Effects Of AGEs And MSCs On The VEGF Signal Pathway Of HUVECs And Its Mechanism

Objective: This study was performed to discuss the pathological change of VEGF(vascular endothelial growth factor) signal pathway in diabetic angiopathy and the mechanism on MSC promoting angiogenesis in patients with diabetes mellitus. For this purpose, we examined the expression change of VEGFR and PI3K/AKT/e NOS pathway in human umbilical vein endothelial cells(HUVECs) after intervented of advanced glycation end products(AGEs) and cocultured with human mesenchymal stem cells(h MSCs).Methods: The human umbilical vein endothelial cells and mesenchymal stem cells were routine cultured. Cells were applied to the experiment when they entered the logarithmic phase. Then, the HUVECs were divided into three groups: normal culture group(NC) 、 AGE intervent group(AGE) and AGE+MSC coculture group(AGE+MSC). HUVECs in NC group were routine cultured. HUVECs in AGE group were stimulated with AGEs at the concentration of 400ng/ml for 36 hours. In AGE+MSC group HUVECs were cocultured with MSCs and also stimulated with AGEs at the concentration of400ng/ml for 36 hours at the same time. PCR and Western blot were used to detect the m RNA and protein expression of VEGF、VEGFR、p-AKT、p-e NOS in HUVECs of all 3 groups 36 hours later. All statistical analysis were performed with SPSS 17.0 statistical software package.Results:1 Effects of AGEs on expression of VEGF-A and VEGFR in HUVECAfter intervented of AGEs, the m RNA expression of VEGFA 、 s Flt-1(3.747±0.415) in HUVECs were both higher than those(1.007±0.139) in NC group(P<0.05), the m RNA expression of VEGFR-2 were lower than NC group(P<0.05).After intervented of AGEs at the concentration of 400ng/ml for 36 hours,the protain expression of VEGFR-1(93.129±4.620) in HUVECs were lower than those(557.395±9.383) in NC group(P<0.05); the protain expression of VEGFR-2(63.791±7.98) in HUVECs were also lower than those(571.995±12.65) in NC group(P<0.05).2 Effects of AGEs on expression of PI3K/AKT/e NOS signal pathway in HUVECsAfter intervented of AGEs at the concentration of 400ng/ml for 36 hours,the protain expression of p-AKT(86.408±3.255) and p-e NOS(187.69±20.732)in HUVECs were both lower than those(482.325±39.859)(426.96±50.063) in NC group(P<0.05).3 Effects of coculture HUVECs with MSCs on expression of VEGFR in HUVECsThrough cocultured with h MSCs, the m RNA expression of s Flt-1(2.048±0.245) in HUVECs decreased than those in AGE group(P<0.05);Through cocultured with h MSCs, the protain expression of VEGFR-1(319.43±3.649) in HUVECs were higher than those in AGE group(P<0.05)and the protain expression of VEGFR-2(202.81±48.879) in HUVECs also increased than those in AGE group(P<0.05).4 Effects of coculture HUVECs with MSCs on expression of PI3K/AKT/e NOS signal pathway in HUVECsThrough cocultured with h MSCs, the protain expression of p-AKT and p-e NOS in HUVEC both increased compared with those(238.385±30.384)( 255.545±9.227) in AGE group(P<0.05).Conclusions:1 AGE could cause damage to the VEGF receptor and make the expression of inhibitory receptor increase, weakening the signal transduction of its downstream pathways, resulting in endothelial cells dysfunction, which can further affect angiogenesis.2 The effect of MSCs promoting angiogenesis in patients with diabetes mellitus may partly through affecting on VEGF receptor and its pathways.