ObjectiveTo explore the involvement of miR-126 and the role of mammalian target of rapamycin (mTOR)/hypoxia-induced factor 1 α (HIF-1 α) pathway in regulating human umbilical cord mesenchymal stem cells (hUCMSCs) exosomes (Exo) on vascular endothelial growth factor (VEGF)-A levels in high glucose-induced human retinal vascular endothelial cells (HRECs). MethodsThe hREC was cultured in EGM-2-MV endothelial cell culture medium with 30 mmol/L glucose and placed in hypoxic cell incubator by 1% oxygen concentration. The cell model of high glucose and low oxygen was established. After modeling, divided HRECs into Exo group, phosphate buffer saline (PBS) group, PBS+anti-miR126 group, Exo+anti-miR126 group, PBS+anti-mTOR group, and PBS+anti-HIF-1 α group. High-glucose and hypoxia-induced hREC in the PBS and Exo groups were respectively co-cultured with PBS and 100 μg/ml hUCMSC Exo. PBS+anti-mTOR group, PBS+anti-HIF-1 α group: 500 nmol/L mTOR inhibitor ADZ2014, 25 μmol/L HIF-1 α inhibitor YC-1 pretreatment for hREC 12 h, and then co-culture with PBS after High-glucose and hypoxia-induced. PBS+anti-miR126 group, Exo+anti-miR126 group: miR-126 LNA power inhibitor probe was transfected with high glucose, and co-cultured with PBS and hUCMSC Exo 6 h after transfection. Real-time polymerase chain reaction (qPCR) measured miRNA-126 expression levels in PBS, and Exo groups for 0, 8, 16 and 24 h. After 24 hof co-culture, the levels of mTOR and HIF-1 α in the cells of PBS and Exo groups were detected by immunofluorescence, Western blot and qPCR, respectively. Western blot, qPCR detection of VEGF-A expression levels in cells of the PBS+anti-mTOR and PBS+anti-HIF-1 α groups. The expression of VE GF-A, mTOR, and HIF-1 α mRNA was measured in cells of PBS+anti-miR126 group and Exo+anti-miR126 group by qPCR. Comparison between two groups was performed by t-test; one-way ANOVA was used for comparison between multiple groups. ResultsAt 0, 8, 16 and 24 h, the relative mRNA expression of miR-126 gradually increased in the Exo group (F=95.900, P＜0.05). Compared with the PBS group, The mTOR, HIF-1 α protein (t=3.466, 6.804), mRNA in HRECs in the Exo group, VEGF-A mRNA expression (t=8.642, 7.897, 6.099) were all downregulated, the difference was statistically significant (P＜0.05). The relative expression level of VEGF-Aprotein (t=3.337, 7.380) and mRNA (t=8.515, 10.400) was decreased in HRECs of the anti-mTOR+PBS group and anti-HIF-1 α+PBS group, differences were statistically significant (P＜0.05). The relative expression of VEGF-A, mTOR, and HIF-1 α mRNA was significantly increased in the cells of the Exo+anti-miR126 group, the differences were all statistically significant (t=4.664, 6.136, 6.247; P＜0.05). ConclusionsmiR-126 plays a role in regulating the effect of hUCMSCs exosomes on VEGF-A levels in high glucose-induced HRECs via mTOR-HIF-1 α pathway.
ObjectiveTo observe the effect and mechanism of human umbilical cord blood mesenchymal stem cells-derived microvesicles (hUMSCs-MVs) on the injury of the primary rat retinal ganglion cells (RGCs) by high glucose environment. Methods The primary RGCs of Sprague Dawley rats were cultured in vitro, hUMSCs-MVs were isolated and extracted by ultra-centrifugation. hUMSCs-MVs were internalized with RGCs. The RGCs were divided into 4 groups under the conditions below: normal control group (group A), high glucose condition group (group B, RGCs+glucose 33 mmol/L), normal RGCs co-cultured with hUMSCs-MVs group (group C, RGCs+hUMSCs-MVs), and RGCs co-cultured with hUMSCs-MVs in high glucose condition group (group D, RGCs+hUMSCs-MVs+glucose 33 mmol/L). The cell activity was detected by CCK-8 test. Annexin Ⅴ/PI staining detected the cell apoptosis rate by flow cytometry. And the relative expression levels of the genes such as Bcl-2, Bax and Caspase-3 were detected by fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Statistical analysis was performed by using One-way analysis of variance and SNK-q test was used for the comparison between groups. Results The hUMSCs-MVs were extracted by ultra-centrifugation, which were characterized as single or cluster of circular membranous vesicle-like structure with diameter ranging from 100 nm to 1000 nm. The flow cytometry analysis showed that hUMSCS-MVs were highly positived by the surface markers of CD44, CD29, CD73, and CD105 whereas been poorly expressed the integrin (CD49f), HLA class Ⅱ, CD34, CD45. There were significant differences in the cell activity and the apoptosis rate among 4 groups, the cell apoptosis rate of group B was higher significantly than that of group A and group D (F=107.92, P=0.000), the cell activity of group B was lower than that of group A and group D (F=382.11, P=0.000). The results of RT-PCR and Western blot showed that the relative mRNA (F=219.79, 339.198, 1 071.21; P=0.000, 0.000, 0.000) and protein (F=544.28, 295.79, 224.75; P=0.000, 0.000, 0.000) expression of Bcl-2, Bax, Caspase-3 and the protein expression of cleaved Capspase-3 (F=533.18, P=0.000) in group B and D were higher significantly than those in group A and C. The relative expression of Bcl-2 mRNA and protein in group B was significantly lower than that of in group D (P＜0.05). The relative expression of Bax, Caspase-3 mRNA and protein in group B was higher than that in group D (P＜0.05). The relative expression of cleaved Caspase-3 protein in group B was higher significantly than that in group D (P＜0.05). Conclusion The hUMSCs-MVs can protect the cultured rat RGCs from the damage of the high glucose condition through increasing the cell activity and reducing the apoptosis rate of RGCs by promoting the Bcl-2 expression, decreasing the expression of Bax and Caspase-3 and inhibiting the Caspase-3 into the activity form of cleaved Caspase-3.
ObjectiveTo observe the changes of macular microvessels in patients with retinal vein occlusion (RVO) and macular edema (ME) after intravitreal injection of aflibercept (IVA), and analyze its correlation with best corrected visual acuity (BCVA).MethodsA retrospective case study. Thirty patients (30 eyes) with monocular RVO with ME (RVO-ME) who were diagnosed in the clinical examination of Tianjin Eye Hospital from April 2019 to February 2020 were included in the study. Among them, there were 12 males (12 eyes) and 18 females(18 eyes); the average age was 54.30±13.17 years. The average course of disease was 3.43±1.97 months. Both eyes were examined by BCVA and optical coherence tomography (OCTA). The on-demand injection was adopted after the first injection in IVA treatment regimen. The macular area 6 mm×6 mm in both eyes was scanned with an OCTA instrument, and the area of the foveal avascular area (FAZ), FAZ circumference (PERIM), and out-of-roundness were measured at baseline and 1, 3, and 6 months after treatment. Index (AI), blood flow density within 300 μm width of FAZ (FD-300), foveal retinal thickness (CMT), superficial retinal capillary plexus (SCP), deep retinal capillary plexus (DCP) blood flow density. The paired t test was used to compare the quantitative parameters of the affected eye and the contralateral healthy eye at baseline; the changes of the quantitative parameters at baseline and 1, 3, and 6 months after treatment were analyzed by repeated measures analysis of variance. Pearson correlation analysis was used to analyze the correlation between BCVA, retinal perfusion, and macular blood supply parameters at 6 months after IVA treatment.ResultsAt baseline, compared with the contralateral healthy eye, the FAZ area (t=−4.091), PERIM (t=−5.098) and AI (t=−9.093) of the RVO-ME eye were enlarged, and FD-300 (t=7.237) and overall SCP and DCP blood flow density (t=8.735, 9.897) decreased, the difference was statistically significant (P＜0.001). Six months after treatment, the BCVA of RVO-ME eyes was significantly increased, CMT decreased, FAZ area expanded, and AI decreased (t=8.566, 16.739, −6.469, 9.719; P＜0.001), the difference was statistically significant. There was no significant change in the blood flow density of FD-300 and overall SCP and DCP, and the difference was not statistically significant (t=1.017, 1.197, 0.987; P＞0.05). Compared with baseline, the FAZ area of RVO-ME eyes gradually expanded at 3 and 6 months after treatment, and the difference was statistically significant (F=21.979, P＜0.001). Correlation analysis results showed that BCVA at 6 months after treatment was positively correlated with the overall SCP and DCP blood flow density at baseline and 6 months after treatment (r=−0.538, −0.484, −0.879, −0.854; P＜0.05). There was a negative correlation with the area of FAZ 6 months after treatment (r=0.544, P=0.001). The number of ME recurrences was negatively correlated with BCVA and overall SCP and DCP blood flow density 6 months after treatment (r=0.604, −0.462, −0.528; P＜0.05), it was positively correlated with FAZ area (r=0.379, P=0.043).ConclusionWithin 6 months of IVA treatment in RVO-ME eyes, ME is significantly reduced and visual acuity is improved; SCP blood flow density decreases, and FAZ area expands.