Monomeric C-reactive protein and Notch-3 co-operatively increase angiogenesis through PI3K signalling pathway

• Published in: Cytokine (Philadelphia, Pa.) Vol. 69; no. 2; pp. 165 - 179
• Main Authors: Boras, Emhamed, Slevin, Mark, Alexander, M. Yvonne, Aljohi, Ali, Gilmore, William, Ashworth, Jason, Krupinski, Jerzy, Potempa, Lawrence A., Al Abdulkareem, Ibrahim, Elobeid, Adila, Matou-Nasri, Sabine
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.10.2014
• Subjects: Angiogenesis; Animals; Blotting, Western; C-reactive protein; C-Reactive Protein - pharmacology; Cadherins - metabolism; Cattle; Cell Movement - drug effects; Cell Proliferation - drug effects; Chickens; Chromones - pharmacology; Coculture Techniques; Dipeptides - pharmacology; Down-Regulation - drug effects; Electrophoresis, Agar Gel; Endothelial Cells - cytology; Endothelial Cells - drug effects; Endothelial Cells - metabolism; Gamma-secretase; Morpholines - pharmacology; Myocytes, Smooth Muscle - cytology; Neovascularization, Physiologic - drug effects; Nitric Oxide Synthase Type III - metabolism; Notch; Phosphatidylinositol 3-Kinases - metabolism; PI3K/Akt pathway; Proto-Oncogene Proteins c-akt - metabolism; Rats; Receptors, Notch - metabolism; Signal Transduction - drug effects; Spheroids, Cellular - cytology; Spheroids, Cellular - drug effects; Up-Regulation - drug effects; Gamma-secretase; Angiogenesis; C-reactive protein; PI3K/Akt pathway; Notch
• ISSN: 1043-4666; 1096-0023; 1096-0023
• DOI: 10.1016/j.cyto.2014.05.027

•Monomeric CRP up-regulates endothelial Notch-3 receptor gene expression.•mCRP and Notch3 activator cooperatively stimulate angiogenesis through PI3K pathway.•mCRP with Notch-3 activator together stabilize the co-vascular structures.•Potential therapeutic combination to reduce risk of hemorrhage from unstable plaque. C-reactive protein (CRP) is the most acute-phase reactant serum protein of inflammation and a strong predictor of cardiovascular disease. Its expression is associated with atherosclerotic plaque instability and the formation of immature micro-vessels. We have previously shown that CRP upregulates endothelial-derived Notch-3, a key receptor involved in vascular development, remodelling and maturation. In this study, we investigated the links between the bioactive monomeric CRP (mCRP) and Notch-3 signalling in angiogenesis. We used in vitro (cell counting, wound-healing and tubulogenesis assays) and in vivo (chorioallantoic membrane) angiogenic assays and Western blotting to study the angiogenic signalling pathways induced by mCRP and Notch-3 activator chimera protein (Notch-3/Fc). Our results showed an additive effect on angiogenesis of mCRP stimulatory effect combined with Notch-3/Fc promoting bovine aortic endothelial cell (BAEC) proliferation, migration, tube formation in MatrigelTM with up-regulation of phospho-Akt expression. The pharmacological blockade of PI3K/Akt survival pathway by LY294002 fully inhibited in vitro and in vivo angiogenesis induced by mCRP/Notch-3/Fc combination while blocking Notch signalling by gamma-secretase inhibitor (DAPT) partially inhibited mCRP/Notch-3/Fc-induced angiogenesis. Using a BAEC vascular smooth muscle cell co-culture sprouting angiogenesis assay and transmission electron microscopy, we showed that activation of both mCRP and Notch-3 signalling induced the formation of thicker sprouts which were shown later by Western blotting to be associated with an up-regulation of N-cadherin expression and a down-regulation of VE-cadherin expression. Thus, mCRP combined with Notch-3 activator promote angiogenesis through the PI3K/Akt pathway and their therapeutic combination has potential to promote and stabilize vessel formation whilst reducing the risk of haemorrhage from unstable plaques.