The Experimental Study Of Intercellular Adhesion Molecule-1(ICAM-1) Chorused Cohesion Between Mesenchymal Stem Cells(MSCs) And Endothelial Progenitor Cells(EPSs)

Objective 1. To investigate the mechanism of intercellular adhesion molecule-1(ICAM-1) chorused cohesion between mesenchymal stem cells(MSCs) and endothelial progenitor cells(EPCs).Methods 1. MSCs and EPCs were isolated and cultured successfully from six to eight weeks C57BL/6 murine bone marrow by in vitro amplification; 2. Determination of IL-1β expression levels in the culture medium by Elisa. 3. Cell immunofluorescence was used to detect the expression of ICAM-1 in normal group, IL-1β group and P38 MAPK inhibitor SB203580 group; 4. To observe the ICAM-1 chorused cohesion between MSC and EPC by supplement with anti-ICAM-1 neutralizing antibody, IL-1β or P38 MAPK inhibitor SB203580.Results 1. In this study, To determine the IL-1β expression level in MSCs, EPCs, MSCs&EPCs, EPC-CM-MSCs and MSC-CM-EPCs, all the groups were seeded with same-cell-density(2×105 cells/well) and treated with serum- and factors- free medium in 6-well plate for 24 h. The results showed that both of the MSCs and EPCs can secrete IL-1β, and the MSCs&EPCs(49.13±6.21 ng/ml) group was detected the IL-1β with higher expression than MSCs(8.96±2.70 ng/ml, *P<0.05) and EPCs(22.14±1.83 ng/ml, *P<0.05) group, respectively. The EPC-CM-MSCs(35.02±1.19 ng/ml) and MSC-CM-EPCs(33.85±1.37 ng/ml) also showed higher level of IL-1β than MSCs(8.96±2.70 ng/ml, *P<0.05) and EPCs(22.14±1.83 ng/ml, *P<0.05) group, but not for MSCs&EPCs(49.13±6.21 ng/ml, P>0.05). 2. The coverslips for adhesion(2×104 cells/sample) were cultured in 6 mm dish with each corresponding medium 2 ml to culture for 24 h. Then, replace medium was replaced as serumand factors- free and supplement with IL-1β(25 μg/ml) or p38 MAPK inhibitor SB203580(20 μmol/ml) to stimulate for another 24 h, respectively. Our research showed that both of MSCs and EPCs can express ICAM-1 in low level, but MSCs&EPCs can promote the expression of ICAM-1 whether through pre-treated or not. All of the IL-1β groups showed higher expression of ICAM-1 than normal groups and SB203580 groups between the corresponding groups, respectively. SB203580 groups also showed lower expression of ICAM-1 than other two groups between the corresponding groups, respectively. 3. MSCs, EPCs and MSCs&EPCs expressed ICAM-1 protein and p38 MAPK protein which were analyzed by western blotting and the results showed by ICAM-1/GAPDH IOD relative value and p-p38MAPK/total-p38 MAPK IOD relative value for statistical analysis. The dates showed that normally cultured MSCs&EPCs(0.21±0.01, *P<0.05) group increased the expression of ICAM-1 compared with the corresponding MSCs(0.11±0.02, *P<0.05) group and EPCs(0.11±0.02, *P<0.05) group, but there was no significant difference between MSCs and EPCs group. The IL-1β stimulated MSCs&EPCs(0.38±0.02, *P<0.05) groups increased the expression of ICAM-1 compared the corresponding MSCs(0.23±0.02, *P<0.05) group and EPCs(0.25±0.02, *P<0.05) group. The p38 MAPK inhibitor SB203580 stimulated MSCs&EPCs(0.10±0.02, *P<0.05) groups increased the expression of ICAM-1 than the corresponding MSCs(0.05±0.01, *P<0.05) group and EPCs(0.05±0.01, *P<0.05) group. All of IL-1β stimulated groups(*P<0.05) were presented on an apparent up-regulated, but the SB203580 stimulated groups(*P<0.05) were opposites on the expression of ICAM-1. The normally cultured MSCs&EPCs(0.52±0.02, *P<0.05) group increased the expression of phosphorylation p38 MAPK than the corresponding MSCs(0.35±0.02, *P<0.05) group and EPCs(0.36±0.01, *P<0.05) group, but there was no significant difference between MSCs and EPCs group. The IL-1β stimulated MSCs&EPCs(0.74±0.06, *P<0.05) groups increased the expression of phosphorylation p38 MAPK than the corresponding MSCs(0.54±0.04, *P<0.05) group and EPCs(0.47±0.03, *P<0.05) group. The p38 MAPK inhibitor SB203580 stimulated MSCs&EPCs(0.37±0.02, *P<0.05) groups increased the expression of phosphorylation p38 MAPK than the corresponding MSCs(0.26±0.03, *P<0.05) group and EPCs(0.17±0.01, *P<0.05) group. The p38 MAPK phosphorylation level increased after being stimulated by IL-1β(*P<0.05) and declined after induced by p38 MAPK inhibitor SB203580(*P<0.05), respectively. The nonphosphorylation level of p38 MAPK remained constant in all groups after different treated. 4. MSCs were labeled with DAPI and EPCs were labeled with Mitotracker red. The MSCs-DAPI and EPCs-Mitotrack-red were co-cultured in Matrigel(5×104 cells/well, 1:1) with EPCs culture medium, supplemented with anti-ICAM-1 neutralizing antibody(Abcam, ab171123, USA, 1:50), IL-1β(25 μg/ml) and p38 MAPK inhibitor SB203580(20 μmol/ml) at 24 wells plate, respectively. Then, the cells were cultured for 6-8 h at 37°C in a 5% CO2 humidified incubator. The results showed that anti-ICAM-1 neutralizing antibody and p38 MAPK inhibitor SB203580 can decline the adhesion between MSCs and EPCs, but supplement with IL-1β significantly increased this adhesion phenomenon.Conclusion 1. Using the method of whole bone marrow adherent with the different rates of attachment can successfully isolate and culture MSCs and EPCs. 2. ICAM-1 had participated in the adhesion between MSCs and EPCs. 3. Interleukin-1 beta induces intercellular adhesion molecule-1 expression enhancing the adhesion between mesenchymal stem cells and endothelial progenitor cells through the p38 MAPK signaling pathway. 4. The adhesion of MSCs and EPCs can secrete IL-1β through direct and indirect secretion.