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

Abstract

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.

Antioxidant Effects of Bovine Lactoferrin on Dexamethasone-Induced Hypertension in Rat

“…It has been suggested that the vasodilatory action of LF is strongly mediated by NO production because of complete blockade of this effect by a NO synthase inhibitor [19]. Ikeda and coworkers have reported significant phosphorylation of Src, Akt, and endothelial nitric oxide synthase (eNOS) after treatment with LF suggesting a Src Akt eNOS-dependent pathway in promotion of vascular endothelial cell function by LF [31]. …”

Endothelial dysfunction in individuals born after fetal growth restriction: cardiovascular and renal consequences and preventive approaches

“…Breastfeeding could improve endothelial function, primarily due to the presence of lactoferrin, an iron-binding glycoprotein with antioxidant, anti-inflammatory, pro-angiogenic and NO-dependent vasodilator properties. Daily treatment with lactoferrin after unilateral hind limb surgery-induced ischemia in C57BL/6J mice promoted angiogenesis, activated endothelial function via an NO-dependent mechanism 171 and protected HUVECs against hydrogen peroxide-induced oxidative stress 172 …”

Bilirubin exerts pro-angiogenic property through Akt-eNOS-dependent pathway

“…20 Capillary density and arterioles number Capillary density was estimated by CD31 immunohistochemistry. 20,21 Briefly, 28 days after surgery, adductor muscles were snap frozen in liquid nitrogencold isopentane containing optimal cutting temperature compound and cut into 8 μm cryosections. Capillary density was expressed as the number of CD31-positive cells corrected for the number of muscle fibers.…”

“…20,21 Briefly, 1 × 10 4 HAECs were cultured per well of Matrigel-coated 96-well plate in the presence of each concentration of bilirubin or vehicle, with or without 2 μM LY294002. Cells were then incubated at 37°C for 18 h. The formation of tube-like structures were observed using an inverted contrast microscope and captured with a CCD camera.…”