Endothelial progenitor cells (EPCs) play an important role in the restore of damaged retinal blood vessels in physiological and pathological conditions. It has been suggested that the number and function of circulating EPCs could be reduced in diabetic. We consider that the development of Diabetic retinopathy (DR) may closely related to the dysfunction of EPCs, but the definite mechanism is not clear so far. Hyperglycemia is the main sign of diabetes, to explore the effect of high glucose on the biological characteristics of EPCs may lay a foundation for the study of the pathogenesis and the regulating pathways of DR, and may provide new targets for the treatment of DR.
Objective To investigate the effects of high glucose on the biological characteristics of EPCs in vitro, and further to study the related mechanisms.
Methods ①EPCs were isolated by density gradient centrifugation from human umbilical cord blood and were cultured in vitro, then EPCs were identified by flowcytometry and confocal microscope. ②EPCs were cultured in normal and high glucose condition respectively. The proliferation and adhesion capacity of EPCs were detected by MTT assay and cell counts. Flow cytometry was used to detect apoptosis of EPCs in each group. RT-PCR was used to detect the mRNA levels of Bcl-2 and Bax. The acLDL uptake ability of EPCs was detected by confocal microscope. Fluo3/AM staining was used to detect the [Ca2+]i level in each group. Patch clamp technology was introduced to analysis the effect of glucose on electrophysiological properties of EPCs. Western Blot was used to detect the influence of high glucose on the expression of p-eNOS and p-AKT on protein level.
Results 1. EPCs expressed CD133, CD34, VEGFR2 and CD31 at different levels on 10d, and most of the cells were double positive to DiI-acLDL and FITC-UEA-I..2.① Compared with normal group, the proliferation activity of EPCs was enhanced in high glucose at 1d, but decreased obviously at 7d. ②The adhesion property of EPCs decreased by treatment with high glucose for 1d and 3d compared with normal condition. ③After high glucose treatment for 7d, the number of pro-apoptotic EPCs increased, and the mRNA level of Bax raised, while the Bcl-2 mRNA level decreased. ④After high glucose culture for 3d, the [Ca2+]i in the two groups had no significant difference. In calcium free medium, the [Ca2+]i presented an rapid increase in both group after administration of CaCl2, the [Ca2+]i in normal group return to the basal level gradually, while the [Ca2+]i in some cells of high glucose group failed to recover to the basal line. ⑤The resting membrane potential of EPCs remained at about -50mv in normal conditions, which had no significant difference between high glucose group and control group. The membrane current of EPCs decreased under the action of TEA, indicating the presence of calcium-dependent potassium current in the composition of membrane potential. There was no significant difference between high glucose group and control group on membrane currents. ⑥The phosphorylation level of eNOS and AKT decreased obviously after high glucose culture compared with normal circumstance.
Conclusions ①High glucose can affect EPCs on proliferation, adhersion and mobilization, which may be caused by inhibition of PI3K/AKT/eNOS pathway. High glucose may promote apoptosis by upregulation of Bax gene and downregulation of Bcl-2 gene on mRNA level. ②High glucose may affect calcium homeostasis, the EPCs were more likely to suffer calcium overloading under high glucose condition, the abnormal calcium signal transduction pathway caused by calcium homeostasis disorder may be responsible for the dysfunction of EPCs. ③Calcium-dependent potassium channel (KCa) exist on the membrane of EPCs, and KCa may take part in the maintaining of membrane potential of EPCs. |
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