Expression And Prognostic Relevance Of Circulating Endothelial Progenitor Cells For Severe Traumatic Brain Injury

BackgroundEndothelial progenitor cells(EPCs) come from mesoblast of embryo and postnatal bone marrow. Recent research showed that EPCs participated embryonic vessel formation and postnatal angiogenesis. EPCs indwelt in fetal liver, cord blood and peripheral blood and had multipotentiality, such as endothelia cells. The population of cells has the function of repairing injured endothelial cells and vasculogenesis and angiogenesis, suggesting critical role in ischemia and vessel injury. Recent researchs showed that EPCs had the ability to regenerate vessel and nerve cells, which contributed the further studies.Objective1. To investigate the isolation,culture, proliferation and identification of endothelial progenitor cells(EPCs) from peripheral blood of patients.2. To investigate protective mechanism of EPCs preventing nerve cells from being subjected to secondary injury after severe traumatic brain injury (TBI).3. To investigate the effect of mobilization and prognostic relevance of circulating EPCs for severe traumatic brain injury.Methods1.cells from peripheral blood of TBI patients were isolated by Fercoll gradient centrifbgation.Isolated cells were cultured in EBM medium. EPCs were identified by means of immunofluorescence and FACS(fluorescence activated cell sorter). The biological functions of endothelial cells were examined by the adsorption of ulex-agglutinin(UEA) labeled by fluorescein isothiacyanate(FITC) and Dil-ac-LDL internalization.2. From January 2009 to April 2010, adult patients with severe traumatic brain injury without body injury were recruited from a single neurotrauma center and demographic data, clinical data and peripheral blood samples were collected. The degree of TBI determined by the Glasgow Coma Scale (GCS) score was 3 to 8. EPCs in blood samples from severe TBI patients and healthy controls were quantified by flow cytometry 1,4,7,14 and 21 days after severe TBI. VEGF and Ang-1 were measured by ELISA at the same time points.3. In this study, we collected serum samples from 21 patients with severe TBI (Glasgow Coma Scale scores^8) and 20 healthy subjects. Circulating endothelial progenitor cells number was quantified by flow cytometry and serum VEGF and MMP-9 level measured by ELISA at 1,4,7,14 and 21 days after TBI. The glasgow outcome scale (GOS) score in TBI patienets was prospectively analyzed.Results1. Attached cells exhibited spindled like and clonal morphology after 3 and 7 days' culture respectively.On the 14 days,the network structure were found. The cells could take up Dil-ac-LDL and bind to FITC-UEA-1, and showed double-positive fluorescence. FACS analysis also demonstrated that more than 18.57% attached cells were double positive for CD 133 and CD34.2. Compared with control subjects, circulating EPCs in patients with severe TBI decreased 4 days (p<0.05), but increased 7 and 14 days (p<0.05) after TBI. VEGF increased significantly during the follow up period (p<0.05). Ang-1 increased gradually and reached peak at 7, and 14 days post TBI. The circulating EPCs were significantly correlated with VEGF and Ang-1 at 7 and 14 days after severe TBI.3. circulating EPCs number in the patients decreased to the lowest level 4 days after TBI, then increased to the peak level 7 days after TBI and was significantly correlated with GOS scores (p<0.01) 6 months after TBI. VEGF and MMP-9 levels were significantly increased during the follow-up period after TBI. Circulating EPCs number was positively correlated with GCS score 1 day after TBI, and with MMP-9 and VEGF levels on 7 days and 14 days after TBI.Conclusion1. After induced by EBM medium, EPCs can be obtained from peripheral blood of TBI patients, which provides the basic for EPCs further study.2. VEGF and Ang-1 are two critical proteins in regulating the angiogenic response and vascular integrity. Our results suggest that the increased VEGF and Ang-1 are closely related to increase in circulating EPCs in response to severe TBI, which may be needed for vascular repairs after severe TBI.3. Our data demonstrate that TBI led to an increase of circulating EPCs and serum VEGF and MMP-9 level, suggesting that VEGF and MMP-9 may mediate the recruitment of bone marrow-derived progenitor cells into the circulation in severe TBI condition. Our observation that circulating progenitor cells was associated with nerve functional recovery in patients provides evidence that circulating EPCs may be a potential biomarker to monitor TBI angiogenesis and prognosis.