Antimycotic ciclopirox olamine in the diabetic environment promotes angiogenesis and enhances wound healing

Abstract

Diabetic wounds remain a major medical challenge with often disappointing outcomes despite the best available care. An impaired response to tissue hypoxia and insufficient angiogenesis are major factors responsible for poor healing in diabetic wounds. Here we show that the antimycotic drug ciclopirox olamine (CPX) can induce therapeutic angiogenesis in diabetic wounds. Treatment with CPX in vitro led to upregulation of multiple angiogenic genes and increased availability of HIF-1α. Using an excisional wound splinting model in diabetic mice, we showed that serial topical treatment with CPX enhanced wound healing compared to vehicle control treatment, with significantly accelerated wound closure, increased angiogenesis, and increased dermal cellularity. These findings offer a promising new topical pharmacologic therapy for the treatment of diabetic wounds.

Figure 1. Effect of CPX treatment in vitro.

(A) Treatment with 5–10 µM CPX for 24 hours on bEND.3 endothelial cell line, NIH3T3 cell line, and primary diabetic fibroblasts stimulated HIF-1α activation in a dose-dependent manner (B) Densiometry data showing pixel density of Western blot data calculated using ImageJ software. Pixel density is expressed as a ratio to β-actin and normalized to untreated cells. Astericks indicate statistically significant increase in protein concentration relative to untreated controls. (*p<0.05) (C) Conditioned media of cells treated with 5–10 µM CPX for 24 hours led to a significant increase in VEGF expression as detected by ELISA in bEND.3, NIH3T3, and diabetic primary fibroblast cells. (*p<0.05).

Figure 2. Downstream effects of CPX treatment in vitro.

(A) QRT-PCR analysis on NIH3T3 cells treated with 10 µM CPX for 24 hours versus untreated control. There were 3 to10-fold increases in the mRNA expression of multiple angiogenesis-related genes relative to that of untreated control cells. Asterisks indicate *p<0.05. (B) Angiogenesis antibody array using condition media from NIH3T3 cells treated with 10 µM CPX for 24 hours versus condition media from untreated control cells. Membrane images of antibody array treated with condition media of treated and control cells (top). Specific proteins captured on the array are labeled 1 to 8. Semi-quantitative profiles of proteins 1 to 8 on the angiogenesis antibody array using reverse image scanning densitometry (bottom). There was a significant increase in the expression of multiple angiogenic proteins by NIH3T3 cells treated with CPX compared to untreated control cells. Asterisks indicate *p<0.05. (C) The ability of CPX (10 µM) in inducing in vitro tubule formation with or without VEGF (10 ng/ml) treatment. Representative cell images showing field of view as indicated (left). The angiogenic effect was quantified by counting the number of branch points per field of view (right). Endothelial cells treated with CPX (10 µM) increased the number of branch point cells with or without concomitant VEGF (10 ng/ml) treatment compared to those cells treated with DMSO control (40X). White arrows indicate examples of branch points (*p<0.05).

Figure 3. Topical CPX enhanced wound healing in diabetic mice.

Full-thickness skin wounds on diabetic mice were treated topically with 50 mM CPX, DMSO as vehicle control, and no treatment every other day until wound closure. (A) Wound closure rate over time. There was a significant acceleration of wound healing in the CPX treatment group (n = 10; *p<0.05) (top). Representative photographs of wounds treated with DMSO vehicle control, no treatment, or 50 mM CPX (bottom). Treatment with CPX healed wounds significantly faster with increased vascularity (grossly represented in red color) in the wound bed compared to vehicle control. (B) Western blot showing statistically significant HIF-1α expression in tissue lysates harvested from wounds treated with CPX compared to those harvested from unwounded untreated skin, untreated wounded skin, and vehicle-treated wounded skin. (*p<0.01)

Figure 4. Histological analysis of wounded skin.

(A) Effects of CPX on wound vascularity. Wound sections were stained with an anti-CD31 antibody and detected with Fluor 594 (red). Software-assisted quantification of vessel density in the entire area of residual wounds at day 22, as percent fluorescence. Wounds from CPX-treated mice had significantly higher vessel density compared with that from DMSO-treated mice (*p<0.05). (B) Hematoxylin and Eosin stains of wound tissue after full closure at 22 days post-wounding (left). CPX-treated wounds have higher dermal cellularity in the wound bed compared to DMSO-treated control wounds. The black box indicates the area of healed wound depicted under high power view. Quantification of cellularity in the wound by counting total cell number per high-powered field views (10×40) (right).