Repression of retinal microvascular endothelial cells by transthyretin under simulated diabetic retinopathy conditions

AIM： To investigate biological effects of transthyretin （TTR） on the development of neovascularization under simulated diabetic retinopathy （DR） condition associated with high glucose and hypoxia. METHODS： Human retinal microvascular endothelial cells （hRECs） were cultured in normal and simulated DR environments with high glucose and hypoxia. The normal serum glucose concentration is approximately 5.5 mmol/L; thus, hyperglycemia was simulated with 25 mmol/L glucose, while hypoxia was induced using 200 μmol/L CoCI. The influence of TTR on hRECs and human retinal pigment epithelial cells （hRPECs） was determined by incubating the cells with 4μmol/L TTR in normal and abnormal media. A co -culture system was then employed to evaluate the effects of hRPECs on hRECs. RESULTS： Decreased hRECs and hRPECs were observed under abnormal conditions, including high- glucose and hypoxic media. In addition, hRECs were significantly inhibited by 4 pmol/L exogenous TTR during hyperglycemic culture. During co-culture, hRPECs inhibited hRECs in both the normal and abnormal environments. CONCLUSION： hREC growth is inhibited by exogenous TTR under simulated DR environments with highglucose and hypoxic, particularly in the medium containing 25 mmol/L glucose, hRPECs, which manufacture TTR in the eye, also represses hRECs in the same environment. TTR is predicted to inhibit the proliferation of hRECs and neovascularization.

Evans blue staining to detect deep blood vessels in peripheral retina for observing retinal pathology in early-stage diabetic rats

AIM: To observe and compare the statistical significance of superficial and deep vascular leakage in the pathological changes of the diabetic rats retina after the Evans blue(EB) perfusion, and utilize the modified whole-retina spreading method to make the slides while protecting the periphery of the retina. METHODS: The Sprague-Dawley(SD) rats were randomly divided into 6 groups. Each group named as the normal groups for 4, 8, and 12 wk and the diabetic groups for 4, 8, and 12 wk. The EB was injected into the cardiovascular system of the rats at the different time points. The retina of each group was obtained for observation.RESULTS: The superficial vascular leakage was found in all 6 groups. The size of leakage area of superficial retinal blood vessels was(0.54±0.23)%,(0.65±0.11)%,and(0.58±0.10)% in normal group. No notable leakage was found in the deep blood vessels [(0.03±0.04)%,(0.03±0.05)%, and(0.03±0.05)%]. The deep retinal vascular leakage was found in the peripheral retina of diabetic rats. The size of leakage area of superficial retinal blood vessels in diabetic group were(0.53±0.22)%,(0.69±0.16)%, and(0.52±0.11)%. The leakage areas of deep blood vessels were(0.54±0.50)%,(1.42±0.16)%, and(1.80±0.07)% at 4, 8, and 12 wk, respectively. There was a statistically difference of the leakage area between the 8 th week and the 4 th week of diabetes group(P=0.003). The statistically significant difference between the diabetes and the control groups was noted at 4 wk and 8 wk(P<0.001).CONCLUSION: The main retinal pathological changes of early-stage diabetic rats are the vascular leakage of the periphery of deep retina. Diabetic rats modeled after 8 wk have semi-quantitative statistical difference compared with the normal rats, thus early intervention treatment research can start at this time point.

Protective effect of human umbilical cord mesenchymal stem cell-derived exosomes on rat retinal neurons in hyperglycemia through the brain-derived neurotrophic factor/TrkB pathway

AIM:To explore whether human umbilical cord mesenchymal stem cell(hUCMSC)-derived exosomes(hUCMSC-Exos)protect rat retinal neurons in high-glucose(HG)conditions by activating the brain-derived neurotrophic factor(BDNF)-Trk B pathway.METHODS:h UCMSC-Exos were collected with differential ultracentrifugation methods and observed by transmission electron microscopy.Enzyme-linked immunosorbent assays(ELISAs)was used to quantify BDNF in hUCMSC-Exos,and Western blot was used to identify surface markers of hUCMSC-Exos.Rat retinal neurons were divided into 4 groups.Furthermore,cell viability,cell apoptosis,and TrkB protein expression were measured in retinal neurons.RESULTS:hUCMSCs and isolated hUCMSC-Exos were successfully cultured.All hUCMSC-Exos showed a diameter of 30 to 150 nm and had a phospholipid bimolecular membrane structure,as observed by transmission electron microscopy.ELISA showed the BDNF concentration of hUCMSCs-Exos was 2483.16±281.75.hUCMSCs-Exos effectively reduced the apoptosis of retinal neuron rate and improved neuron survival rate,meanwhile,the results of immunofluorescence verified the fluorescence intensity of TrKB in neurons increased.And all above effects were reduced by treated hUCMSCs-Exos with BDNF inhibitors.hUCMSC-Exos effectively reduced the apoptosis rate of retinal neurons by activating the BDNF-TrkB pathway in a HG environment.CONCLUSION:In the HG environment,hUCMSC-Exos could carry BDNF into rat retinal neurons,inhibiting neuronal apoptosis by activating the BDNF-TrkB pathway.