CREB-binding protein silencing inhibits thrombin-induced endothelial progenitor cells angiogenesis

Abstract

Endothelial progenitor cells (EPCs) are known to promote neovascularization in ischemic diseases. Recent evidence from our group suggested that CREB-binding protein (CBP) plays an important role in thrombin-induced EPCs migration. However, whether CBP could regulate EPCs angiogenic properties is unknown. In the present study, we investigated whether CBP silencing could inhibit thrombin-induced EPCs angiogenesis. EPCs isolated from the bone marrow of Sprague-Dawley rats were cultured and identified, and then were treated by thrombin alone or combined with CBP-shRNA lentivirus. The effect of CBP silencing on EPCs proliferation was assessed using BrdU incorporation assay. Cell adhesion and tube formation were detected to evaluate the angiogenic functions. Finally, mRNA and protein expression of relevant angiogenic genes were examined by real-time PCR, western-blot, and enzyme-linked immunoassay respectively. Luciferase reporter gene assay was performed to evaluate NF-κB activity. Administration of thrombin significantly promoted EPCs proliferation and adhesion. Thrombin also increased the tube formation in Matrigel assay. However, these effects of thrombin were abolished by CBP gene silencing. CBP silencing also abrogated thrombin-induced increases of integrin β2 expression. In thrombin-induced EPCs, CBP silencing significantly decreased the secretion of VEGF, IL-6 and suppressed NF-κB activity. In conclusion, thrombin-induced EPCs proliferation, adhesion, and tube formation were inhibited by CBP silencing, indicating that CBP plays an important role in thrombin-induced EPCs neovascularization.

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“…RSK2 is a Ser/Thr kinase that regulates cell proliferation, cell survival and cycle by phosphorylating cAMP response element binding (CREB) proteins ( 62 – 64 ). As a transcription factor, CREB participates in the signal pathway related to promoting tumor progression, stimulating growth, giving apoptosis resistance and promoting angiogenesis ( 65 , 66 ). Li et al.…”