Blocking RAGE improves wound healing in diabetic pigs

Abstract

Receptor for Advanced Glycated End-products (RAGE) is highly expressed in diabetes and impairs wound healing. We proposed that administering an antibody that blocks RAGE will hasten the healing of dorsal wounds in diabetic pigs compared with a non-immune IgG. Two purpose-bred diabetic (D) Yucatan minipigs (Sinclair, Auxvasse MO) each underwent 12 2 × 2 cm full thickness dorsal wounds: four wounds received decellularized porcine skin patches (Xylyx Bio, Bklyn NY): four anti-RAGE Ab (CR-3) infused patches, four saline infused patches and four wounds were left open. One pig received anti-RAGE Ab (CR-3) 1 mg/kg IM q 10 days and other received non-immune IgG. Wounds were measured at 2 and 4 weeks followed by euthanasia and wound harvesting. At 2 weeks few of the patches appeared to be incorporated into the wound. By 4 weeks all patches in pigs treated systemically with CR-3 were detached and the wounds almost healed. For all 24 wounds for both pigs regardless of presence of patch or type of patch, the average IgG treated pig wound size at 4 weeks was 69.2 ± 14.6% of initial size and the average CR-3 treated pig wound size was 40.9 ± 11.3% of initial size (P = 0.0002). Quantitative immunohistology showed greater staining for collagen in the CR-3 treated wounds compared with IgG treated. Staining was positive for RAGE, Mac, and IL-6 in the IgG treated wounds and negative in the CR-3 treated wounds. From these pilot experiments, we conclude that a RAGE blocking antibody given parenterally improved wound healing in a diabetic pig while patches were not effective.

Keywords: RAGE; diabetes; wound healing.

FIGURE 1

Digital photographs of the dorsum of each pig focused on the area of wounding and patch placement. Row (A) patch plus CR‐3, row (B) patch alone, (C) wound without patch. Left images from pig receiving q 10 days IM injections of 1 mg/kg IgG and right images from pig receiving same dosing of CR‐3. The top panels show baseline photos and bottom panels photos taken at 4 weeks

FIGURE 2

Top graphs shows plot of serial values for average % wound size from baseline, 2 and 4 weeks for IgG treated pig on left and CR‐3 treated pig on right. Legend identifies the three groups: patch + CR‐3 (blue line), patch alone (orange line), and no patch (grey line). The tables on the bottom show the numerical values (average ± SD) for the data points plotted in the graphs for 2 and 4 weeks for the IgG treated pig on the left and the CR‐3 treated pig on the right

FIGURE 3

Bars represent average ± SD for all wounds on the IgG treated pig (n = 12) (light grey) and for the CR‐3 treated pig (n = 12) (dark grey)

FIGURE 4

Pentachrome IHC staining on wound sections taken at necropsy on the two pigs. The % collagen (yellow stain) for each section is shown on the image. The yellow staining was higher in the CR‐3 treated pig for wounds receiving no patch, patch alone, and patch plus Cr‐3. The bottom images show “stitched” together sections to give an overall view of wound size and collagen content from IgG treated (left) and CR‐3 treated pig (right)

FIGURE 5

Pentachrome ICH from IgG treated pig wound on left and CR‐3 treated pig on right with pentachrome on top and RAGE staining on bottom

FIGURE 6

IHC stained tissue sections for macrophages (Mac), TNF‐α in middle, and IL‐6 on right for diabetic pig treated with IgG top, and diabetic pig treated with CR‐3 on bottom row