cAMP-Response-Element-Binding-Protein-Binding Protein Silencing Inhibits Thrombin-Induced Endothelial Progenitor Cell Migration via Downregulation of CXCR4 Expression

Abstract

Previous studies have suggested that bone marrow-derived endothelial progenitor cell (EPC) could migrate to the ischemic tissue and incorporate into sites of neovascularization.1) Transplantation of ex vivo expanded EPC could successfully enhance the neovascularization process.2) As EPC migration from peripheral blood to ischemic region is a key procedure for neovascularization, the mediators involved in the migration of EPC appear to be important. Thrombin is a multifunctional coagulation factor that involved not only in post injury inflammatory responses but also in promoting angiogenesis.3) It appears that thrombin plays an important role in the induction of EPC migration because activation of proteinase activated receptor-1 (PAR-1), the main thrombin receptor expressed on EPC, is associated with increased migration of EPC. 4) Howerver, the underlying mechanism remains unknown.cAMP-response-element-binding-protein-binding protein (CBP) has been shown to anticipate in many cellular biological processes. It functions as a coactivator of DNA binding, regulating transcriptional activity of many transcriptional factors. 5,6) We hypothesized that thrombin might enhance EPC migration by upregulating CBP expression. Therefore, in the present study, we examined the role of CBP in the regulation of EPC migration and CXCR4 expression induced by thrombin. MATERIALS AND METHODS Cell Culture and IdentificationMononuclear cells (MNCs) were isolated from the bone marrow of femurs and tibias of Sprague-Dawley rats by density gradient centrifugation with Ficoll separating solution as previously described. 2)Briefly, MNCs were plated on culture dishes and maintained in endothelial basal medium (Cambrex) supplemented with 5% fetal bovine serum, human vascular endotheial growth factor (VEGF)-A, human fibroblast growth factor-2, human epidermal growth factor, insulin-like growth factor-1, and ascorbic acid. After cultured for 4 d, non-adherent cells were removed by washing with phosphate buffered saline (PBS), and the adherent cells were maintained in new media for 7 d. The experiments were performed in accordance with the guidelines specified by the Animal Care and Use Committee of Wuhan University, China.To confirm the EPC phenotype, direct fluorescent staining was used to detect dual binding of fluorescein isothiocyanate (FITC)-labeled Ulex europaeus agglutinin (UEA)-1 (SigmaAldrich, St. Louis, MO, U.S.A.) and (DiI)-labeled acetylated low density lipoprotein (Biomedical Technologies Inc., Stoughton, MA, U.S.A.). Doublepositive cells were identified as EPC by laser scanning confocal microscope. Lentiviral Construction and Transduction To obtain silencing of CBP gene (GenBank Accession NM_133381), the dominant CBP sequence and negative control sequence were constructed by Genehem (Shanghai, China). A BLAST search was performed to make sure that short interference RNA (siRNA) duplexes were specifically directed against the targeted genes. The sense siRNA sequence targeting CBP was GCAAACAGAGCATGGTCAA, and the negative control sequ...

Beta‐Adrenoceptor Activation Reduces Both Dermal Microvascular Endothelial Cell Migration via a cAMP‐Dependent Mechanism and Wound Angiogenesis

“…β 1 -AR, β 2 -AR ( Ponicke et al, 2006 ) and β 3 -AR ( Soeder et al, 1999 ) can all couple to Gαs activating the membrane effector enzyme adenylate cyclase (AC) which generates the secondary messenger molecule cyclic adenosine monophosphate (cAMP) by catalysing the conversion of adenosine triphosphate to cAMP ( Gilman, 1987 ; Hamm, 1998 ). Intracellular changes in cAMP levels can alter a multitude of cell functions including rat bone marrow progenitor EC migration ( Chen et al, 2010 ), rat aortic EC proliferation ( Torella et al, 2009 ) and chick chorioallantoic membranes (CAM) angiogenesis ( Pacini et al, 2011 ) through the downstream action of cAMP-dependent protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC) ( de Rooij et al, 1998 ). Prior to 1998, any intracellular cAMP effects were attributed solely to PKA; the discovery that the small GTPase Rap1 can be directly activated by EPAC, a family of cAMP-activated guanine nucleotide exchange factors (GEFs), heralded a new era in cAMP research ( Kawasaki et al, 1998 ; de Rooij et al, 1998 ).…”