Tree shrew model of early diabetic retinopathy reveals microvascular dysfunction and identifies phosphoserine aminotransferase 1 as a novel therapeutic target.

BACKGROUND: Vascular endothelial growth factor (VEGF) protein levels in diabetes mellitus (DM) patients with ulcerative foot will tend to decrease. Matrix metalloproteinases (MMPs) and their inhibitors have also been identified in regulating capillary tubes formation (morphogenesis) with the collagen matrix, associated with the formation and regression of granulation tissue during the wound healing process. AIM: This study was aimed to determine the relationship between gene polymorphism VEGF rs699947 with VEGF and MMP-14 protein levels in cases of diabetic foot ulcers (DFUs). METHODS: This study was an observational research with cross-sectional comparative study design. The population in this study were type-2 DM patients who met the inclusion criteria. According to the Meggitt-Wagner classification, the study sample was divided into two groups: Type 2 DM group without DFU and type 2 DM group with DFU Grades 1–3. RESULTS: In this study, there were differences in the protein levels of MMP-14 (p = 0.039) VEGF (p = 0.002) between type-2 DM patients with and without FDU. However, there was no difference in the VEGF gene polymorphism rs6999947 between type-2 DM patients with and without FDU (p = 0.099). In addition, the results showed that type-2 DM patients with MMP-14 protein levels ≤ 3.864 had a 3.6 times greater risk of suffer FDU compared to type-2 DM patients with MMP-14 protein levels > 3.864 but not significant (PR = 3.600 (IK 5 % 1.142–11.346); p = 0.052). Meanwhile, type 2 DM patients with VEGF protein levels ≤567.42 were significantly more at risk of 9048 times to suffer FDU compared to type 2 DM patients with VEGF protein levels > 567.42 (PR = 9.048 (CI 5% 2.571–31.842); p = 0.001). CONCLUSION: In type 2 DM patients with FDU, there were lower levels of MMP-14 and VEGF compared to patients without FDU. There is a significant relationship between VEGF protein levels and the incidence of FDU in type 2 DM patients, but there is no relationship between MMP-14 and the gene polymorphism VEGF rs6999947 with the incidence of FDU in type 2 DM patients.

3029 Background: Treatment with the trifunctional anti-EpCAM x anti-CD3 antibody catumaxomab efficiently eliminates tumor cells from the peritoneal cavity (Jäger et al., ASCO 2007) and led to clinically relevant prolongation of puncture-free survival (PuFS) in patients with malignant ascites (MA) in a pivotal phase II/III trial (Parsons et al., ASCO 2008). As vascular endothelial growth factor (VEGF) levels are markedly elevated in MA in comparison to cirrhotic ascites the question was addressed whether catumaxomab treatment impacts the expression or accumulation of VEGF within MA. Here we report that in addition to tumor cell depletion, VEGF protein levels in MA significantly decreased upon catumaxomab therapy. We propose that the strongly correlated tumor cell elimination and reduced VEGF protein levels are causative for the prolonged PuFS of patients suffering from MA. Methods: VEGF and total protein levels were measured by ELISA and BCA from MA supernatants before catumaxomab therapy, after the 1st infusion (10μg; day 3) and after the 4th infusion (150μg; day 11). Data were statistically analysed for the ratio of the VEGF protein concentration versus the total protein concentration for the MA treatment groups with ovarian (OC) or nonovarian cancer (NC) as underlying disease and the corresponding control groups that received paracentesis only. Results: One day after the last catumaxomab infusion 46 or 47 patients analysed in the OC or NC treatment group showed a statistically significant decrease in VEGF to total protein ratio when compared to the measurement before catumaxomab therapy (ANOVA p=0.034 for OC and p<0.001 for NC). These results are consistent with the tumor cell elimination previously assessed in these patients. In contrast, the OC control group showed a statistically significant increase of VEGF to total protein ratio (p=0.009), which is accompanied by an increase in tumor cell numbers. In the NC control group VEGF to total protein ratio remained unaffected (p=0.096). Conclusions: Catumaxomab therapy significantly reduced VEGF protein levels correlating with tumor cell elimination in MA, which in turn led to the prevention of fluid accumulation in the peritoneal cavity and finally to prolonged PuFS of patients suffering from MA. [Table: see text]

PurposeThis study investigated changes in the transcript levels of genes related to glutamate neurotransmission and transport as diabetes progresses in the Long-Evans rat retina. Transcript levels of vascular endothelial growth factor (VEGF), erythropoietin, and insulin-like growth factor binding protein 3 (IGFBP3) were also measured due to their protective effects on the retinal vasculature and neurons.MethodsDiabetes was induced in Long-Evans rats with a single intraperitoneal (IP) injection of streptozotocin (STZ; 65 mg/kg) in sodium citrate buffer. Rats with blood glucose >300 mg/dl were deemed diabetic. Age-matched controls received a single IP injection of sodium citrate buffer only. The retinas were dissected at 4 and 12 weeks after induction of diabetes, and mRNA and protein were extracted from the left and right retinas of each rat, respectively. Gene expression was analyzed using quantitative real-time reverse-transcription PCR. Enzyme-linked immunosorbent assay was used to quantify the concentration of VEGF protein in each retina. Statistical significance was determined using 2×2 analysis of variance followed by post-hoc analysis using Fisher’s protected least squares difference.ResultsTranscript levels of two ionotropic glutamate receptor subunits and one glutamate transporter increased after 4 weeks of diabetes. In contrast, 12 weeks of diabetes decreased the transcript levels of several genes, including two glutamate transporters, four out of five N-methyl-D-aspartate (NMDA) receptor subunits, and all five kainate receptor subunits. Diabetes had a greater effect on gene expression of NMDA and kainate receptor subunits than on the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits, for which only GRIA4 significantly decreased after 12 weeks. VEGF protein levels were significantly increased in 4-week diabetic rats compared to age-matched control rats whereas the increase was not significant after 12 weeks. Transcript levels of VEGF and VEGF receptors were unchanged with diabetes. Erythropoietin and IGFBP3 mRNA levels significantly increased at both time points, and IGFBP2 mRNA levels increased after 12 weeks.ConclusionsDiabetes caused significant changes in the transcriptional expression of genes related to ionotropic glutamate neurotransmission, especially after 12 weeks. Most genes with decreased transcript levels after 12 weeks were expressed by retinal ganglion cells, which include glutamate transporters and ionotropic glutamate receptors. Two genes expressed by retinal ganglion cells but unrelated to glutamate neurotransmission, γ-synuclein (SNCG) and adenosine A1 receptor (ADORA1), also had decreased mRNA expression after 12 weeks. These findings may indicate ganglion cells were lost as diabetes progressed in the retina. Decreased expression of the glutamate transporter SLC1A3 would lead to decreased removal of glutamate from the extracellular space, suggesting that diabetes impairs this function of Müller cells. These findings suggest that ganglion cells were lost due to glutamate excitotoxicity. The changes at 12 weeks occurred without significant changes in retinal VEGF protein or mRNA, although higher VEGF protein levels at 4 weeks may be an early protective response. Increased transcript levels of erythropoietin and IGFBP3 may also be a protective response.

This Issue At A Glance

Angiogenesis in diabetic retinopathy (DR) is a multifactorial process regulated by hypoxia-induced growth factors and inflammatory cytokines. In addition to the angiogenic switch, the proteolytic processing and altered synthesis of the extracellular matrix are critical steps in this disease. This study was performed to evaluate the levels of matrix metalloproteinase-2 and matrix metalloproteinase-9 (MMP-2 and MMP-9), angiopoietin-1 and angiopoietin-2 (Ang-1 and Ang-2), vascular endothelial growth factor (VEGF), erythropoietin (EPO) and transforming growth factor-β1 (totalTGFβ1) in the vitreous of diabetic eyes undergoing vitrectomy compared with control eyes operated because of macular hole or pucker. Prospective consecutive controlled observational study performed in the unit of vitreoretinal surgery in Finland during the years 2006-2008. Vitreous samples were collected before the start of the conventional 3-ppp vitrectomy. Vitreous MMP-2 and MMP-9, Ang-1 and Ang-2, VEGF, EPO and TGFβ1 concentrations were measured from 69 patients with Type 1 or 2 diabetes and 40 controls. Comparison of eyes with DR with controls revealed that the mean vitreous concentrations of proMMP-2 (p = 0.0015), totalMMP-2 (p = 0.0011), proMMP-9 (p = 0.00001), totalMMP-9 (p < 0.00001), Ang-2 (p < 0.00001), VEGF (p < 0.00001), EPO (p < 0.00001) and totalTGFβ1 (p = 0.000026) were significantly higher in the former group. A multivariate logistic regression analysis suggested intravitreal Ang-2 concentration being the key marker of PDR (p = 0.00025) (OR = 1507.9). The main new finding is that the intravitreal concentrations of Ang-2 correlated significantly with MMP-9, VEGF, EPO and TGFβ1 levels in diabetic eyes undergoing vitrectomy. Thus, these factors could promote retinal angiogenesis synergistically.

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for vascular endothelial cells and promotes neovascularization in vivo. To determine whether interleukin-1 beta (IL-1 beta), which is present in atherosclerotic lesions, induces VEGF gene expression in vascular smooth muscle cells, we performed RNA blot analysis on rat aortic smooth muscle cells (RASMC) with a rat VEGF cDNA probe. IL-1 beta increased VEGF mRNA levels in RASMC in a time- and dose-dependent manner. As little as 0.1 ng/ml IL-1 beta increased VEGF mRNA levels by 2-fold and 10 ng/ml IL-1 beta increased VEGF mRNA by 4-fold. We also measured the half-life of VEGF mRNA and performed nuclear run-on experiments before and after addition of IL-1 beta to see if IL-1 beta increased VEGF mRNA levels by stabilizing the mRNA or by increasing its rate of transcription. The normal, 2-h half-life of VEGF mRNA in RASMC was lengthened to 3.2 h (60%) by IL-1 beta, and IL-1 beta increased the rate of VEGF gene transcription by 2.1-fold. In immunoblot experiments with an antibody specific for VEGF, we found that IL-1 beta increased VEGF protein levels in RASMC by 3.3-fold. Together these data indicate that IL-1 beta induces VEGF gene expression in smooth muscle cells. This IL-1 beta-induced expression of VEGF may accelerate the progression of atherosclerotic lesions by promoting the development of new blood vessels.

Objective To determine the intraocular levels of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) in patients with neovascular glaucoma (NVG) and to evaluate the relationship between probable clinical diathesis and associated levels. Methods Experimental study. Fifty-four NVG eyes of 54 patients and 10 fresh healthy donor eyes for corneal transplantation as controls were selected. The levels of VEGF and PDGF in aqueous humor and vitreous liquid aspirates from them were measured. Of the 54 eyes, 17 had central retinal vein occlusion (CRVO), 22 had diabetic retinopathy (DR), 4 had retinal vasculitis (Eales disease),4 had retinal detachments (RD) and 7 had unidentified NVG (NA). Among them, the number of NVG cases with iris neovascularization grades Ⅰ , Ⅱ, Ⅲ and Ⅳ were 17, 12, 13 and 12, respectively,and 36 eyes were treated with prophylactic retinal photocoagulation and/or cryotherapy. The levels of VEGF and PDGF were measured using an enzyme-linked immunosorbent assay (ELISA) method. The differences in VEGF and PDGF levels between the NVG and control groups were analyzed with a Mann-Whitney U test. The differences in the various primary causes, in the iris neovascularization grades and between the prophylactic-treated and untreated groups were analyzed with ANOVA, LSD-t and independent samples t test, respectively. The correlation analysis between VEGF and PDGF levels in each group were checked with a Pearson test. Results The free VEGF and PDGF concentrations in aqueous humor from NVG patients were (926.3±223.5)ng/L and (226.2±81.5)ng/L and the concentrations in vitreous liquid were (1096.1±235.9)ng/L and (375.3±141.5)ng/L, which were higher than concentrations in normal control eyes (aqueous humor: ZVEGF=-4.993, ZPDGF=-4.891, vitreous liquid: ZVEGF=-4.991, ZPDGF=-4.992, all P=0.000). The free VEGF concentrations in aqueous humor and vitreous liquid from NVG secondary to CRVO were higher than those in the NA group (aqueous humor: t=1.746, P=0.033; vitreous liquid: t=1.917, P=0.027). There were no differences in VEGF between CRVO, DR, Eales, and RD eyes. The PDGF concentrations in aqueous humor and vitreous liquid from NVG with diabetic retinopathy (DR) were higher than the concentrations in NVG secondary to Eales disease (aqueous humor: t=1.697, P=0.043; vitreous liquid: t=1.762, P=0.038).There were no statistical differences between VEGF in aqueous humor and vitreous liquid among the various iris neovascularization grades. The vitreal PDGF level in iris neovascularization grade Ⅳ was higher than that in grade Ⅲ (t=1.740, P=0.049). The VEGF and PDGF concentrations in aqueous humor and vitreous liquid in NVG with previous retinal photocoagulation and/or cryotherapy were lower than those in non-intervention NVG (aqueous humor: ZVEGF=2.945, PVEGF=0.003; tPDGF=3.199,PPDGF=0.002; vitreous liquid:ZVEGF=3.165, PVEGF=0.002; tPDGF=2.984, PPDGF=0.004). Correlation analysis showed that there was a positive correlation between the VEGF and PDGF levels in aqueous humor (r=0.305, P=0.025) and vitreous liquid (r=0.303, P=0.026). In NVG secondary to CRVO, the VEGF level in vitreous liquid was positively correlated with PDGF (r=0.503, P=0.040), while the VEGF level in aqueous humor from NVG with DR was positively correlated with PDGF (r=0.462, P=0.030).Conclusion VEGF and PDGF levels are related to primary causes of NVG and iris neovascularization grading, and their release may be inhibited after retinal photocoagulation and/or cryotherapy in NVG eyes. Key words: Glaucoma,neovascular; Vascular endothelial growth factors; Platelet-derived growth factor; Neovascularization; Laser therapy

Vascular endothelial growth factor and basic fibroblast growth factor expression in esophageal adenocarcinoma and Barrett esophagus

Interleukin-6 Receptor-Mediated Activation of Signal Transducer and Activator of Transcription-3 (STAT3) Promotes Choroidal Neovascularization

Diabetic retinopathy, a severe complication of diabetes, is the leading cause of blindness in the developed world. This study investigated the effects of quercetin on levels of monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) in serum of rats with diabetic retinopathy, and explored the functional mechanisms of quercetin in the treatment of diabetic retinopathy. Twenty rats with induced diabetes were divided into a model group and a quercetin group, with 10 rats in each group. Ten healthy rats were also included to serve as a control group. Rats in the quercetin group were treated with an intragastric injection of quercetin (150 mg/kg), while the same amount of sodium carboxymethyl cellulose (CMCNa) was used for rats in the model group and the control group. The treatment was performed once per day and blood glucose was measured in each group at 0, 10 and 20 weeks after the first treatment. Blood glucose tests showed that quercetin did not reduce blood glucose in rats with diabetes. However, pathological examination showed that quercetin could relieve pathological changes caused by diabetes, such as retinal edema and vacuoles. ELISA results showed that, compared with the control group, levels of MCP-1, MMP-9 and VEGF in the model group were significantly increased (P<0.01). No significant difference in serum MCP-1 content was found between the model group and the quercetin group, but levels of MMP-9 and VEGF were significantly decreased in the quercetin group (P<0.01). Results of RT-PCR and western blot analysis showed that, compared with the control group, levels of MCP-1, MMP-9 and VEGF mRNA and protein in the retinal tissue of rats in the model group were significantly increased (P<0.01). No significant differences in expression levels of MCP-1 mRNA and protein were found between the model group and the quercetin group, but levels of MMP-9 and VEGF mRNA and protein were significantly decreased in the quercetin group (P<0.01). Quercetin has a certain therapeutic effect on rats with diabetic retinopathy and its effect may be achieved by reducing the expression of MMP-9 and VEGF, but not the inflammatory mediator, MCP-1.

Background. The study of the role of transforming growth factor (TGF) and vascular endothelial growth factor (VEGF) in the development of prenatal pathologies in calves, especially in the context of complicated pregnancy in cows, is relevant due to the high incidence of morbidity and mortality in newborn young animals, which significantly reduces the economic efficiency of the livestock industry. The study is based on analyzing placental tissue collected immediately after calving to identify relationships between the levels of these proteins and the incidence of pathologies in newborn calves. Changes in TGF and VEGF concentrations can serve as predictors of feto- and uteroplacental insufficiency, which in turn negatively affects the health of calves. Correlation analysis revealed statistically significant relationships between TGF and VEGF levels and clinical disease manifestations, highlighting their role in prenatal disorder pathogenesis. These studies underscore the importance of monitoring TGF and VEGF levels in veterinary practice, potentially enabling early risk identification and the development of effective disease prevention strategies in young animals. Purpose. The objective of the present study is to develop new methods for predicting prenatal diseases in calves based on the correlation analysis of TGF and VEGF protein levels. Materials and methods. At the SPC "Plemzavod Vtoraya Pyatiletka", in the period from 2023 to 2024, a correlation analysis of the proteins TGF and VEGF in the context of complicated pregnancy was carried out. For the experiment, 200 dry first-calf heifers aged 24 to 48 months were randomly selected. The animals were divided into two groups: Group A included 100 cows with a physiological pregnancy and uncomplicated births, and Group B consisted of 100 animals with complicated pregnancy, accompanied by feto- and uteroplacental insufficiency. The distribution of livestock into groups was carried out on the basis of anamnesis data and clinical examination results. Placental tissue collected immediately after calving in compliance with the temperature (cold) regime was used as research material. The study analyzed the protein level of transforming growth factor (TGF), which regulates various cellular functions such as growth, development, immune responses, and tissue remodeling. The protein level of vascular endothelial growth factor (VEGF), which is involved in angiogenesis, was also assessed using enzyme immunoassay kits from the biotechnological company Cutimmune systems, located in the United States. The licensed program "Primer of Biostatistics 4.03. For Windows" was used to process the data in the study. The homogeneity of variances was assessed using the Fisher criterion, which allows determining the uniformity of variances in a group. To test the statistical significance of differences between the compared groups, two criteria were used: the Student criterion and the nonparametric Mann-Whitney criterion. The Student criterion is a parametric method and is designed to compare the average values ​​of two groups. The nonparametric Mann-Whitney criterion, in turn, is designed to compare two independent and unrelated small samples by a quantitative feature of two groups. Differences were considered reliable at p&lt;0.05. To conduct a correlation analysis, the Spearman method was used, which allows determining the strength and direction of the relationship between two features. Results. During the study, it was found that prenatal disorders in calves born from cows with feto- and uteroplacental insufficiency have a direct correlation with the level of protein transforming growth factor (TGF) and protein vascular endothelial growth factor (VEGF). Conclusion. The results of the studies revealed significant differences in the levels of VEGF and TGF proteins in cows with complicated pregnancy. Data analysis showed that the level of VEGF in animals from group B was 24% higher compared to the indicators of group A (p&lt;0.05). At the same time, a significant decrease in the concentration of TGF in the placentas of cows with complicated pregnancy was observed - 2.4 times lower than in animals with a physiological course of pregnancy (p&lt;0.01). These changes may indicate a compensatory mechanism in which an increase in VEGF production is aimed at leveling out the consequences of a sharp decrease in the level of TGF. Also, special attention was paid to the ratio of VEGF and TGF in the placenta. In group A, this coefficient was 1.1%, which indicates a balance of the processes regulated by these growth factors. At the same time, a significant increase in the VEGF/TFR ratio was observed in Group B – approximately 3.2 times compared to Group A. This indicates a pronounced imbalance in the regulation of angiogenesis and metabolic processes in cows with complicated pregnancy. The obtained VEGF/TFR ratio can become a valuable tool for predicting physiological disorders in calves during postembryonic ontogenesis. Its use will optimize veterinary strategies and, possibly, prevent the development of diseases in the long term. This is important not only for ensuring animal welfare, but also for increasing the economic efficiency of animal husbandry by reducing losses associated with morbidity and mortality of young animals. EDN: VGYUTD

Background The primary pathological basis of diabetic retinopathy (DR) is new blood formation due to anoxia and inflammation,which results in breakdown of blood-retinal barrier (BRB).Vascular endothelial growth factor (VEGF) is a key factor promoting neovascularization.Researches determined that lycium barbarum polysaccharides (LBP) can protect cells against oxidative damage.However,the study of LBP in ophthalmology is lack.Objective This study was to investigate the effects of LBP on the dynamic pathological change of retinal vessels and expression of VEGF in retina of diabetic rats.Methods One hundred and seventeen SPF male SD rats were randomly divided into the normal control group,the diabetic mellitus (DM) group and the LBP group according to random number table.Type 1 DM models were induced by intraperitoneal injection of streptozotocin (STZ,55 mg/kg) in the rats of the DM group and the LBP group,and then 250 mg/kg LBP was intragastically administered in the rats of the LBP group.The morphological change of retinal vessels was dynamically observed by retinal stretched preparation with Evans blue (EB) in 4,10 and 16 weeks after modeling.E B (45 mg/kg) was slowly injection via jugular vein,and 1％ polyoxymethylene was infused into the left ventricule.The eyeballs were extracted and retina were isolated.EB content in the retinas (mg/g) was calculated using retinal stretched preparation method at the time points mentioned above.Expressions of VEGF protein and mRNA in the retinas were detected by immunohistochemistry and real-time quantitative PCR at various time points,respectively.Results Retinal stretched preparation with EB exhibited that the abnormal degree in the shape,diameter of vessels and leakage of the retinal blood vessels were significantly slighter in the LBP group than those of the DM group in 4,10,16 weeks after modeling.At 4,10,16 weeks,EB content in the retinas was (12.17±1.55),(16.46±1.60) and (19.55±1.49) mg/g,which was significantly lower than (15.76± 1.90),(21.61 ±2.05) and (26.30±2.28) mg/g of the DM group (P＜0.05).Immunochemistry showed that the expression of VEGF protein primarily located at retinal ganglion cells (RGCs) layer.The staining intensity for VEGF protein was weaker in the LBP group than that of the DM group.The expression levels of VEGF protein (A value) in the LBP group were 0.234±0.011,0.331±0.023 and 0.536±0.031at various time points,with significant decline in comparison with 0.281±0.018,0.533±0.055 and 0.765±0.075 of the DM group (all at P＜0.05).Real-time quantitative PCR revealed that the expression levels of VEGF mRNA were 0.157±0.013,0.505 ±0.114 and 1.577±0.074 in the LBP group at various time points,which were significantly lower than 0.235±0.209,1.043±0.084 and 2.446±0.061 of the DM group (all at P＜0.05).Conclusions LBP can alleviate the DM-induced retinal vasculopathy,lessen the leakage of vessels well,and further protect the BRB. Key words: Diabetes mellitus/complication; Retina/diabetic retinopathy; Lycium barbarum polysaccharide; Vascular endothelial growth factor

Background: Type 1 diabetes mellitus (DM) is a chronic metabolic disorder that causes hyperglycemia and increases the risk of morbidity and mortality. Diabetic retinopathy, a microvascular complication that often occurs in DM patients, can cause visual impairment and even blindness. Regular eye examinations are important for early detection of diabetic retinopathy. Optical coherence tomography (OCT) is a non-invasive method that can be used to measure the thickness of retinal layers, including RGC and RNFL. It is thought that thinning of the retinal layer can be a sensitive biomarker in detecting diabetic retinopathy in type 1 DM patients. This study aims to determine changes in RGC and RNFL thickness in children with type 1 DM. Methods: This cross-sectional design analytical observational study was conducted at the eye polyclinic of Dr. M. Djamil General Hospital Padang in November 2023-March 2024. A total of 46 eyes from 46 people, divided into two groups: the type 1 DM group and the control group, were recruited in this study. RGC thickness was measured using AS-OCT GC-IPL thickness analysis and RNFL with optic disc RNFL thickness analysis. Data analysis was carried out using the unpaired T-test. Results: The results showed RGC depletion in the type 1 DM group (RGC 83.48 ± 3.75) compared to the control group (RGC 86.70 ± 4.87) with a value of p = 0.016 (p &lt; 0.05). There was no statistical difference in RNFL thickness between the type 1 DM group (RNFL 102 ± 11.80) and the control group (RNFL 100.96 ± 10.97) with a value of p = 0.581 (p&gt; 0.05). Conclusion: This study found RGC thinning in type 1 DM patients, but did not find differences in RNFL thickness between the two groups. This RGC depletion is thought to be caused by apoptosis of retinal neuronal cells due to chronic hyperglycemia. Examination of RGC thickness with OCT can be developed as an early detection of diabetic retinopathy in children with type 1 DM.

To examine the association of diabetes and diabetic retinopathy (DR) with retinal ganglion cell (RGC) loss. Observational case-control study. Type 2 diabetes cases and age-gender matched controls without diabetes. Spectral-domain optical coherence tomography (OCT) parameters of RGCs were calculated after automated segmentation of macular scans. DR severity was graded on fundus photographs using the modified Airlie House Classification system. Generalized estimating equation was used to compare OCT parameters between cases and controls, adjusted for covariates. Average ganglion cell-inner plexiform layer (GC-IPL) and average retinal nerve fibre layer (RNFL) thicknesses. We analyzed 227 cases and 227 controls. The mean age (years) of cases was 58.3 and controls was 58.1 (P = 0.13). Among cases, 101 had none, 25 had mild and 101 had moderate or severe DR. Compared with controls, GC-IPL and RNFL were thinner in all cases [mean difference (95% confidence interval [CI]): GC-IPL -4.49 µm (-2.92; -6.06), RNFL -0.93 µm (-0.09; -1.85)], including cases with no DR [mean difference (95% CI), GC-IPL -4.37 µm (-2.72; -6.02), RNFL -1.06 µm (-0.10; -2.02)]. Cases with any DR had thinner GC-IPL than controls [mean difference (95% CI): GC-IPL -4.81 µm (-2.12; -7.50)]. Among cases, subjects with moderate or severe DR had thinner GC-IPL than subjects with no DR [mean difference (95% CI): GC-IPL -2.07 µm (-0.08; -4.07)]. RGC loss is present in subjects with diabetes and no DR, and is progressive in moderate or severe DR. RGC neuronal damage in diabetes and DR can be clinically detected using OCT.

Objective To study the effect of down-regulation of Claudin-3 mediated by adeno-associated virus (AAV) of shRNA on the cultured retinal ganglion cells (RGCs) in vitro. Methods RGCs isolated from mouse eyes were divided into normal control group, AAV-shScramble group, and AAV-shClaudin-3 group. The RGCs in AAV-shScramble group and AAV-shClaudin3 group were treated with AAV-shScramble and AAV-shClaudin-3 respectively 24 hours after cell seeding. Dynamic live cell fluorescence microscopy was used to observe the transfection efficiency 96 hours after transfection. Immunofluorescent staining of β-tubulin was used to measure the length of RGCs' axon. 4', 6-diamidino-2-phenylindole staining was used to observe the nuclei of apoptotic cells. The mRNA level of Claudin-3 and VEGF was measured by real-time polymerase chain reaction. The protein levels of Claudin-3, vascular endothelial growth factor (VEGF), Bcl-2 and Caspase-3 was determined by Western blot. Results The positive transfection rate was more than 50% in both AAV-shScramble group and AAV-shClaudin-3 group. The length of RGCs' axon in AAV-shClaudin-3 group was shorter than that in normal control group and AAV-shScramble group (F=22 363.274,P<0.05). Down-regulation of Claudin-3 accelerated RGCs' apoptosis with nuclei shrinkage, tapering, and nucleolus formation of apoptotic bodies. The mRNA levels of Claudin-3 and VEGF in AAV-shClaudin-3 group were lower than those in normal control group and AAV-shScramble group (F=257.408, 160.533;P<0.05). The protein levels of Claudin-3, VEGF and Bcl-2 in AAV-shClaudin-3 group were lower than those in normal control group and AAV-shScramble group (F=129.671, 420.552, 62.669;P<0.05), while the protein level of Caspase-3 in AAV-shClaudin-3 group was higher than that in normal control group and AAV-shScramble group (F=231.348,P<0.05). Conclusion Down-regulation of Claudin-3 increases the expression of Caspase-3, reduces the expression of VEGF and Bcl-2, accelerates RGCs' apoptosis and inhibit the RGCs' axon growth. Key words: Retinal ganglion cells/physiology; Claudins; RNA, small interfering; Animal experimentation