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

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 Matrigel(TM) 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.

Monomeric C-reactive protein-a key molecule driving development of Alzheimer’s disease associated with brain ischaemia?

“…1D ) indicating a potential mechanism for amyloid cleavage. mCRP also phosphorylated ERK and AKT as shown previously (data not included; 13 ).…”

“…Recombinant forms of both mCRP and nCRP (0.5 mg/mL in 25 mM NaPBS, pH 7.4) was produced in the laboratory of Dr. L.A. Potempa as previously described 8 9 10 13 . The mCRP solution contained an endotoxin concentration lower than 0.125 EU/mL and all cell culture medium was endotoxin-free.…”

Biological actions of pentraxins

“…We have demonstrated mCRP ability to promote angiogenesis either in vitro (endothelial cell migration, proliferation and tubulogenesis) or in vivo (chorioallantoic membrane assay) and have determined the main CRP-induced pro-angiogenic genes [34]. In addition, a recent study has demonstrated the ability of CRP to promote angiogenesis via activation of the PI3K/Akt survival pathway [60].…”

Combretastatin A4 Regulates Proliferation, Migration, Invasion, and Apoptosis of Thyroid Cancer Cells via PI3K/Akt Signaling Pathway

“…Final concentration of 0.5 mg/mL MTT solution (Sigma Aldrich, St. Louis, MO) was added to each well and incubated for 4 h. After the incubation, formazan crystals were dissolved by addition of 150 μl dimethyl sulfoxide per well. The absorbance was measured at 570 nm using an automated ELISA plate reader (Thermo Scientific ® , Rockford, IL) [16]. …”