Monomeric C-reactive protein and Notch-3 co-operatively increase angiogenesis through PI3K signalling pathway |
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| Institution: | 2. Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University, Latina, Italy;3. Department of Experimental Medicine, Sapienza University, Rome, Italy;4. Department of Women’s and Children’s Health, University of Padova, Padova, Italy;5. Molecular Oncology Program, DeWitt Daughtry Family, Department of Surgery; Miami, FL, USA;11. Institute Pasteur-Foundation Cenci Bolognetti, Sapienza University, Rome, Italy;1. Department of Neurology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-0811, Japan;2. Department of Morphological and Physiological Sciences, Graduate School of Health Sciences, Kumamoto University, Kumamoto 862-0976, Japan;3. Department of Neurosurgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-0811, Japan;4. Department of Pathology and Experimental Medicine, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-0811, Japan;1. German Center for Neurodegenerative Diseases (DZNE) Dresden, 01307 Dresden, Germany;2. CRTD — Center for Regenerative Therapies Dresden, Genomics of Regeneration, Technische Universität Dresden, 01307 Dresden, Germany;3. German Center for Neurodegenerative Diseases (DZNE) Bonn, 53175 Bonn, Germany;4. Department of Neurology, Charité University Medicine Berlin, 10117 Berlin, Germany |
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| Abstract: | 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. |
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| Keywords: | C-reactive protein Notch Angiogenesis PI3K/Akt pathway Gamma-secretase |
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