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

Abstract

α2-macroglobulin (α2M) is an acute-phase protein released upon challenges like cardiac hypertrophy and infarction. α2M signals via the low density lipoprotein receptor-related protein (LRP-1) and may induce stem cell activation. In the present study, the effects of α2M 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. α2M 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 α-smooth muscle actin. Enhancement of vasculogenesis/angiogenesis by α2M was abolished by the LRP-1 antagonist receptor-associated protein (RAP) and LRP-1 blocking antibody. Notably, α2M stimulated vascular growth in the chicken chorioallantois membrane assay, but not in a human umbilical vein endothelial cell spheroid model. α2M 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 α2M were blunted by the NO synthase inhibitor L-NAME, the ERK1/2 inhibitor PD98059, and the PI3K inhibitor LY294002. Furthermore, vasculogenesis/angiogenesis by α2M was inhibited in the presence of the FGF receptor 1 antagonist SU5402. In conclusion, α2M 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.

LDL receptor-related protein-1 regulates NFκB and microRNA-155 in macrophages to control the inflammatory response

“…EI-tPA and α 2 M* had the entirely opposite effect on BMDM physiology, suppressing NFκB activation in response to LPS and decreasing expression of proinflammatory mediators. In previous studies with different cell types, RAP and LF have been shown to competitively antagonize LRP1-dependent cellular responses triggered by ligands like α 2 M* or tPA (15)(16)(17)(33)(34)(35). We hypothesized that, under basal conditions, macrophages express one or more agonistic ligands that bind to LRP1 in an autocrine pathway, suppressing NFκB activation.…”

Cardiogel: A Nano-Matrix Scaffold with Potential Application in Cardiac Regeneration Using Mesenchymal Stem Cells

“…Tmsb4x and A2m have been reported to enhance the differentiation of Embryonic Stem Cells (ESCs) into functional cardiomyocytes [31] , [32] . Cardiogel has also been shown to promote early differentiation and maturation of ESC-derived cardiomyocytes [14] , [33] .…”

“…Cardiogel has also been shown to promote early differentiation and maturation of ESC-derived cardiomyocytes [14] , [33] . Tmsb4x and A2m have also been shown to stimulate angiogenesis by promoting endothelial cell migration, survival and differentiation [32] , [34] , [35] . Tmsb4x and A2m were identified in the proteomic analysis, suggesting a role for these proteins in the differentiation and angiogenic potential of cardiogel.…”

“…RT-PCR analysis showed an increased expression of cardiac transcription factors such as Gata4 and Mef2c, the major cardiac gap junction protein Cx43, a cardiac hormone BNP, the adrenergic receptor Adra1a and Adra1b and the muscarinic acetylcholine receptor, Chrm1 while Western Blotting showed an increase in the expression of GATA4, ACTA1 and CX43 in BMSCs cultured on cardiogel compared to those cultured on gelatin coated controls. The cardiomyogenic differentiation of BMSCs on cardiogel could be contributed to the presence of proteins such as Tmsb4x, A2m, laminin and fibronectin, which are known to enhance cardiomyogenic differentiation of stem cells [31] , [32] , [58] , [59] .…”

“…In addition, RT-PCR analysis showed that there was an increased expression of angiogenic markers such as FLT1, CD31 and CDH5 in EA.hy926 cells cultured on cardiogel in comparison to gelatin coated controls. The angiogenic potential of cardiogel could be attributed to the synergistic effect of various proteins such as collagens I and III, laminin, fibronectin, Tmsb4x and A2m which are known to promote endothelial cell proliferation, migration and neo-vasculogenesis [32] , [34] , [35] , [61] – [68] .…”