α2-Macroglobulin Enhances Vasculogenesis/Angiogenesis of Mouse Embryonic Stem Cells by Stimulation of Nitric Oxide Generation and Induction of Fibroblast Growth Factor-2 Expression

alpha(2)-macroglobulin (alpha M-2) is an acute-phase protein released upon challenges like cardiac hypertrophy and infarction. alpha M-2 signals via the low density lipoprotein receptor-related protein (LRP-1) and may induce stem cell activation. In the present study, the effects of alpha M-2 on vasculogenesis/angiogenesis and underlying signaling cascades were investigated in mouse embryonic stem (ES) cells. LRP-1 was expressed in ES cells and upregulated during differentiation. alpha M-2 dose dependently increased CD31-positive vascular structures in ES cell-derived embryoid bodies, the early cardiovascular markers isl-1, Nkx-2.5, and flk-1 as well as numbers of VE-cadherin and flk-1-positive cells, but downregulated alpha-smooth muscle actin. Enhancement of vasculogenesis/angiogenesis by alpha M-2 was abolished by the LRP-1 antagonist receptor-associated protein (RAP) and LRP-1 blocking antibody. Notably, alpha M-2 stimulated vascular growth in the chicken chorioallantois membrane assay, but not in a human umbilical vein endothelial cell spheroid model. alpha M-2 increased fibroblast growth factor-2 (FGF-2) protein expression, which was abolished by RAP, induced nitric oxide (NO) generation as determined by 4,5-diaminofluorescein diacetate microfluorometry, and activated nitric oxide synthase-3 (NOS-3) as well as extracellular-regulated kinase 1,2 (ERK1/2) and phosphatidyl inositol 3-kinase (PI3K). NO generation, the increase in FGF-2 expression, and the stimulation of vasculogenesis/angiogenesis by alpha M-2 were blunted by the NO synthase inhibitor L-NAME, the ERK1/2 inhibitor PD98059, and the PI3K inhibitor LY294002. Furthermore, vasculogenesis/angiogenesis by alpha M-2 was inhibited in the presence of the FGF receptor 1 antagonist SU5402. In conclusion, alpha M-2 stimulates endothelial and early cardiac, but not smooth muscle differentiation of ES cells through generation of NO, activation of ERK1/2 as well as PI3K, and induction of FGF-2 expression.