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 this 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) after 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 and the phosphorylation of Src, Akt, and endothelial nitric oxide synthase (eNOS) were 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. The action of bLF on eNOS phosphorylation was abolished by both LY294002, a phosphatidylinositol 3-kinase inhibitor, and 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo [3,4-d]pyrimidine (PP2), an Src inhibitor. Similarly, bLF-induced acceleration of tube formation, cell proliferation, and cell migration in human aortic endothelial cells 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.
Department of Pharmacology, University of Tokushima Graduate School of Health Biosciences, Tokushima, Japan. yasuike@tokushima-u.ac.jp
Yasumasa
Y
Ikeda
Soichiro
S
Tajima
Yuki
Y
Izawa-Ishizawa
Yoshitaka
Y
Kihira
Keisuke
K
Ishizawa
Sumiko
S
Yoshida
Ken-ichi
K
Aihara
Koichiro
K
Tsuchiya
Toshiaki
T
Tamaki
D000818:Animals; D001011:Aorta / Q000166:cytology; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000502:physiology; D004730:Endothelium, Vascular / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D006614:Hindlimb / Q000098:blood supply / Q000503:physiopathology; D006801:Humans; D066298:In Vitro Techniques; D007511:Ischemia / Q000503:physiopathology; D007781:Lactoferrin / Q000494:pharmacology*; D017078:Laser-Doppler Flowmetry; D051379:Mice; D008810:Mice, Inbred C57BL; D008892:Milk / Q000737:chemistry; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015427:Reperfusion Injury / Q000378:metabolism / Q000503:physiopathology*; D015398:Signal Transduction; D019061:src-Family Kinases / Q000378:metabolism*
C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D019061:src-Family Kinases; D051057:Proto-Oncogene Proteins c-akt; D007781:Lactoferrin