| The incidences of Diabetes, hypercholesterolemia, hypertension, atherosclerosis andcoronary artery disease increase year by year and their vascular complications can causeserious damage to the body and even life-threatening. Endothelial progenitor cells (EPCs)from Type I and Type II diabetes patients have reduced cell number and impaired functions,and the cell number and function would be further reduced when accompanied byhyperlipidemia, cardiovascular disease and cardiovascular risk factors such as hypertensionand smoking et al[1-4]. Type II diabetes is often accompanied by hyperlipidemia, high levelplasma lipids can cause accumulation of oxidized low-density lipoprotein (ox-LDL) inplasma[5]. Lysophosphatidylcholine (LPC) is the major component of Ox-LDL. Severalstudies have shown that LPC can induce apoptosis of mature endothelial cells[6]; inhibitionof mature endothelial cell migration and proliferation[7]; recuiting mononuclear cells to thearterial wall in the early stage of atherosclerosis[8]; inducing the expression of leukocyteadhesion molecules in mature endothelial cells[9]; inducing the expression of growth factorspromoting migration and proliferation of smooth muscle cells and fibroblasts in matureendothelial cells response to trauma[10]; inhibiting generation of endothelin-1(ET-1) matureendothelial cells[11]. LPC acts through G protein-coupled receptors on differentiatingmonocytes to generate mature dendritic cells[12]. However, for the process of differentiatingearly EPC from human CD34positive hematopoietic stem cells, the effect of LPC treatmentis unclear.1. In vitro isolation and expansion of human CD34+hematopoietic stem cellsObjective: To isolate CD34+hematopoietic stem cells from human peripheral blood andabtain a certain number of CD34+hematopoietic stem cells by effectively expanding it invitro. Methods: Human peripheral blood mononuclear cells (PBMNCs) were isolated fromhealthy human peripheral blood by density gradient centrifugation. CD34+cells wereisolated by magnetic beads-based selection. The purity of CD34+cells was analyzed in aFACS flow cytometer. A nonadhesive expansion method is applicated for expansion ofCD34+hematopoietic stem cells using Stem II serum-free medium with addition of severaleffective stimulating factors in ultra-low attachment6-well plates. Results: After positive selection, single staining flow cytometry revealed that approximately70%of the cellsexpressed CD34after positive selection process. The selected cells were first cultured onultra-low attachment6-well plates in serum free Stem II medium supplemented with rh-SCF（100ng/ml）, rh-FLT-3L（50ng/ml）, rh-IL-3（20ng/ml）, rh-TPO（100ng/ml）and rh-GCSF（100ng/ml）in a humidified incubator at37°C with5%CO2for eight days. After eightdays, the cells had expanded60fold at average (59.4±4.1, Mean±SD, n=9). Conclusion: Thenonadhesive expansion method using serum-free Stem II medium supplemented witheffective stimulating factors to culture CD34+hematopoietic stem cells is an effective wayto expand CD34+hematopoietic stem cells in vitro, and a certain number of CD34+hematopoietic stem cells are obtained from this method.2. Effect of LPC on the process of differentiating CD34+hematopoietic stem cells to earlyEPC2.1Differentiation of CD34+hematopoietic stem cells to early EPCObjective: To differentiate CD34+hematopoietic stem cells to early EPC in vitro.Methods: After the eight day expansion period the CD34+hematopoietic stem cells werecollected and counted. Thereafter the cells were cultured in endothelial cell growthmedium-2(EGM-2). After three to four days of culture, nonadherent cells were removed andadherent cells were further cultivated for another three to four days in fresh EGM-2.Phenotypical chatacterisation of early EPCs were analyzed by FACS flow cytometry.Expression of endothleial cell specific markers on early EPC was analyzed byimmunofluorescence staining. Results: early EPC differentiating from CD34+hematopoieticstem cells showed adherent spindle-like morphology; early EPC expressed CD31and KDR,wheareas the expression level of CD34is rather low. Positive expression of endothelialcell-specific markers such as vascular endothelial cadherin (VE-Cadherin), von Willebrandfactor (Vwf) and endothelial nitric oxide synthase (eNOS) were found on early EPC afterimmunofluorescence staining. Conclusions: CD34+hematopoietic stem cells can be inducedto differentiate to early EPC in vitro; these early EPCs positively expressed endothelialcell-specific markers such as CD31，KDR，VE-Cadherin, vWF and eNOS.2.2Effect of LPC on gene expression of early EPC derived from CD34+hematopoietic stemcellsObjective: To evaluate the effect of LPC on the process of CD34+hematopoietic stem cellsdifferentiating to early EPC and the molecular mechanism. Methods: Treat the cells withLPC（10ug/ml） during the late stage of the differentiation period, and cells were further cultivated for1h,5h or24h. The cells were collected and prepared for real-time quantitativeRT-PCR to examine gene expressions of vascular endothelia growth factor (VEGF),angiopoietin-1(Ang-1), chemokine CCL2(CCL2) and Interleukin-8(IL-8). early EPCstreated with LPC (10ug/ml)/phosphatidylcholine (PC)(10ug/ml) and further cultivated for1h, gene expression levels of VEGF, Ang-1and CCL2showed no statistically significantdifference when compared among the negative control group, LPC group and PC group;however IL-8gene expression level was significantly up-regulated (p <0.05) in LPC groupcompared with the negative control group. early EPCs treated with LPC(10ug/ml)/PC(10ug/ml) and further cultivated for5h, gene expression levels of VEGF,CCL2and Ang-1showed no statistically significant difference when compared among thenegative control group, LPC group and PC group; IL-8gene expression level wassignificantly up-regulated (p <0.05) in LPC group compared with the negative control group.early EPCs treated with LPC (10ug/ml)/PC(10ug/ml) and further cultivated for24h, geneexpression levels of VEGF, CCL2and IL-8showed no statistically significant differencewhen compared among the negative control group, LPC group and PC group; Ang-1geneexpression level was significantly down-regulated (p <0.01) in LPC group compared withthe negative control group; Ang-1gene expression level was significantly down-regulated (p<0.05) in LPC group compared with PC group. Conclusion: LPC could downregulateangiogenic Ang-1gene expression of early EPC derived from CD34+hematopoietic stemcell. LPC could upregulate proinflammatory IL-8gene expression of early EPC derived fromCD34+hematopoietic stem cell.2.3Effect of LPC on the capability of early EPC to support endothelial tube network formedby human umbilical vein endothelial cells (HUVECs) in vitroObjective: To evaluate the effect of LPC on the capability of early EPC to supportendothelial tube network formed by HUVECs in vitro. Method: The capability of early EPCto support endothelial tube formation was assessed in a co-culture systerm with humanumbilical vein endothelial cells (HUVECs)(Passages P3-P5) using MatrigelTM, and earlyEPCs were treated with/without LPC (10ug/ml). Results: early EPCs didn’t form tubuli likestructures when cultured alone on the MatrigelTM, they exhibited an angiogenic stimulatoryeffect on the ability of HUVECs to form tubuli and they could well incorporate into the tubenetwork. After treatment with LPC, the above proangiogenic ability of early EPC reduced,they couldn’t well incorporate into the tube network formed by HUVECs and the tubenetwork was impared and looked like loose and incomplete. Conlusion: early EPC derived from CD34+hematopoietic stem cells has the ability to promote tube network formation bythe endothelial cells (HUVECs) in vitro; LPC exhibits inhibitory effect on this proangiogenicability of early EPC. |
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