Combination therapy accelerates diabetic wound closure

Abstract

Background: Non-healing foot ulcers are the most common cause of non-traumatic amputation and hospitalization amongst diabetics in the developed world. Impaired wound neovascularization perpetuates a cycle of dysfunctional tissue repair and regeneration. Evidence implicates defective mobilization of marrow-derived progenitor cells (PCs) as a fundamental cause of impaired diabetic neovascularization. Currently, there are no FDA-approved therapies to address this defect. Here we report an endogenous PC strategy to improve diabetic wound neovascularization and closure through a combination therapy of AMD3100, which mobilizes marrow-derived PCs by competitively binding to the cell surface CXCR4 receptor, and PDGF-BB, which is a protein known to enhance cell growth, progenitor cell migration and angiogenesis.

Methods and results: Wounded mice were assigned to 1 of 5 experimental arms (n = 8/arm): saline treated wild-type, saline treated diabetic, AMD3100 treated diabetic, PDGF-BB treated diabetic, and AMD3100/PDGF-BB treated diabetic. Circulating PC number and wound vascularity were analyzed for each group (n = 8/group). Cellular function was assessed in the presence of AMD3100. Using a validated preclinical model of type II diabetic wound healing, we show that AMD3100 therapy (10 mg/kg; i.p. daily) alone can rescue diabetes-specific defects in PC mobilization, but cannot restore normal wound neovascularization. Through further investigation, we demonstrate an acquired trafficking-defect within AMD3100-treated diabetic PCs that can be rescued by PDGF-BB (2 μg; topical) supplementation within the wound environment. Finally, we determine that combination therapy restores diabetic wound neovascularization and accelerates time to wound closure by 40%.

Conclusions: Combination AMD3100 and PDGF-BB therapy synergistically improves BM PC mobilization and trafficking, resulting in significantly improved diabetic wound closure and neovascularization. The success of this endogenous, cell-based strategy to improve diabetic wound healing using FDA-approved therapies is inherently translatable.

Figure 1. AMD3100 treatment (10 mg/kg IP), but not PDGF-BB (2 μg/wound topically), rescues the BM PC mobilization defect in wounded diabetic mice.

Circulating(c) PC (L^-S⁺K⁺) from wounded AMD3100-treated (A+), PDGF-BB treated (P+), or saline-treated (DB) db/db or wild-type (WT) mice were FACS-sorted from the circulating blood volume and quantified. A) Systemic AMD3100 mobilizes diabetic PCs at or above wild-type levels within the first two weeks post-injury while PDGF-BB does not alter diabetic PC mobilization. B) Over 3 weeks, area-under-curve analysis demonstrates a 6.2-fold increase in AMD3100-mediated BM PC-mobilization compared to saline-treated controls. (*p<0.05, **p<0.01 compared to wild-type control, values represent mean +/− SEM, 8 animals/group.)

Figure 2. Combination therapy restores wound neovascularization partially though PC-mediated vasculogenesis.

Wounds from AMD3100-treated (A+), AMD3100/PDGF-treated (A+P+), saline-treated diabetic (DB), and wild-type (WT) mice were harvested at post wounding day 21 for analysis. A,B) Immunofluorescent-staining of 28-day-old wounds for endothelial marker, CD31, demonstrates that only A+P+ therapy normalizes wound neovascularization to wild-type levels. C) BrdU-labeling of PCs in the presence of AMD3100 demonstrates only minor, non-significant, inhibition in PC proliferation (15.4±3.3% decrease, p = 0.06). D) When 2.5×10⁴ DiI-labeled PCs were intravascularly delivered to wounded animals on post-wounding day 1 and wounds were then harvested on post-wounding day 28 after lectin perfusion, we observe direct incorporation of PCs (red) into wound neovasculature (green) with DAPI counterstain (blue). (*p<0.05, **p<0.01 compared to wild-type control, values represent mean +/− SEM, 8 animals/group.)

Figure 3. AMD3100 is PC-specific, altering PC migration towards SDF1α, but not towards PDGF-BB.

0.5×10⁴ PCs or primary db/db fibroblasts were plated in a 96-well plate, cultured in AMD3100-supplemented or control media for 3 days. A) BrdU staining of these cells reveals that AMD3100 does not significantly alter their proliferative capacity (p>0.05). B) Additionally, AMD3100 treatment of either cell line did not alter adhesion to fibronectin-coated chamber slides. C) After 5×10⁴ diabetic PCs were seeded on a fibronectin-coated 24-transwell insert with a receiver compartment containing media supplemented with either SDF1α (100 mg/ml) or PDGF-BB (100 ng/ml) and allowed to migrate for 20 hours, we found that AMD3100 treatment significantly impaired PC migration towards SDF1α (25% decrease, p<0.05) but not towards PDGF-BB (8.4% decrease, p>0.05). (*p<0.05, **p<0.01 compared to wild-type control, values represent mean +/− SEM, 8 animals/group.)

Figure 4. Combination therapy restores diabetic wound closure.

Stented dorsal full-thickness dermal wounds were created on wild-type (WT) mice and diabetic mice treated with either saline (DB), AMD3100 (A+), PDGF-BB (P+), or a combination of both AMD3100 and PDGF-BB daily (A+P+). A) Representative photographs from each treatment group at days 0, 7, 14, 21, and 28 post-wounding. B) Using photogrammetric analysis, the percent wound closure was measured and compared between groups, with A+P+ mice showing wound healing rates comparable to WT mice. (*p<0.05, **p<0.01 compared to wild-type control, values represent mean +/− SEM, 8 animals/group.)