Effects Of Visfatin On The Function Of Endothelia Progenitor Cells And The Possible Mechanisms

Objective: Endothelial dysfunction and inflammation are the first step ofAtherosclerosis(AS). Endothelial progenitor cells (endothelial progenitor cells,EPCs) are precursor cells of endothelial cells that can repair the damage ofendothelial cells. Visfatin can increase the mRNA expression of endothelialinflammatory factor of intercellular adhesion molecule-1(intercellularadhesion molecule1, ICAM-1) and vascular cell adhesion molecule-1(vascular cell adhesion molecule1, VCAM-1), but whether the Visfatin couldincrease the expression of ICAM-1and VCAM-1on the surface of EPCs andaffect the repair ability of EPCs remains uncertain. In this study, we use therecombinant human Visfatin to stimulat EPCs and nuclear factor kB (Nuclearfactor kappa B, NF-kB) channel inhibitor pyrrolidine dithiocarbamate(pyrrolidine two dithiocarbamate, PDTC) to treated cells. We observe theexpression of ICAM-1and VCAM-1on the cell surface, aiming to explore theeffect and the related mechanism of exogenous Visfatin on proinflammatoryrole of EPCs.Methods:1Endothelial progenitor cells were isolated and cultured in vitro.Fresh cord blood from healthy full-term pregnant women by Cesareansection diluted with an equal volume PBS, added a certain volume oflymphocyte separation medium, at the room temperature, with the speed of2000rpm/min under the centrifugal25minutes to separate mononuclear cells(MNCs). Density of1×106/ml were seeded in fibronectin coated cultureflasks.Cultured the cells with EPCs special medium, incubered at thecondition of37℃,5%CO2. Morphological changes of cell growth wasobserved and recorded daily under an inverted microscope. In the3rd day, wechanged the medium and discarded the non-adherent cells. Passage was on the 7th day. We detected cell surface CD34, CD133, CD309three positive cellswith flow cytometry and Dil acetylated low-density lipoprotein (Dil-acLDL)and FITC-Ulex lectin (IFITC-UEA-I) double staining of cells under a laserscanning confocal microscopy.2EPCs stimulated with Visfatin.After7-10days of culture EPCs planted in6-well plates. The experimentwas divided into4groups ofâ‘ Control groupâ‘¡Visfatin (10ng/ml)â‘¢Visfatin (20ng/ml)â‘£Visfatin (50ng/ml).Visfatin incubated EPCs for24hours. Then cell surface ICAM-1and VCAM-1mRNA of each group wereassayed with reverse transcription polymerase chain reaction (RT-PCR). Cellsurface ICAM-1and VCAM-1expression of each group were detected withflow cytometry.3The effect of pyrrolidine dithiocarbamate (PDTC) on the stimulation ofEPCs to Visfatin.After7-10days of culture EPCs planted in6-well plates. The experimentwas divided into4groups ofâ‘ Control groupâ‘¡Visfatin (50ng/ml)â‘¢PDTCgroup (100uM)â‘£PDTC（100uM）+Visfatin（50ng/ml）. PDTC (100uM)pretreated EPCs for2hours, then Visfatin incubated EPCs for24hours.Cellsurface ICAM-1and VCAM-1mRNA of each group were assayed with areverse transcription polymerase chain reaction (RT-PCR), and cell surfaceICAM-1and VCAM-1expression of each group were detected with flowcytometry.Results:1Cultured after24-48hours, mononuclear cells were round andsuspended in the medium. After3-4days, cells were gradually adherent, about40-50%of the adherent cells were spindle.After5-7days, adherent cellsappeared colony. Cultured after10-14, cells showed cobblestone-like, withhigh proliferation ability.2After cultured for7days, most of the cells were capable of bindingFITC-UEA-I and uptaking Dil-ac-LDL.Double-staining experiments werepositive. 3By flow cytometry:cultured for7days, cells surface CD34, CD133,CD309expression positive rate were43.12%，36.03%å'Œ67.76%, respectively.4Compared with control group, cell surface ICAM-1and VCAM-1mRNA increased in Visfatin group. Each group cell surface ICAM-1andVCAM-1mRNA made difference with defferent concentrations of Visfatin ina dose-and effect-relationship.5The effects of Visfatin on the expression of ICAM-1and VCAM-1proteins on EPCs cell surface: with the stimulation of EPCs, we measure theexpression of ICAM-1on the cell surface in4groups(0,10ng/ml,20ng/ml,50ng/ml) by flow cytometry. The results were18.25±3.77%,25.40±4.25%,38.88±4.02%and48.83±5.12%, respectively, and there were significantdifferences among the4groups(p<0.05). The expression of VCAM-1were6.75±2.07%,10.65±2.33%,17.11±2.37%and24.82±3.19%, respectively,and there also were significant differences among the4groups(p<0.05). Itshowed a dose-effect relationship.6The effects of PDTC on the transcription level of ICAM-1andVCAM-1on EPCs cell surface: Compared with Visfatin (50ng/ml) group,ICAM-1and VCAM-1mRNA of PV group decreased.7The effects of PDTC on the protein level of ICAM-1and VCAM-1onEPCs cell surface: the ICAM-1expression of PV group was lower than that ofVisfatin (50ng/ml) group (45.25±2.95%vs18.44±1.97%), and there wassignificant difference (p<0.05). The VCAM-1expression of PV group waslower than that of Visfatin (50ng/ml) group （22.92±2.46%vs6.82±1.79%）,and there was significant difference(p<0.05). There was no significantdifference in the expression of ICAM-1and VCAM-1bewteen Control groupand PDTC group (p>0.05).Conclusions:1It is feasible to obtain EPCs from cord blood in vitro.2Compared with control group, cell surface ICAM-1and VCAM-1mRNA and protein expression increased in Visfatin group in a dose-andeffect-relationship. 3PDTC inhibits the mRNA and proterin expression of ICAM-1andVCAM-1on EPCs surface, which suggested the inflammatory effect ofVisfatin to EPCs may be mediated by NF-kB signaling pathway.