Research About The Effects And Its Signal Transduction Pathway Of GM-CSF And VEGF On Functions Of The Endothelial Progenitor Cells Of Rat In Vitro

IntroductionThe occurrence and development of atherosclerosis (AS) is a very complicated dynamic pathologic process concerned with many factors, and its serious clinical complications such as acute coronary syndrome(ACS) are the principal diseases and causes of death in most of developed countries and some developing countries, which have been gradually been the focus of health problems all over the world. Injury of endothelial monolayer by conventional and unconventional cardiovascular risk factors induces a cascade of infiltration of monocytic cells and proliferation of smooth muscle cells. These processes can lead to the formation of atherosclerotic lesions, plaque rupture, and finally a series of clinical complications such as ACS. The maintenance of the endothelial integrity and functional activity is therefore of crucial importance in postponing thrombosis, intima proliferation, prevention and therapy of ischemic diseases such AS.Because of limited functions of mature endothelial cells, the endothelial cells and growth factors of blood vessels alone cannot effectively complete the regeneration and reparation of blood vessels, establish collateral circulation, and postpone the development of lesion. It has been shown by numerous studies in recent years that bone marrow derived/tissue specific endothelial progenitor cells (EPCs) can be activated, get into the ischemic area, participate in the dynamic maintenance and reconstruction of blood vessels after birth, and its potential of high proliferation and high differentiation makes the quick recovery of the structure and functions of damaged blood vessels possible.Not only inflammatory factor, granulocyte-macrophage colony stimulating factor (GM-CSF) can also stimulate proliferation and differentiation of stem cells. Many researches have shown that the number of EPCs in the circulation positively correlates with the level of vascular endothelial growth factor (VEGF). PI3K/Akt signal transduction pathway is an important intra-cellular signal transduction pathway. Apoptosis protein Bcl-2/Bax, NO are the downstream targets of it.Currently, researches about the influence brought by GM-CSF on functions of EPCs are quit rare, so bone marrow-and spleen-derived EPCs of rats were respectively isolated and cultured in vitro in order to investigate and compare the effects of GM-CSF on the functions of two kinds of EPCs. In addition, bone marrow-derived EPCs and cardiac microvascular endothelial cells (CMECs) of rats were respectively isolated and cultured in vitro, and the model of CMECs incorporated with EPCs was established in order from cell, protein, gene level to investigate the effects of VEGF on the functions of EPCs and its signal transduction pathway, and provide theoretical and experimental basis for the exploration on the regenerative and reparative mechanism of blood vessels and the treatment of many ischemic diseases, such as AS.Part one. The Effects of GM-CSF on functions of EPCs of Rat1. Methods(1) Isolation, culture, purity and identification of EPCsEPCs were isolated from bone marrow and spleen of rats with density gradient centrifugation method and prepared into cell suspension with M200 culture liquid containing LSGS.24h later, the culture liquid was added into another culture flask for continuous culture. The culture liquid was first changed 5d later, and then the liquid was changed every 2d. Then observed growth and differentiation process of EPCs with inverted phase contrast microscope. EPCs were identified via dual fluorescent staining of FITC-AC133 and PE-vWF.(2) Experimental grouping Concentration effect group:control group (serum-free DMEM culture fluid), groups using different concentrations of RatGM-CSF (25,50, 100ng/ml),96hours' treatment; groups receiving different time (24,48,72,96h) of treatment using the same concentration of Rat GM-CSF (100ng/ml).(3) Detection of the proliferation, migration and angiogenesis of EPCsDetected the effects of RatGM-CSF on proliferation, migration, and blood vessels formation of EPCs with MTT colorimetry, Transwell chamber, Matrigel.(4) Statistical analysisThe tests of each group were repeated for 3 times. The experimental data were expressed with x±s , and One-way ANOVA was used for statistical analysis. P<0.05 was considered to be statistically significant.2. Results(1) Morphologic observation and identification of EPCsAfter 5 days' culture, many cell colonies adhering to the wall formed, there were round cells in the center of colonies, and many spindle cells spread from the center to the peripheral area, which were similar to the blood islands during embryogenesis. After 7 days'culture, round cells in the center of colonies floated, spindle cells of peripheral area continued growing rapidly. After 14 days' culture, endothelial cells monolayer was formed. AC133 positive cells were labeled with green fluorescence, vWF positive cells were labeled with red fluorescence, and the cells with dual positive staining had yellow fluorescence, which were considered to be EPCs under differentiation.(2) Effects of GM-CSF on the proliferation, migration and angiogenesis of EPCsCompared control, RatGM-CSF significantly increased OD490, the number of migratory cells and tubes in vitro in certain rages showing a dose-dependent and time-dependent manner. Part two. Effects of VEGF on the Functions of Bone Marrow-Derived Endothelial Progenitor Cells and its Signal Transduction Pathway1. Methods(1) Isolation, culture, purity and identification of EPCsThe method is same as part one.(2) Isolation, culture and identification of CMECsEPCs were isolated from bone marrow of rats with trypsin and collagenase II, and prepared into DMEM culture fluid containing 20% fetal bovine serum. Immunocytochemical method was used to identify vWF of CMECs, and light microscope observation was performed.(3) Experimental groupingConcentration effect group:control group (serum-free DMEM culture fluid), groups using different concentrations of RatVEGF (10,25,50ng/ml),48 hours' treatment; groups receiving different time (12,24,36,48h) of treatment using the same concentration of RatVEGF (50ng/ml).(4) Establishment of CMECs model incorporated with EPCsLabeled the nuclei of EPCs which have been stimulated by RatVEGF with 2-(4-Amidinophenyl)-6 -indolecarbamidine dihydrochloride (DAPI), digested EPCs and CMECs, mixed them with an appropriate proportion, and established angiogenesis in vitro model with Matrigel to observe the number of EPCs incorporated into CMECs monolayer.(5) Detection the proliferation, migration and angiogenesis of EPCsDetected the effects of RatVEGF on proliferation, migration, and blood vessels formation of EPCs with MTT colorimetry, Transwell chamber, Matrigel. The method is same as part one.(6) Detection of the expression of EPCs apoptosis proteins Bcl-2 and Bax, integrinP2 and endogenous VEGFDetected the expression of EPCs apoptosis protein Bcl-2 and Bax, integrinβ2, endogenous VEGF with Western blotting.(7) Detection of the signal transduction pathway of VEGF in EPCs①Detection of the expression of eNOSmRNA and eNOS proteinEPCs were divided into the control group (serum-free DMEM culture fluid), RatVEGF group (50ng/ml), and RatVEGF (50ng/ml)+LY294002 (50μM) group, and received treatment for 1h and 48h respectively. RT-PCR and Western blotting were respectively used to detect the expression of eNOS mRNA and eNOS protein.②Detection of the effects of eNOS inhibitors on the functions of EPCsEPCs were divided into the control group (serum-free DMEM culture fluid), RatVEGF group (50ng/ml), and RatVEGF (50ng/ml)+L-NAME (lOμM) group, and received treatment for 48h respectively. MTT colorimetry, Transwell cell culture chamber, Matrigel angiogenesis in vitro model were respectively used to detect the proliferation, migration and angiogenesis capability of EPCs. The method is same as part one.(8) Statistical analysisThe tests of each group were repeated for 3 times. The experimental data were expressed with x±s, and One-way ANOVA was used for statistical analysis. P<0.05 was considered to be statistically significant.2. Results(1) Morphologic observation and identification of CMECsAfter about 4hours'culture, CMECs adhered to the wall, looked like short spindles, and then grew like "paving stone". vWF positive cells were considered to be CMECs.(2)Effects of VEGF on EPCs incorporated into CMECs monolayerCompared control, RatVEGF significantly facilitated the number of EPCs incorporated into CMECs monolayer.(3) Effects of VEGF on the proliferation, migration and angiogenesis of EPCs Compared control, RatVEGF significantly increased OD490, the number of migratory cells and tubes in vitro in certain rages showing a dose-dependent and time-dependent manner.(4) Effects of VEGF on the expression of EPCs apoptosis proteins Bcl-2 and Bax, integrinβ2 and endogenous VEGFCompared control, RatVEGF significantly upregulated the expression of apoptosis protein Bcl-2, integrinβ2 and endogenous VEGF; downregulated the expression of apoptosis protein Bax in vitro in certain rages showing a dose-dependent manner.(5) Effects of VEGF on PI3K/Akt→eNOS/NO signal transduction pathwayCompared with the control group, the expression of eNOSmRNA and eNOS protein in the RatVEGF group significantly increased, and after PI3K inhibitor LY294002 was added, the expression of eNOSmRNA and eNOS protein significantly decreased when compared with the RatVEGF group. PI3K inhibitor LY294002 could weake the expression of eNOSmRNA and eNOS protein caused by RatVEGF.(6)The relationship between PI3K/Akt→eNOS/NO signal transduction pathway and contribution of VEGF to the functions of EPCsCompared with the control group, RatVEGF could significantly enhance the capabilities of proliferation, migration and angiogenesis of EPCs, and after eNOS inhibitor L-NAME was added, the functions of EPCs significantly decreased when compared with the RatVEGF group. eNOS inhibitor L-NAME could weake the contribution of RatVEGF to the functions of EPCs.Conclusions1. GM-CSF can significantly facilitate proliferation, migration and blood vessels formation of EPCs in vitro in certain rages showing a dose-dependent and time-dependent manner.2. VEGF can significantly facilitate the number of EPCs incorporated into mature blood vessel endothelial cells monolayer in vitro in certain rages showing a dose-dependent and time-dependent manner. 3. VEGF has the stimulant effects on the functions of EPCs, such as proliferation, survival, migration, blood vessels formation, adhesion, autocrine in vitro in certain rages showing a dose-dependent and time-dependent manner.4. The effects of VEGF on EPCs can be implemented by PI3K/Akt→eNOS/NO signal transduction pathway.