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  • Endothelial cells receive a variety of information from the


    Endothelial cells receive a variety of information from the environment which finally guides them through all phases of neovascularization. Angiogenic signals enhance the proliferation of endothelial cells, upregulated resistance to apoptosis, alterations within proteolytic balance, reorganization of cytoskeletons, the capacity to migrate and eventually to differentiate and to form a new vascular cavity [19]. The adult endothelial cells are considered a rather inactive cell type [20]. Nevertheless, under the stimulation of angiogenesis, endothelial cells enter into a state of active proliferation. The pathways into the sto products are dependent upon angiogenesis signals that are received, including VEGF/VEGFR, FGF/FGFR, ERK/MAPK, and PI3K/AKT [[21], [22], [23], [24], [25], [26], [27], [28], [29], [30]]. Thus, we examined the changes in the key factors in angiogenesis and in the related signaling pathway; furthermore, the dynamic effects of Mg alloy extracts and signaling pathway inhibitor on HUVEC angiogenesis were detected. Herein, Mg–Zn–Mn alloy microstructure and mechanics performance could be examined and presented. Mg alloy extracts at different concentrations were prepared and added to the HUVEC culture mediums; the DNA synthesis, cell viability, and tube formation of HUVECs under different concentrations of Mg alloy extracts were examined to select an appropriate extract concentration for further experiments. The mRNA expression and protein levels of angiogenesis key regulators, including eNOS, FGF, HIF1α, and MMP2 were determined under Mg alloy extract treatment; the changes in the factors of FGF/FGFR signaling pathway were examined under Mg alloy extract treatment. Finally, we treated HUVECs in Mg alloy extract with or without FGF pathway suppressor BFJ398, then evaluated FGF/FGFR signaling pathway activation and the tube formation capacity of HUVECs. In summary, we examined the effects of Mg alloy extracts on HUVEC angiogenesis in vitro and further investigated the signaling pathway that might be involved.
    Materials and methods
    Discussion Mg has been reported to exert a strong protective effect on the skeleton. The positive function of Mg scaffolds of Mg ion on osteogenic differentiation has been previously reported [7,[32], [33], [34], [35]]. Over the past few years, magnesium and its alloys have been extensively studied as underlying biodegradable implants [8,[36], [37], [38], [39], [40], [41], [42]]. Comparing to pure Mg implants, biodegradable Mg alloys have excellent mechanics performance, which makes them better suit the application of load-bearing implants. In addition, due to their elastic performance which is similar to that of bone, stress shielding can be prevented and bone regeneration can be improved, so they have emerge as ideal materials of hard tissue implants for the stability of fracture [36,38]. Herein, we selected a novel type of Mg-based alloy, namely Mg–Zn–Mn alloy, and examined its effects on endothelial cell angiogenesis, a critical issue in delayed union or nonunion [43]. In addition to the promotive effects reported previously, Mg–Zn–Mn alloy extracts significantly promoted the DNA synthesis, cell viability, and tube formation capacity; therefore, Mg–Zn–Mn alloy extracts could enhance the angiogenesis of HUVECs to improve the fracture healing. Regarding the molecular mechanism, several signaling molecules can act as mitogens, increasing the quantity of cells generated in bone or promoting vascularization, such as FGF [44]. One of the main pathways resulting in endothelial cell proliferation is triggered via the dimerization and autophosphorylation of FGF receptors (especially FGFR1) after FGF2 binding [19]. VEGF binding to the endothelial-specific receptor VEGFR2 has emerged as the major extracellular signal which initiates an angiogenesis response. VEGF-induced mitogenic response can be amplified via NO, possibly via the enhancement of ERK activation. Actually, the VEGF mitogenic effects are significantly reduced by NO production suppressors [45,46]. VEGF enhances eNOS activity, NO-synthase endothelial-specific isoform, via a Src-mediate pathway [47]. In the present study, 6.25% Mg–Zn–Mn alloy extracts significantly increased the mRNA and protein expression of FGF, HIF1α, and MMP2, as well as the ratio of p-eNOS/NOS, indicating the activation of FGF signaling by alloy extracts in HUVECs.