The Study Of Progesterone-mediated Angiogenic Activity Of Endothelial Progenitor Cells (EPCs) And Treatment Of Severe Brain Injury Patients By Decompressive Craniectomy

Objectives:1.The aim of the project is to investigate whether progesterone could augment angiogenic potential of endothelial progenitor cells（EPCs）and what role is progesterone receptor（PR）played in the whole process.2.The project is to investigate if CXCL12/CXCR4（CXC chemokine ligand 12/CXC chemokine receptor 4）involved in progesterone-induced EPCs viability.3.The aim of our study was to evaluate effectiveness of decompressive craniectomy（DC）treatment for traumatic brain injury（TBI）and seek some prognostic predictors.Materials and methods:1.EPCs were isolated from bone marrow-derived mononuclear cells（BM-MNCs）.EPCs were identified by uptake of diI-acetylated low-density lipoprotein（DiI-acLDL）and binding with ulex europaeus agglutinin-1 labeled with fluorescein isothiocyanate（UEA-1-FITC）.Endothelial phenotype was detected by using primary antibodies against von Willebrand Factor（vWF）,CD31 and kinase domain receptor（KDR）.The stem cell marker,CD34 was examined using anti-CD34 antibody.Besides,tube formation capability was tested using Matrigel assay.EPCs derived from rats were stimulated with graded concentrations(0,10^-10M,10^-9M,10^-8M,10^-7M)of PROGESTERONE or 10^-6M ulipristal acetate（UPA,a PR antagonist）.Tube formation assay,adhesion assay,transwell migration assay were employed to investigate the angiogenic activity of EPCs.Vascular endothelial growth factor（VEGF）level in the supernatant was measured by ELISA kit.Western blot assay was utilized to verify the expression of progesterone receptor（PR）.2.EPCs were treated with progesterone（5,10 and 100nM）.MTS[colorimetric3-（4,5-dimethylthia-zol-2-yl）-5-（3-carboxy-methoxyphenyl）-2-（4-sulfophenyl）-2H-tetra-zo lium]assay was used to investigate EPCs viability.CXCL12 secreted by EPCs in the supernatant was measured by using a CXCL12 ELISA kit.To detect CXCR4 on cell membranes,flow cytometric analysis was performed.Cell membrane and cytoplasm proteins were extracted with membrane and cytoplasm protein extraction kits.To explore the mechanisms of progesterone-mediated EPCs viability,CXCR4 inhibitor AMD3100 and PI3K inhibitor LY294002 were used.EPCs were pretreated with AMD3100 or LY294002 for 2h followed by stimulation with 5nM progesterone for6h.Western blotting assay was performed to detect CXCL12,CXCR4,Akt and pAkt（phosphorylated Akt,also known as protein kinase B）.In addition,EPCs were treated with or without appropriate concentrations of CXCL12（10–120 ng/ml）for 6h.3.According to the therapy method,we divided the patients into 2 groups:DC group and standard care group.Between 2010 and 2014,a total number of 223 severe TBI patients,containing 112 patients undergoing DC and 111 patients undergoing standard care,were enrolled into the study according to Glasgow Coma Scale（GCS）.The long-term prognosiswas evaluated by Extended Glasgow Outcome Scale（GOSE）12months after discharging from hospital.We used univariate analysis and receiver operating characteristic curves to explore prognostic predictors.Results:1.Fourteen days in culture,cells exhibited homogeneous and cobblestone-like morphology and showed the ability of forming capillary networks in Matrigel matrix.EPCs were characterized as cells coexpressing the endothelial cell markers（vWF,CD31 and KDR）and stem cell marker（CD34）,and exhibiting the ability of binding with UEA-I-FITC and uptaking acLDL-DiI.The quantification of tube numbers and length revealed that tube formation capability of EPCs-treated by low concentration of progesterone(10^-10,10^-9 and 10^-8M)increased in a time-dependent fashion,achieving the best state after 6h of stimulation.Progesterone also improved other the angiogenic potential,including adhesion,migration,viability and VEGF secretion of EPCs,in a dose-dependent fashion with the maximal effect achieved at 10^-9M progesterone.High concentration(10^-7M)of progesterone impaired the angiogenic potential of EPC.Western blot showed that 10^-9M progesterone increased the content of progesterone receptor A and B（PRA and PRB）of EPCs while 10^-7M progesterone reduced the content of both PR isoforms.Notably,10^-6M UPA antagonized the stimulatory effects of 10^-9M progesterone.2.Progesterone-induced EPCs viability was time-and dose-dependent.EPCs were treated with low concentrations of progesterone（5 and 10nM）and showed progressive increase in viability starting at 6h and reaching a peak by 12h.Notably,the 5nM progesterone treatment significantly increased the viability at 12h,compared to control,10nM or 100nM progesterone treatment groups respectively.High-dose progesterone treatment（100nM）dramatically impaired EPCs viability compared to control.As shown by western Blot,progesterone significantly increased CXCL12expression instead of CXCR4.Western blotting was performed to confirm that progesterone did not induce differential expression of CXCR4 in either cytomembrane or cytoplasmic proteins.Flow cytometry indicated that expression of CXCR4 in membrane proteins was not changed by progesterone treatment.After treated with or without appropriate concentrations of CXCL12（10–120 ng/ml）for 6 h,the data indicated that CXCL12 induced a dose-dependent increase in EPC viability.Progesterone significantly increased pAkt levels as well as EPCs viability compared to the nontreatment control group.Inhibition of PI3K/Akt activity by LY294002treatment significantly attenuated progesterone-induced EPCs viability and increased expressionof pAkt when compared to the progesterone alone treatment group.Inhibition of the CXCL12/CXCR4 signalling pathway by high dose AMD3100（60uM）significantly attenuated progesterone-induced EPCs viability andreduced the level of pAkt compared to the progesterone treatment alone group.Low dose of ADM3100（20uM）did not reduce EPCs viability or pAkt expression.There was no potential effect of the inhibitors alone in EPCs viability and pAkt.3.The results showed that patients in the DC group had a lower mortality,but there was no statistical significance in long-term prognosis between these 2 groups.It seemed that admission GCS,platelet,neutrophile granulocyte,total protein,and albumin were associated with long-term prognosis in DC group and reactivity of pupils in standard care group.Simultaneously,using the multivariable logistic regression model,we confirmed that admission GCS and albumin were independent prognostic predictors for patients undergoing DC,and reactivity of pupils for those undergoing standard care.Conclusion:1.Taken together,this preliminary study provides evidence that short-term progesterone stimulation maybe improve angiogenic activity of EPC through a classical PR-dependent pathway.The safety and efficacy of progesterone intervention in human EPCs warrants further investigation.2.Progesterone does dependently augmented EPCs viability and increased CXCL12expressionandPI3K/Aktsignallingpathwayactivity.The CXCL12/CXCR4/PI3K/Akt signaling pathway contributed to progesterone-induced EPC viability.3.Our data suggested that DC was an effective therapy for severe TBI patients in reducing mortality,but it failed to improve long-term prognosis.Through our study,we could comprehend the characteristics of the two treatments and provide more scientific individuation therapy for severe TBI patients.