Various effects of 11,12 EET rescue wound healing in a combined model of diabetes and ischemia

Abstract

Chronic non healing wounds in diabetic patients still impose a major problem in modern medicine. Especially additional peripheral vascular disease complicates treatment success in these patients. Thus, we analyzed the effects of 11,12 epoxyeicosatrienoic acid (EET) in a combined model of hyperglycemia and ischemia in mice. Hyperglycemia was induced by Streptozotozin 2 weeks prior to wounding. 3 days before wound creation 2 of the 3 suppling vessels of the moue ear were cautherized for ischemia. Either 11,12 EET or solvent for control was applied. Wound closure as well as TNF-α, TGF-β, SDF-1α, VEGF, CD31, and Ki67 were measured. The wounds closed on day 14.4 ± 0.4 standard deviation (SD). 11,12 EET treatment enhanced healing to 9.8 ± 0.6 SD. TNF-α level was augmented on day 9 compared to control and receded on day 18. TGF-β seemed to be elevated all days observed after 11,12 EET treatment. SDF-1α was enhanced on day 6 and 9 by 11,12 EET, and VEGF on day 6 and 18 as well as CD13 on day 3, 6, and 18. 11,12 EET did not alter Ki67. 11,12 EET are able to rescue deteriorated wound healing in a combined model of hyperglycamia and ischemia by resolution of inflammation, augmentation of neovascularization and increasing expression of TGF-β as well as SDF-1α.

Figure 1

(a) Photograph of the wound on the mouse ear. Black arrows indicating the two ligated vessels. (b) Day of wound closure. Comparison between non-treated and 11,12 EET treated ischemic wounds in diabetic mice. (c) Percentage of closed wound area from day 0–16. Comparison between ischemic wounds of diabetic animals with or without 11,12 EET treatment. (d) Representative pictures of ischemic wounds throughout the wound healing process in diabetic mice and after treatment with 11,12 EET (data is shown as mean ± SD; n = 10). **p < 0.01, ***p < 0.001.

Figure 2

(a) Percentage of TNF-α positive area on day 3, 6, 9, and 18 of ischemic wounds of diabetic mice with 11,12 EET treatment and solvent control; on the right, representative pictures of the staining. (b) Percentage of TGF-β positive cells on day 3, 6, 9, and 18 of ischemic wounds of diabetic mice with 11,12 EET treatment and solvent control, representative pictures of the staining (data is shown as mean ± SD; n = 8 for day 3,6, an 9 n = 10 for day 18). *p < 0.05, **p < 0.01.

Figure 3

(a) Percentage of SDF-1α positive cells on day 3, 6, 9, and 18 of ischemic wounds of diabetic mice with 11,12 EET treatment and solvent control; on the right, representative pictures of the staining (n = 12). (b) Percentage of Ki-67 positive area on day 3, 6, 9, and 18 of ischemic wounds of diabetic mice with 11,12 EET treatment and solvent control; on the right, representative pictures of the staining (data is shown as mean ± SD; n = 8 for day 3,6, an 9 n = 10 for day 18). **p < 0.01.

Figure 4

(a) Percentage of VEGF positive area on day 3, 6, 9, and 18 of ischemic wounds of diabetic mice with 11,12 EET treatment and solvent control; on the right, representative pictures of the staining. (b) Percentage of CD31 positive cells on day 3, 6, 9, and 18 of ischemic wounds of diabetic mice with 11,12 EET treatment and solvent control; on the right, representative pictures of the staining (data is shown as mean ± SD; n = 8 for day 3,6, an 9 n = 10 for day 18). *p < 0.05, **p < 0.01, ***p < 0.001.