FTO fuels diabetes-induced vascular endothelial dysfunction associated with inflammation by erasing m6A methylation of TNIP1.

Endothelial dysfunction is a critical and initiating factor of the vascular complications of diabetes. Inflammation plays an important role in endothelial dysfunction regulated by epigenetic modifications. N6-methyladenosine (m6A) is one of the most prevalent epigenetic modifications in eukaryotic cells. In this research, we identified an m6A demethylase, fat mass and obesity-associated protein (FTO), as an essential epitranscriptomic regulator in diabetes-induced vascular endothelial dysfunction. We showed that enhanced FTO reduced the global level of m6A in hyperglycemia. FTO knockdown in endothelial cells (ECs) resulted in less inflammation and compromised ability of migration and tube formation. Compared with EC Ftofl/fl diabetic mice, EC-specific Fto-deficient (EC FtoΔ/Δ) diabetic mice displayed less retinal vascular leakage and acellular capillary formation. Furthermore, methylated RNA immunoprecipitation sequencing (MeRIP-Seq) combined with RNA-Seq indicated that Tnip1 served as a downstream target of FTO. Luciferase activity assays and RNA pull-down demonstrated that FTO repressed TNIP1 mRNA expression by erasing its m6A methylation. In addition, TNIP1 depletion activated NF-κB and other inflammatory factors, which aggravated retinal vascular leakage and acellular capillary formation, while sustained expression of Tnip1 by intravitreal injection of adeno-associated virus alleviated endothelial impairments. These findings suggest that the FTO-TNIP1-NF-κB network provides potential targets to treat diabetic vascular complications.

The cause of redetachment after vitrectomy with air tamponade for a cohort of 1715 patients with retinal detachment: an analysis of retinal breaks reopening.

BACKGROUND: To investigate the prevalence and predictors of retinal breaks reopening after vitrectomy with air tamponade in rhegmatogenous retinal detachment (RRD). METHODS: A retrospective cohort study was conducted in Shanghai General Hospital. Chart review was performed among 1715 patients with primary RRD who received pars plana vitrectomy (PPV) with air tamponade as initial management. Patients were followed up for recurrence. The clinical features of the eyes with retinal breaks reopening were recorded. Logistic regression was constructed to investigate the predictors for breaks reopening. RESULTS: A total of 137 (7.99%) patients had recurrent retinal detachment after PPV with air tamponade. The causes of surgery failure included new or missed retinal breaks (48.9%), reopening of original tears (43.8%) and proliferative vitreoretinopathy (7.3%). The median time to recurrence for the patients with breaks reopening was 18.0 days. Multivariate logistic regression indicated that the presence of retinal break(s) ≥ 1.5 disc diameters (DD) (odds ratio [OR]: 2.68, 95% confidence interval [CI]: 11.04-6.92, P = 0.041), and shorter period for restricted activities (OR: 0.94, 95% CI: 0.89-0.99, P = 0.020) were the independent predictors for breaks reopening. CONCLUSIONS: Breaks reopening is an important cause for retinal redetachment after PPV with air tamponade in primary RRD. The first 2-4 weeks after surgery is the "risk period" for breaks reopening. Special attention should be paid for patients with retinal break(s) ≥ 1.5 DD. A prolonged period for restricted activities is recommended.

Differences in Vitreous Protein Profiles in Patients With Proliferative Diabetic Retinopathy Before and After Ranibizumab Treatment.

Proliferative diabetic retinopathy (PDR) accounts for severe impact on vision, its mechanism is still poorly understood. To compare the differences of vitreous protein profiles in PDR patients before and after a complete anti-vascular endothelial growth factor (VEGF) loading dose with ranibizumab treatment. Twelve vitreous humor (VH) samples were collected from six PDR patients before (set as pre group) and after (set as post group) intravitreal injection of ranibizumab (IVR) treatment. LC-MS/MS and bioinformatics analysis were performed to identify differentially expressed proteins. Proteins were validated with targeted proteomics using parallel reaction monitoring (PRM) in a validation set consisting of samples from the above patients. A total of 2680 vitreous proteins were identified. Differentially expressed proteins were filtrated with fold change ≥2.0 (post group/ pre group protein abundance ratio ≥2 or ≤ 0.5) and p-value <0.05. 11 proteins were up-regulated and 17 proteins were down-regulated, while consistent presence/absence expression profile group contains one elevated protein and nine reduced proteins, among which seven proteins were identified as potential biomarkers for IVR treatment through PRM assays. Bioinformatics analysis indicated the up-regulated proteins were significantly enriched in "GnRH secretion" and "Circadian rhythm" signaling pathway. This report represents the first description of combined label-free quantitative proteomics and PRM analysis of targeted proteins for discovery of different proteins before and after IVR treatment in the same patient. IVR treatment may protect against PDR by promoting SPP1 expression through "GnRH secretion" and "Circadian rhythm" signaling pathway.

Dihydroartemisinin ameliorates retinal vascular dysfunction in diabetes mellitus via the FASN/Kmal-mTOR/SREBP1 feedback loop.

Microvascular dysfunction is the primary aetiology of visual impairment caused by diabetic retinopathy (DR). Dihydroartemisinin (DHA), the active metabolite of the antimalarials artemisinins, exhibits antiangiogenic properties in numerous diseases. Here, we investigated the function and mechanisms of DHA as a vasculoprotective agent in DR. DHA exerted its protective effect on vascular injuries in diabetic mice and inhibited cell proliferation and tube formation in human retinal microvascular endothelial cells by decreasing the level of fatty acid synthase (FASN), enhancing the malonylation of mechanistic target of rapamycin (mTOR) at lysine 1218 (K1218) and attenuating the activation of mTOR complex 1 (mTORC1). Impressively, a chemosynthetic small interfering RNA against FASN and mutagenesis of K1218 of mTOR showed therapeutic potential in suppressing cell proliferation and tube formation induced by high glucose. Notably, suppression of mTORC1 kinase activity further inhibited FASN by reducing p70S6K phosphorylation to subsequently reduce the expression of sterol regulatory element binding protein 1, which interacted directly with the FASN promoter at nucleotide positions -64 and -55. In conclusion, our study elucidated the promising effects of FASN and malonylation on vascular injuries of DR and indicated the great potential of DHA as a therapeutic approach.

A Higher Serum Calcium Level is an Independent Risk Factor for Vision-Threatening Diabetic Retinopathy in Patients with Type 2 Diabetes: Cross-Sectional and Longitudinal Analyses.

OBJECTIVE: An elevated serum calcium level is associated with a higher risk of type 2 diabetes (T2D), but its role in microvascular complications remains unclear. This study was conducted to investigate the association between serum calcium levels and vision-threatening diabetic retinopathy (VTDR). METHODS: This study employed a cross-sectional and longitudinal design. The cross-sectional part included all patients treated for T2D at Shanghai General Hospital between 2007 and 2016, while the longitudinal part involved an overlapping cohort of diabetic patients without VTDR who were followed from their admission until December 2019. Multivariable logistic and Cox proportional hazard regression analyses were performed, respectively. VTDR was defined as severe nonproliferative diabetic retinopathy, proliferative diabetic retinopathy, or clinically significant macular edema. RESULTS: A total of 3269 patients were included in the cross-sectional analysis, and 649 patients were included in the longitudinal analysis. In the cross-sectional analysis, higher corrected serum calcium (odds ratio: 1.31 per 0.1 mmol/L, 95% confidence interval: 1.16-1.49), younger age, longer diabetes duration, albuminuria, impaired renal function, and lower serum magnesium were independently associated with VTDR. In the longitudinal analysis, 95 subjects developed VTDR during follow-up (9.7 years, interquartile range: 7.4-10.9 years). Higher corrected serum calcium (hazard ratio: 1.38 per 0.1 mmol/L, 95% confidence interval: 1.10-1.72), younger age, longer diabetes duration, sub-VTDR, albuminuria, lower serum magnesium, and higher glycated hemoglobin were identified as independent risk factors for VTDR. CONCLUSIONS: A higher serum calcium level may be an independent risk factor for VTDR in patients with T2D.

Higher Serum Uric Acid Levels Are Associated With an Increased Risk of Vision-Threatening Diabetic Retinopathy in Type 2 Diabetes Patients.

Purpose: To investigate the association between serum uric acid (SUA) levels and vision-threatening diabetic retinopathy (VTDR) in patients with type 2 diabetes. Methods: This cross-sectional study evaluated 3481 patients with type 2 diabetes from four communities in China between 2016 and 2019. VTDR was defined as severe nonproliferative, proliferative diabetic retinopathy, or clinically significant macular edema evaluated by fundus photography and optical coherence tomography. Potential association between SUA and VTDR was examined using multivariable logistic regression. Sub-group analyses based on sex were constructed. Results: A total of 305 participants had VTDR. Both higher SUA (odds ratio [OR], 1.22 per 100 µmol/L; 95% confidence interval [CI], 1.04-1.44; P = 0.013) and hyperuricemia (OR, 1.47; 95% CI, 1.07-2.04; P = 0.019) were positively associated with VTDR after adjustment for relevant covariates. Compared with those in the lowest SUA quartile, participants in the third (OR, 1.60; 95% CI, 1.07-2.39; P = 0.022) and fourth (OR, 2.05; 95% CI, 1.37-3.08; P = 0.001) sex-specific SUA quartiles showed a significantly increased risk of VTDR after adjustment. No sex-related difference was observed. Conclusions: Higher SUA levels were associated with an increased risk of VTDR in patients with type 2 diabetes in both sexes, although females seemed to be more sensitive to high SUA than males. Prospective cohort studies are needed to verify SUA as a biomarker for predicting the risk of VTDR. Whether decreased SUA levels could decrease the risk of VTDR also requires further investigation.

Difference in the Vitreal Protein Profiles of Patients with Proliferative Diabetic Retinopathy with and without Intravitreal Conbercept Injection.

PURPOSE: To examine the difference in the vitreal protein profiles of patients with proliferative diabetic retinopathy (PDR) with and without preoperative intravitreal conbercept (IVC) treatment. METHODS: Liquid chromatography-tandem mass spectrometry- (LC-MS/MS-) based proteomic methods were used to determine the protein profiles of the vitreous humor in patients with PDR treated with (IVC group; n = 9) and without (PDR group; n = 8) preoperative IVC. Gene ontology (GO) annotation and REACTOME pathway analysis were obtained to overview differentially expressed proteins between each group. Intravitreal levels of apolipoprotein A-II (APOA2) and ceruloplasmin (CP) were measured using enzyme-linked immunosorbent assays. RESULTS: 307 proteins were expressed differentially between PDR and IVC groups, including 218 proteins downregulated in response to IVC. The most notable GO annotations in level 3 and REACTOME pathways describing the differentially expressed proteins were "innate immune response" and "platelet degranulation." The intravitreal levels of APOA2 and CP were lower in the IVC group than in the PDR group (p < 0.01). CONCLUSIONS: In addition to decreasing the intravitreal vascular endothelial growth factor level, IVC may alter the vitreal protein profile in patients with PDR, with the differentially regulated proteins involved in the immune response, platelet degranulation, complement activation, and inflammation.

The role of uric acid in the pathogenesis of diabetic retinopathy based on Notch pathway.

OBJECTIVE: Uric acid has been proposed as an independent risk factor of diabetic retinopathy. Although Notch signaling was reported to be affected in the presence of high concentrations of uric acid or glucose, the underlying mechanisms of hyperuricemia through the Notch signaling pathway to promote the development of diabetic retinopathy remain unknown. METHODS: We incubated human retinal endothelial cells (HRECs) with high glucose, high uric acid and high glucose plus high glucose respectively and evaluated the apoptosis rate in different treated cells by Tunel staining. We induced diabetic model by intraperitoneally streptozotocin. Then healthy rats and diabetic rats were given with adenine and oteracil potassium by gavage. Using automatic biochemical analyzer to detect blood glucose, uric acid, urea nitrogen, creatinine levels, to verify the success of modeling. The expression and mRNA levels of ICAM-1, IL-6, MCP-1, TNF-a, receptors Notch 1, ligands Dll 1, Dll 4, Jagged 1, Jagged 2 were detected by RT-PCR and Western-Blot. Notch1 siRNA was used to interfere Notch signaling pathway, the expression and mRNA levels of ICAM-1, IL-6, MCP-1 and TNF-α was detected by RT-PCR and Western blot respectively. RESULTS: In vitro models, the apoptosis of HRECs cells in high uric acid plus high glucose group was the most significant. In vitro and vivo models, detection of inflammatory cytokines revealed that the expression of inflammatory cytokines increased most significantly in high uric acid plus high glucose group. Notch signaling pathway activity was also increased most significantly in high uric acid plus high glucose group. After Notch 1 siRNA transfection in high glucose and high glucose plus uric acid group, the activity of Notch signaling pathway was successfully down-regulated. We found that the apoptosis of HRECs was significantly decreased in cells transfected with Notch 1 siRNA compared to the blank vector group, and the expression of inflammatory cytokines in cells was also significantly decreased. CONCLUSION: Our study reported that high uric acid can promote the inflammation of the retina and increase the activity of Notch signaling pathway on the basis of high glucose. Hyperuricemia promotes the development of diabetic retinopathy by increasing the activity of Notch signaling pathway. Notch signaling pathway is a potential therapeutic target for diabetic retinopathy.

[In vitro effect of photoactivated hypericin on anti-Schistosoma japonicum adult male worms].

OBJECTIVE: To investigate the in vitro effect of photoactivated hypericin on anti-Schistosoma japonicum adult male worms. METHODS: Kunming mice were infected with 60-80 Schistosoma japonicum single-sex cercariae. At 6 weeks post-infection, the mice were sacrificed and adult male worms of S. japonicum were collected. The worms were incubated in DMEM medium containing different concentrations of hypericin (0.1, 0.2, 0.5, 1.0, 1.5, 2.0, and 2.5 micromol/L) in the presence or absence of light. In photoactivated hypericin groups, after 6 h of dark incubation the worms were exposed to LED light irradiation (590 nm) for 30, 60, 90, and 120 min, respectively, and then cultured overnight in darkness (16h). In the next morning, the parasites were washed, resuspended in drug-free medium, and incubated in the dark for 48 h. These worms were observed with stereomicroscopy and scanning electron microscopy (SEM). RESULTS: Photoactivated hypericin showed the ability to kill Schistosoma japonicum in vitro. The death rate was 20% in 0.1 micromol/L photoactivated hypericin group under 30 min irradiation, and 100% in 2 micromol/L under 90 min irradiation and 2.5 micromol/L under 60 min irradiation, respectively. In blank control group, DMSO control group, and hypericin groups without light irradiation, worms were alive. After 60 min irradiation, the worms in 1.0, 2.5, 5.0 micromol/L photoactivated hypericin groups showed spastic paralysis characterized by reduced body length, pronounced tight curl, body stiffness, and complete cessation of movement. Surface tegumental damages of adult worms in 2.0 micromol/L photoactivated hypericin group for 60 min irradiation were observed under SEM, such as vacuole formation, erosion and peeling of the tegument, collapse of the sensory papillae, and even the normal structure disappeared completely. Both death rate and morphological damage of the worms treated by photoactivated hypericin were positively correlated with hypericin dose and light irradiation time. CONCLUSION: Photoactivated hypericin has anti-Schistosoma japonicum adult male worms effect in vitro.