Effects Of High Glucose On The Function Of Endothelial Progenitor Cells And The Related Mechanisms

Endothelial progenitor cells (EPCs), as the precursor cells of vascular endothelial cells, play an important role in the repair of adult vascular endothelium and angiogenesis. It has been suggested that the number and function of circulating EPCs could be reduced in diabetic, and there is a significant relationship between the development of diabetic vasculopathy and the dysfunction of EPCs. Diabetic retinopathy (DR) is the most common ocular diabetic microvascular complication, which is characterized by the disorder of retinal microcirculation in early stage and the formation of retinal neovascularization in late stage. The main pathological basis of DR is the dysfunction of vascular endothelium and the injury of retinal capillary. However, EPCs play an important role in the restore of damaged retinal blood vessels in physiological and pathological conditions. Therefore, we consider that the development of DR is closely related to the dysfunction of EPCs caused by diabetes, but the definite mechanism is not clear so far. Hyperglycemia is the main sign of diabetes, it is very important to explore the effect of high glucose on the biological characteristics of EPCs and further to study the related mechanisms, which may play important roles in the study of the etiology and prevention of DR.Objective To investigate the effects of high glucose on the proliferation, apoptosis, adhesion and acLDL uptake ability of EPCs in vitro, and further to study the related mechanisms.The study 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.Methods①EPCs were isolated by density gradient centrifugation from human umbilical cord blood and were cultured in vitro, then EPCs were identified by flow cytometry and confocal microscope.②EPCs were cultured in normal and high glucose condition respectively. The effects of high glucose on the proliferation and adhesion capacity of EPCs were detected by MTT assay and cell counts respectively. 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 effect of high glucose on 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, as well as the dynamic variation of [Ca2+]i level after dropping of CaCl2. 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.③Photodynamic method was used to establish retinal vascular injury model in rat. Fundus photography and fundus fluorescein angiography were used to investigate the degree of retinal vascular damage. RT-PCR was used to detecte the SDF-1 mRNA expression after retinal vascular injury.Results1. Mononuclear cells were isolated from human umbilical cord blood, which were then cultured and induced into EPCs in vitro. After culturing for 3 days, EPCs formed small colonies. After culture about 10 days, the cells were highly proliferative and presented spindle shape. The EPCs exhibited cobblestone morphology on about 2-3w. Flow cytometry analysis showed that 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. Immunocytochemistry stain showed that VEGF was expressed in the EPCs.2.①The proliferation activity of EPCs was enhanced after cultivation with high glucose for 1d compared with the normal condition, and the proliferation activity of EPCs in high glucose group had no significant difference with normal group at 3d, while the proliferation activity decreased obviously after treatment with high glucose for 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 in EPCs 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, and there was no significant difference on the membrane potential between the high glucose group and the 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.⑥Western blot showed that the phosphorylation level of eNOS and AKT decreased obviously after high glucose culture compared with normal circumstance.3. Rat retinal microvascular damage can be established by photodynamic coagulation of retinal vein. Occlusion, leakage and hemorrhage of retina can be observed. The SDF-1 mRNA level increased after retinal vascular injury. Conclusions①High 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 expression and downregulation of Bcl-2 gene expression 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. High glucose had no notable effect on the level of membrane potential and the amplitude of membrane current flow.④Retinal vascular injury can cause increased SDF-1 expression in the retina, the SDF-1/CXCR4 axis may related to the retinal vascular repair and angiogenesis mediated by EPCs.