Cutaneous structural and biochemical correlates of foot complications in high-risk diabetes

Abstract

Objective: Impairment of skin quality may contribute to diabetic foot ulceration (DFU). Our goal was to determine whether high-risk patients exhibited specific skin structural and metabolic deficits that could predispose to foot complications.

Research design and methods: A total of 46 patients comprising 9 diabetic control subjects, 16 with diabetic peripheral neuropathy (DPN) alone, and 21 with recurrent DFUs (including 9 with Charcot neuroarthropathy [CNA]) were recruited and compared with 14 nondiabetic control (NDC) subjects. DPN was assessed using the Michigan Neuropathy Screening Instrument (MNSI). Skin punch biopsies (3 mm) were performed on upper and lower leg skin for measurements of intraepidermal nerve fiber density (IENFD), structural analysis, type 1 procollagen abundance, tissue degrading matrix metalloproteinases (MMPs), and poly(ADP-ribose) (PAR) immunoreactivity.

Results: MNSI scores were comparable across DPN groups. IENFD was decreased by diabetes and DPN but did not differ between neuropathic groups. Skin structural deficit scores were elevated in all neuropathic subjects, particularly in the DFU group. Type 1 procollagen abundance was reduced in DFU subjects 387 ± 256 units (mean ± 1 SD) compared with NDC subjects (715 ± 100, P < 0.001). MMP-1 and MMP-2 were activated by diabetes. PAR immunoreactivity was increased in DFU (particularly in the CNA group; P < 0.01) compared with other DPN subjects.

Conclusions: Increased PAR, reduced type 1 procollagen abundance, and impaired skin structure are associated with foot complications in diabetes. The potential of therapies that improve skin quality to reduce DFU needs to be investigated.

Figure 1

A: Comparison of type 1 procollagen abundance in the lower leg in different subject groups. Skin tissue from nondiabetic and diabetic subjects was maintained in organ culture for 8 days. At the end of the incubation period, sections were dewaxed, blocked with 10% horse serum, and incubated with antibodies to type 1 procollagen, and the immunoreactivity was revealed using a VECTASTAIN Universal Elite ABC Kit and diaminobenzidine. The area and density of staining was determined by image analysis using Scion Image. Data are mean ± SD. P < 0.001 for DFU vs. NDC; P = 0.002 for DFU vs. DC; P = 0.025 for DFU vs. DPN; P < 0.001 for DPN vs. NDC. B: Representative immunohistochemical staining for type 1 procollagen in the skin of healthy subjects and subjects with and without DPN and DFU. Original magnification ×200. (A high-quality color representation of this figure is available in the online issue.)

Figure 2

A: Comparison of percentage of PAR + nuclei in different subject groups. Lower leg skin tissue from nondiabetic and diabetic subjects was maintained in organ culture for 8 days. Paraffin sections (4 μm) were prepared. Mouse monoclonal anti-PAR antibody (1:400) was applied to sections. Immunoreactivity was revealed using diaminobenzidine, and sections were counterstained with hematoxylin. Data are mean ± SD. P < 0.001 for DFU vs. NDC; P = 0.006 for DFU vs. DPN; P = 0.018 for DPN vs. NDC. B: Representative immunohistochemical staining for PAR in the skin in an NDC subject and a patient with DFU (and CNA). (A high-quality color representation of this figure is available in the online issue.)