc produces hydrogen peroxide and triggers proapoptotic signals. Genetic deletion of p66Shc prolongs life span and protects against oxidative stress. In the present study, we evaluated the role of p66Shc in an animal model of diabetic wound healing.

Research design and methods: Skin wounds were created in wild-type (WT) and p66Shc(-/-) control and streptozotocin-induced diabetic mice with or without hind limb ischemia. Wounds were assessed for collagen content, thickness and vascularity of granulation tissue, apoptosis, reepithelialization, and expression of c-myc and beta-catenin. Response to hind limb ischemia was also evaluated.

Results: Diabetes delayed wound healing in WT mice with reduced granulation tissue thickness and vascularity, increased apoptosis, epithelial expression of c-myc, and nuclear localization of beta-catenin. These nonhealing features were worsened by hind limb ischemia. Diabetes induced p66Shc expression and activation; wound healing was significantly faster in p66Shc(-/-) than in WT diabetic mice, with or without hind limb ischemia, at 1 and 3 months of diabetes duration and in both SV129 and C57BL/6 genetic backgrounds. Deletion of p66Shc reversed nonhealing features, with increased collagen content and granulation tissue thickness, and reduced apoptosis and expression of c-myc and beta-catenin. p66Shc deletion improved response to hind limb ischemia in diabetic mice in terms of tissue damage, capillary density, and perfusion. Migration of p66Shc(-/-) dermal fibroblasts in vitro was significantly faster than WT fibroblasts under both high glucose and hypoxia.

Conclusions: p66Shc is involved in the delayed wound-healing process in the setting of diabetes and ischemia. Thus, p66Shc may represent a potential therapeutic target against this disabling diabetes complication.

Show MeSH
Related in: MedlinePlus