Bovine Milk–derived Lactoferrin Exerts Proangiogenic Effects in an Src-Akt-eNOS–dependent Manner in Response to Ischemia

Lactoferrin (LF) exerts a variety of biological effects, including the promotion of angiogenesis by increasing the expression of angiogenesis-related genes and reducing blood pressure via a nitric oxide-dependent mechanism. In the present study, we investigated the effects of LF on angiogenesis using C57BL/6J mice that received daily unilateral treatment with or without bovine milk-derived LF (bLF) following unilateral hindlimb surgery. The analysis of laser speckle blood flow showed that bLF treatment promoted blood flow recovery in response to ischemic hindlimb. The capillary density of ischemic adductor muscles, as well as the phosphorylation of Src, Akt, and endothelial nitric oxide synthase (eNOS) was also significantly higher in bLF-treated mice than in vehicle-treated mice. Furthermore, bLF increased the phosphorylation levels of Src, Akt, and eNOS in in vitro experiments using human aortic endothelial cells (HAECs). The action of bLF on eNOS phosphorylation was abolished by both LY294002, a phosphatidylinositol 3-kinase inhibitor, and PP2, a Src inhibitor. Similarly, bLF-induced acceleration of tube formation, cell proliferation, and cell migration in HAECs were inhibited by LY294002 or PP2. Thus, bLF promotes vascular endothelial cell function via an Src-Akt-eNOS dependent pathway, thereby contributing to revascularization in response to ischemia.