Skin wound healing in diabetic β6 integrin-deficient mice

Abstract

Integrin αvβ6 is a heterodimeric cell surface receptor, which is absent from the normal epithelium, but is expressed in wound-edge keratinocytes during re-epithelialization. However, the function of the αvβ6 integrin in wound repair remains unclear. Impaired wound healing in patients with diabetes constitutes a major clinical problem worldwide and has been associated with the accumulation of advanced glycated endproducts (AGEs) in the tissues. AGEs may account for aberrant interactions between integrin receptors and their extracellular matrix ligands such as fibronectin (FN). In this study, we compared healing of experimental excisional skin wounds in wild-type (WT) and β6-knockout (β6(-/-) ) mice with streptozotocin-induced diabetes. Results showed that diabetic β6(-/-) mice had a significant delay in early wound closure rate compared with diabetic WT mice, suggesting that αvβ6 integrin may serve as a protective role in re-epithelialization of diabetic wounds. To mimic the glycosylated wound matrix, we generated a methylglyoxal (MG)-glycated variant of FN. Keratinocytes utilized αvβ6 and β1 integrins for spreading on both non-glycated and FN-MG, but their spreading was reduced on FN-MG. These findings indicated that glycation of FN and possibly other integrin ligands could hamper keratinocyte interactions with the provisional matrix proteins during re-epithelialization of diabetic wounds.

Fig. 1

(A) Survival data for 50 days pre-wounding and 10 days post-wounding period of wild type (WT; n=9, control; n=14, diabetic) and β6^-/- (n=10, control; n=15, diabetic) mice. (B) Blood glucose levels for WT control vs. diabetic animals and (C) for β6^-/- control vs. β6^-/- diabetic animals. (D) Mean body weight for control vs. diabetic animals and (E) for β6^-/- control vs. β6^-/- diabetic animals. (F) Representative images of mouse tails from the untreated control group compared with streptozotocin-induced diabetes group (G) displaying clinical signs of neuropathy.

Fig. 2

(A) Representative clinical photographs of excisional wounds from WT and β6^-/- mice treated with citrate buffer only (control) or 55mg/kg streptozotocin in citrate buffer (diabetic) at day 0, 5 and 10 after wounding. Wound closure rate in the control mice was similar compared to WT and β6^-/- mice, whereas a delay in wound closure could be observed for the diabetic WT and especially the diabetic β6^-/- mice. Scale bar: 10 mm. (B-F) Relative wound area change over time. Data show mean ± sem percent of wound area relative to wound size at day 0 within each group (n=36-56 wounds per group). Statistical significance between the two groups at each time point was assessed by Student's t-test, * p<0.05, ** p<0.01, *** p<0.001.

Fig. 3

(A) Hematoxylin and eosin (H&E) staining of day 5 mid-wound sections from WT and β6^-/- mice treated with citrate-buffer (control) and 55mg/kg streptozotocin in citrate buffer (diabetic). E, epithelium; GT, granulation tissue; CT, connective tissue; WC, wound crust. Dashed lines indicate epithelial gap distance. Scale bar: 1mm. (B) Epithelial gap distances were measured from day 5 mid-wound H&E sections as the distance between epithelial tongues from all four groups. Mann-Whitney's U-test revealed overall significant differences between control and diabetc animals (p<0.01), and also between diabetic WT and β6^-/- mice, respectively (p<0.05). (C) Histology was scored from 1 (lowest) to 4 (highest) based on the degree of cellular invasion, GT formation, vascularity, and re-epithelialization. n=10-12 sections (two ections per wound) from 5-6 mice per group. Scale bars in B and C show mean ± s.d, * p<0.05, ** p<0.01.

Fig. 4

(A) Movat's modified pentachrome staining of representative day 5 mid-wound sections from WT and β6^-/- mice treated with citrate-buffered (control) and 55mg/kg citrate-buffered streptozotocin (STZ, diabetic). Control animals (WT and β6^-/-) show similar dense and well-organized neo-synthesized collagen fibre bundles (blue-green) in the granulation tissue (GT) Small amounts of mature collagen (yellow) appear near the epidermis (E) and at the wound edge (WE) proximal to the unwounded connective tissue (arrows) and clarified by magnified picture insets. STZ-treated diabetic mice show less formation of GT, especially in the case of the β6^-/- group, and no mature collagen is observed in either WT or β6^-/- mice. Scale bars: 100 μm. (B) The degree of GT formation from 5-6 mice per group was scored by an ordinate scale (1 to 4) according to the degree of density and organization of neo-synthesized collagen fibre bundles as low (1), medium (2), or high (3). Presence of mature collagen among well-granulated wounds yielded a score of 4. No statistical difference could be established, but a tendency towards reduced GT formation among the diabetic animals, especially the β6^-/- mice, was observed. Bars show mean ± s.d, n=10-12 sections (two sections per wound) from 5-6 wounds.

Fig. 5

Immunolocalization of β6-integrin in day-5 mid-wound sections from WT and β6^-/- mice treated with citrate-buffer (control) and 55mg/kg citrate-buffered streptozotocin (diabetic). β6^-/- mice showed a complete lack of β6-integrin staining in all specimens examined, with the exception of limited background staining of hair follicles. In the WT mice, the immunoreactivity of β6 integrin was confined to the wound edge of the epithelium containing populations of proliferating and migrating keratinocytes. WE, wound edge; GT, granulation tissue; CT, connective tissue; HF, hair follicles. Scale bar 200 μm.

Fig. 6

(A) Binding affinity of human fibronectin (FN) and the methylglyoxal treated FN (FN-MG). Immunolabelling with specific antibodies (Ab) showed similar binding affinity to the microtiter plate between the two fibronectin-preparations. (B) SDS-PAGE and (C) Western blot using an antibody that recognizes specifically FN-MG. (D) HaCaT keratinocytes were allowed to spread for 1 hour on various concentrations of native fibronectin or FN-MG or (E) on 20 μg/ml fibronectin or FN-MG over time. In both cases, cells showed reduced spreading on FN-MG (○) compared with FN (●) in a concentration- and time-dependent manner. (F) HaCaT spreading was also quantified in the presence of function blocking anti-integrin antibodies targeted against the αv, β1 and β6 subunits. Data shows mean ± sem of three independent experiments, n=4 (D-E) and n=8 (F). The effect of integrin blocking accounted for 88% of the total variation (p<0.001), whereas no statistical difference was observed between keratinocyte spreading on FN and FN-MG within the same anti-integrin antibody blocking. All keratinocyte spreading data (D-F) were evalutated by a two-way ANOVA with Bonferroni post-tests, *p<0.05, ** p<0.01, *** p<0.001.