Document Type


Publication Date





Objective: 20-Hydroxyeicosatetraenoic acid (20-HETE), an important bioactive lipid metabolite, has recently been identified to be a novel contributor of angiogenesis secondary to ischemia. Moreover, an inflammatory response is required for the initiation of ischemic angiogenesis, in response to ischemic tissue injury. The goal of this study is to investigate the role of inflammation in 20-HETE regulation of ischemia-induced angiogenesis.

Methods: We first established a mouse hind limb ischemia model for immunocompetent Balb/C mice and immunodeficient NOD-SCID mice by femoral artery ligation. Groups of Balb/C and NOD-SCID mice were administered a 20-HETE synthesis inhibitor, DDMS, or saline as a solvent control. Laser Doppler perfusion imaging (LDPI) was used to visualize and quantify blood perfusion on days 0, 1, 3, 7, 14, and 21 post ligation, confirmed by microvessel density analysis. LC/MS/MS analysis was performed on day 3 post ligation on ischemic and non-ischemic control gracilis muscles to measure 20-HETE levels. Additionally, an antibody to lymphocyte antigen 6 complex (Ly6G/C) was administered to neutralize the infiltration of neutrophils, macrophages, and monocytes. 20-HETE levels were again measured on day 3 post ligation in these mice.

Results: Quantification of the compensatory blood perfusion recovery post ischemia by LDPI showed that immunocompetent Balb/C control mice demonstrated a normal course of the compensatory angiogenic response while NOD-SCID immunodeficient mice showed a significantly decreased response. Additionally, DDMS was shown to inhibit the compensatory response in Balb/C mice, while no inhibitory effect was observed in immunodeficient NOD-SCID mice. This observation is confirmed by a marked decrease in microvessel density in SCID mice (1.5±0.2) post ischemia compared to immunocompetent Balb/C mice (2.65±0.32). As expected, ischemia markedly increased 20-HETE levels in the ischemic gracilis muscle of Balb/C mice by 6-fold (6±2 in non-ischemic vs 27±5 pg/mg in ischemic), while levels in NOD-SCID mice showed no change between the ischemic and non-ischemic control. Lastly, Balb/C mice that were treated with Ly6G/C neutralizing antibody exhibited significantly decreased 20-HETE levels in their ischemic gracilis muscle compared to the non-ischemic control.

Conclusion: Inflammation may be an essential contributor in 20-HETE regulation of the ischemia-induced angiogenic response.