Immuno-modulatory effect of local rhEGF treatment during tissue repair in diabetic ulcers

Abstract

Wound healing is a complex process that can be severely impaired due to pathological situations such as diabetes mellitus. Diabetic foot ulcers are a common complication of this pathology and are characterized by an excessive inflammatory response. In this work, the effects of local treatment with recombinant human epidermal growth factor (rhEGF) were studied using a full-thickness wound healing model in streptozotocin-induced diabetic rats. Wound healing process was assessed with different concentrations of rhEGF (0.1, 0.5, 2.0 and 8.0 µg/mL), placebo and both diabetic and non-diabetic controls (n = 53). The macroscopic healing observed in treated diabetic rats was affected by rhEGF concentration. Histologically, we also observed an improvement in the epithelialization, granulation tissue formation and maturation in treated groups, finding again the best response at doses of 0.5 and 2.0 µg/mL. Afterwards, the tissue immune response over time was assessed in diabetic rats using the most effective concentrations of rhEGF (0.5 and 2.0 µg/mL), compared to controls. The presence of macrophages, CD4⁺ T lymphocytes and CD8⁺ T lymphocytes, in the reparative tissue was quantified, and cytokine expression was measured by quantitative real-time PCR. rhEGF treatment caused a reduction in the number of infiltrating macrophages in the healing tissue of diabetic, as well as diminished activation of these leukocytes. These findings show that local administration of rhEGF improves the healing process of excisional wounds and the quality of the neoformed tissue in a dose-dependent manner. Besides, this treatment reduces the local inflammation associated with diabetic healing, indicating immuno-modulatory properties.

Keywords: diabetes mellitus; diabetic foot ulcer; rhEGF; wound healing.

Figure 1

Injection pattern followed in the administration of rhEGF. Perilesional administration (blue spots): 4 subcutaneous injections, 1.1 mL/point. Intralesional administration (white spots): 5 intramuscular injections, 1.1 mL/point.

Figure 2

(A) Complete closure of the defect. Results are expressed as cumulative percentage of animals presenting a closed wound at days 16, 18 and 21 after surgery. (B) Differences in the epidermal thickness between unwounded skin and healed tissue in the different study groups. Results are expressed as mean ± s.d. (µm).

Figure 3

Histological examination of the scar tissue at day 21 with Masson’s trichrome staining. (A) DR-CTRL group, aspect of healing area of one of the animals that did not complete wound closure (OM 5×). (B) DR-CTRL group. Foreign body and inflammatory infiltration in the deep tissue of a fully closed animal (OM 20×). (C) DR-EGF/0.5 group. Epidermis of the repaired tissue (OM 10×). (D) DR-EGF/2.0 group. Dermal–epidermal junction organization (OM 5×). Bar = 200 µm.

Figure 4

Presence of T lymphocytes (CD4⁺ and CD8⁺) in the different groups at day 21: Minimal infiltration was observed in all groups, both for CD4⁺ (left panel) and CD8⁺ (right panel). (A) Presence of CD4⁺ T cells in HR group (OM 64×); (B) presence of CD4⁺ T lymphocytes in DR-PLCB group (OM 40×); (C) presence of CD4⁺ T lymphocytes in DR-EGF/0.5 group (OM 40×); (D) presence of CD8⁺ T lymphocytes in HR group (OM 40×); (E) presence of CD8⁺ T cells in DR-PLCB group (OM 40×); (F) presence of CD8⁺ T lymphocytes in DR-EGF/0.5 group (OM 64×). Bar = 50 µm.

Figure 5

Presence of macrophages in the neo-dermal area: (A, B, C and D) CD68⁺ cells at day 21. Bar = 50 µm. (A) HR group (OM 40×); (B) DR-PLCB group (OM 40×); (C) DR-EGF/0.5 group (OM 40×); (D) DR-EGF/2.0 group (OM 40×); (E) evolution of the wound macrophage presence over time (3–21 day), expressed as number of CD68⁺ cells/field. (δ): * vs HR 3 days; (ϕ): ** vs HR 7 days; (ψ): ** vs DR-0.5 3 dyas; (θ): *** vs DR-0.5 14 days; (γ): ** vs DR-2.0 14 days.

Figure 6

Evolution of the cytokine gene expression over time (3–21 days), measured by qRT-PCR. Results are expressed as arbitrary units. (A) TNF-α; (B) IL-12p40; (C) IL-10.