| PurposeThere are several stages for the endothelial progenitor cells (EPCs) mobilization, such as adhesion, degradation, movement and migration. The number and function of EPCs would change with physiological or pathological conditions in vive. Norepinephrine (NE) is the most important transmitter of human sympathetic nerve system. Up to today, it is not clear whether the EPCs mobilization is regulated by NE. The purpose of this study was to investigate the influence of NE on EPCs mobilization in C57 mouse with limb ischemia, human EPCs proliferation and migration which is cultivated ex vive and the involvement of mitogen-activated protein kinase (MAPK) signal molecular and beta-arrestin 2 in the regulation of EPCs function by NE.MethodsThe regulation of EPCs mobilization by norepinephrine was investigated at molecular level, cellular level and general level.First, at the general levels limb ischemia mouse were prepared with left femoral artery ligation at the proximal and the distal end. Drugs such as NE 10-8mol, alpha adrenoceptor receptor blocker Phentolamine 10-8mol, beta ladrenoceptor blocker Metoprolol 10-8mol and beta 2 adrenoceptor 1127 10-8mol were intraperitoneal injected. The proportions of EPCs (double positive of CD34 and KDR) in bone marrow, spleen and peripheral circulation were analyzed with flow cytometry at the 7th day after the left femoral artery ligation.Second at the cellular levels, after 8 days of cultivation of human EPCs, different concentrations of NE (0,0.01,0.1,1,10,100μM) were added into the media, and the proliferation and migration potential were analyzed with MTT and Transwell chamber respectively.Third, at the molecular levels, the phosphorylation of Mitogen-Activated Protein Kinase (MAPK) signal molecular such as Erk 1/2, JNK and p38 were assay with Western blot.At the last, PBMCs isolated from 30ml peripheral blood of 4 DM-2 patients and 4 healthy subjects were assayed the expression of beta-arrestin2 with Western blot. The ablations of beta-arrestin 2 were confirmed with Realtime PCR and Western blot. The proliferation and migration of EPCs were assayed after the ablation.ResultsThe in vive stydies foud that NE increased mobilization of EPCs from bone marrow to peripheral circulation significantly after limb ischemia in C57 mouse. The proportion of EPCs in bone marrow increased from 2.0±0.4% to 4.7±0.9%, p< 0.05, in the peripheral circulation increased from 1.2±0.6% to 6.2±1.9%, p< 0.05, in spleen increased from 2.5±0.8% to 3.1±1.0%, p< 0.05. The effects of NE could be blocked by Phentolamine and I 127, but could not be blocked by Metoprolol.The cellular studies found that NE dose dependently increased the proliferation potential of EPCs. With the most effective concentration of NE 10μM, EPCs proliferation increased 103.6±54.6%, P< 0.05. This effect could be blocked by Phentolamine,1127, A6355 and L-NAME, but not by Metoprolol, SP600125,, PD169318, LY294002,, RO106-9920 and SNP, P> 0.05.NE significantly increased the migration potential of EPCs (124.1±12.2 VS 71.7±19.6, P< 0.05). This effect could be blocked by Phentolamine and 1127 (57.2±14.3 vs 124.1±12.2, P< 0.05 and 61.3±11.5 vs 124.1±12.2, P<0.05), but not by Metoprolol (112.8±26.0 VS 124.1±12.2,P>0.05).The cell signal studies found that NE significantly increased the phosphorylation of Erk 1/2 and signal molecular Src. This effect could be blocked by 1127, P< 0.05, but not by Phentolamine and Metoprolol, P> 0.05.The cell signal regulation studied found that after the ablation of beta-arrestin 2, EPCs were more sensitive to the addition of VEGF in the media (33.7±6.4% vs 2.1±1.4%, P< 0.05), but less sensitive to the addition of NE (26.6±7.8% vs 64.0±13.5%, P< 0.05). The ablation of beta-arrestin 2 decreased the migration potential and sensitive of EPCs to NE.ConclusionNE increases the mobilization of EPCs in mouse with limb ischemia and proliferation, migration potential of EPCs ex vivo. NE increases the phosphorylation of Erk 1/2 and signal molecular Src. Beta-arrestin 2 participates in the regulation of EPCs proliferation and migration.
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