11,12 and 14,15 epoxyeicosatrienoic acid rescue deteriorated wound healing in ischemia

Abstract

Introduction: Epoxyeicosatrienoic acids (EETs) are able to enhance angiogenesis and regulate inflammation that is especially important in wound healing under ischemic conditions. Thus, we evaluated the effect of local EET application on ischemic wounds in mice.

Methods: Ischemia was induced by cautherization of two of the three supplying vessels to the mouse ear. Wounding was performed on the ear three days later. Wounds were treated either with 11,12 or 14,15 EET and compared to untreated control and normal wounds. Epithelialization was measured every second day. VEGF, TNF-α, TGF-β, matrix metalloproteinases (MMP), tissue inhibitors of metalloproteinases (TIMP), Ki67, and SDF-1α were evaluated immunohistochemically in wounds on day 3, 6, and 9.

Results: Ischemia delayed wound closure (12.8 days ± 1.9 standard deviation (SD) for ischemia and 8.0 days ± 0.94 SD for control). 11,12 and14,15 EET application ameliorated deteriorated wound healing on ischemic ears (7.6 ± 1.3 SD for 11,12 EET and 9.2 ± 1.4 SD for 14,15 EET). Ischemia did not change VEGF, TNF-α, TGF-β, SDF-1α, TIMP, MMP7 or MMP9 level significantly compared to control. Local application of 11,12 as well as 14,15 EET induced a significant elevation of VEGF, TGF-β, and SDF-1α expression as well as proliferation during the whole phase of wound healing compared to control and ischemia alone.

Conclusion: In summary, EET improve impaired wound healing caused by ischemia as they enhance neovascularization and alter inflammatory response in wounds. Thus elevating lipid mediator level as 11,12 and 14,15 EET in wounds might be a successful strategy for amelioration of deranged wound healing under ischemia.

Fig 1

A Day of wound closure. Depicted is day of wound closure of controlwounds, ischemic wounds and ischemic wounds after treatment with 11,12 and 14,15 EET. B Representative in vivo pictures of wound healing taken every second day (data is shown as mean ± SD; n = 10). *p<0.05, **p<0.01, ***p<0.001.

Fig 2

A Percentage of closed wound area from day 0–16 of ischemic and non-ischemic ears. B Percentage of closed wound area from day 0–16 of ischemic and 11,12 EET treated wounds. C Percentage of closed wound area from day 0–16 of ischemic and 14,15 EET treated wounds. (data is shown as mean ± SD; n = 10).*p<0.05, **p<0.01, ***p<0.001.

Fig 3

A Percentage of VEGF positive area on day 3, 6, and 9 after wounding ofcontrol, ischemic and 11,12 as well as 14,15 EET treated ischemic wounds. On the right representative pictures of immunohistological staining. B Percentage of Ki67 positive cells on day 3, 6 and 9 after wounding ofcontrol, ischemic and 11,12 as well as 14,15 EET treated ischemic wounds. On the right representative pictures of immunohistological staining (data is shown as mean ± SD; n = 8). *p<0.05, ***p<0.001.

Fig 4

A Percentage of TNF-α positive area on day 3, 6, and 9 after wounding of control, ischemic and 11,12 as well as 14,15 EET treated ischemic wounds. On the right representative pictures of immunohistological staining. B Percentage of TGF-β positive area on day 3, 6 and 9 after wounding of control, ischemic and 11,12 as well as 14,15 EET treated ischemic wounds. On the right representative pictures of immunohistological staining (data is shown as mean ± SD; n = 8). *p<0.05, ***p<0.001.

Fig 5

A: Percentage of MMP7 positive area on day 3, 6 and 9 after wounding of control, ischemic and 11,12 as well as 14,15 EET treated ischemic wounds. On the right representative pictures of immunohistological staining. B: Percentage of MMP9 positive area on day 3, 6 and 9 after wounding of control, ischemic and 11,12 as well as 14,15 EET treated ischemic wounds. On the right representative pictures of immunohistological staining (data is shown as mean ± SD; n = 8). *p<0.05, **p<0.01, ***p<0.001.

Fig 6

A: Percentage of TIMP1 positive area on day 3, 6 and 9 after wounding of control, ischemic and 11,12 as well as 14,15 EET treated ischemic wounds. On the right representative pictures of immunohistological staining. B: Percentage of SDF-1α positive area on day 3, 6 and 9 after wounding of control, ischemic and 11,12 as well as 14,15 EET treated ischemic wounds. On the right representative pictures of immunohistological staining (data is shown as mean ± SD; n = 8). *p<0.05, **p<0.01, ***p<0.001.