Abstract
Excessive angiogenesis (i.e. neovascularization) in atherosclerotic lesions, sites of dissociation of the inflammatory biomarker pentameric C-reactive protein (pCRP) into monomeric CRP (mCRP), represents a focus of plaque instability with haemorrhagic complications. We previously demonstrated mCRP pro-angiogenic effects on cultured aortic endothelial cells. However, mCRP effects in combination with FGF-2, pro-angiogenic factor released by activated macrophages infiltrating developing lesions, have not yet been described. Here, we examined in vitro the angiogenic capabilities of mCRP combined with FGF-2 by performing endothelial cell proliferation, migration, and differentiation including tube formation and spheroid sprouting assays. The signaling pathways were also investigated by Western blotting and all the cell-based assays were used with or without pharmacological inhibitors of mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 kinase (PI3K) and AƒA½A‚³-secretase, considered as key regulators of angiogenesis. We showed that mCRP-induced endothelial cell proliferation and migration required activation of PI3K pathway. MAPK pathway was essential in mCRP-induced endothelial cell proliferation and spheroid sprouting while AƒA½A‚³-secretase activity was indispensable for mCRP-induced tube formation only. MAPK pathway was required in all FGF-2-stimulated angiogenic assays whereas AƒA½A‚³-secretase slightly inhibited FGF-2 angiogenic effects. PI3K pathway was necessary for FGF-2 angiogenic activities except for cell differentiation. In most of the assays, the additive pro-angiogenic effects of mCRP combined to FGF-2 were mainly attenuated by PI3K and MAPK inhibitors. Altogether, mCRP and FGF-2 have common angiogenic signaling pathways through PI3K and MAPK. Thus, the therapeutic use of PI3K and MAPK inhibitors may inhibit this increased vascularization whilst reducing the haemorrhagic complications from unstable plaques.
Highlights
In Western and newly industrialized countries, atherosclerosis is the underlying cause of about 50% of all deaths related to stroke and cardio-vascular diseases [1,2]
The main interest of this study © Under License of Creative Commons Attribution 3.0 License was to get a better understanding of the in vitro pro-angiogenic effects of the combined factors monomeric CRP (mCRP) with basic fibroblast growth factors (FGF-2) and of the associated signaling pathways targeting ERK1/2, the main key regulator of angiogenic cellular and molecular processes
Using various pharmacological inhibitors blocking mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 kinase (PI3K) pathways and γ-secretase activity, we have demonstrated that MAPK and PI3K pharmacological inhibitors mostly fully decreased the angiogenic processes induced by the combined factors mCRP with FGF-2; suggesting that mCRP and FGF-2 have common signaling pathways engaging mainly PI3K and MAPK
Summary
In Western and newly industrialized countries, atherosclerosis (complex chronic inflammatory disease) is the underlying cause of about 50% of all deaths related to stroke and cardio-vascular diseases [1,2]. Under the stimulation of the most potent angiogenic growth factor FGF-2, activated endothelial cells undergo metabolic modifications associated with the main steps of angiogenesis: i.e. production of matrix metalloproteinases (MMP) that degrade the basement membrane and extracellular matrix, stimulation of endothelial cell migration and proliferation; and differentiation resulting in sprout formation and formation of new blood vessels [14,15] These main FGF-2-induced angiogenic cell events occurred following to FGF-2-FGF receptor (FGFR) interactions that trigger a diverse array of cytoplasmic proteins of signaling pathways including mitogen-activated protein kinase (MAPK) such as extracellular-signal regulated kinase (ERK)1/2, the activation of phosphatidylinositol-3 kinase (PI3K) and involvement of γ-secretase, a transmembrane protein [16,17]. This intensified and uncontrolled angiogenic process contributes to the development of an unstable haemorrhagic rupture-prone environment [18]
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