Glucagon-like peptide-1 receptor agonists rescued diabetic vascular endothelial damage through suppression of aberrant STING signaling.

Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) protect against diabetic cardiovascular diseases and nephropathy. However, their activity in diabetic retinopathy (DR) remains unclear. Our retrospective cohort study involving 1626 T2DM patients revealed superior efficacy of GLP-1 RAs in controlling DR compared to other glucose-lowering medications, suggesting their advantage in DR treatment. By single-cell RNA-sequencing analysis and immunostaining, we observed a high expression of GLP-1R in retinal endothelial cells, which was down-regulated under diabetic conditions. Treatment of GLP-1 RAs significantly restored the receptor expression, resulting in an improvement in retinal degeneration, vascular tortuosity, avascular vessels, and vascular integrity in diabetic mice. GO and GSEA analyses further implicated enhanced mitochondrial gene translation and mitochondrial functions by GLP-1 RAs. Additionally, the treatment attenuated STING signaling activation in retinal endothelial cells, which is typically activated by leaked mitochondrial DNA. Expression of STING mRNA was positively correlated to the levels of angiogenic and inflammatory factors in the endothelial cells of human fibrovascular membranes. Further investigation revealed that the cAMP-responsive element binding protein played a role in the GLP-1R signaling pathway on suppression of STING signaling. This study demonstrates a novel role of GLP-1 RAs in the protection of diabetic retinal vasculature by inhibiting STING-elicited inflammatory signals.

Involvement of IGF1 in endoplasmic reticulum stress contributes to cataract formation through regulating Nrf2/NF-κB signaling.

Endoplasmic reticulum (ER) stress is reportedly involved in the development of ophthalmic diseases. This study aimed to investigate the role and potential mechanism of insulin-like growth factor 1 (IGF1) in ER stress. A mouse cataract model was constructed by subcutaneous injection of sodium selenite, and sh-IGF1 was used to evaluate the effect of silencing IGF1 on cataract progression. Slit-lamp and histological examination of the lens were performed to examine lens damage. The regulatory effects of IGF1 on inflammatory responses, oxidative stress, and ER stress were evaluated using ELISA, reverse transcription-quantitative PCR (RT-qPCR), and immunoblotting analysis. Tunicamycin was used to induce ER stress in the lens of epithelial cells. The NF-E2 related factor-2 (Nrf2) inhibitor ML385 and nuclear factor-κB (NF-κB) agonist diprovocim were used to confirm whether IGF1 regulates inflammation and ER stress through Nrf2/NF-κB signaling. Silencing IGF1 alleviated lens damage and reduced lens turbidity in the cataract mice. Silencing IGF1 inhibited inflammatory response, oxidative stress and ER stress response. Meanwhile, IGF1 was highly expressed in sodium selenite-treated lens epithelial cells. The ER stress agonist tunicamycin suppressed cell viability as well as induced ER stress, oxidative stress and inflammation. Silencing IGF1 increased cell viability, EdU-positive rate and migration. Also, silencing of IGF1 reduced inflammation and ER stress via regulating Nrf2/NF-κB pathway. This study reveals silencing IGF1 attenuated cataract through regulating Nrf2/NF-κB signaling, which shares novel insights into the underlying mechanism of cataract and provides potential therapeutic target for cataract.

Effects of chemokine receptor CCR7 in the pathophysiology and clinical features of the immuno-inflammatory response in primary pterygium.

OBJECTIVE: Chemokine receptor 7 (CCR7) has been considered a critical biomarker in inflammation and the immune response; however, little is known about CCR7 in pterygia. This study aimed to investigate whether CCR7 participates in the pathogenesis of primary pterygia and how CCR7 affects the progression of pterygia. METHODS: This was an experimental study. Slip-lamp photographs of 85 pterygium patients were used to measure the width, extent, and area of pterygia with computer software. Pterygium blood vessels and general ocular redness were quantitatively analyzed with a specific algorithm. The expression of CCR7 and its ligands C-C motif ligand 19 (CCL19) and C-C motif ligand 21 (CCL21) in control conjunctivae and excised pterygia collected during surgery were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence staining. The phenotype of CCR7-expressing cells was identified by costaining for major histocompatibility complex II (MHC II), CD11b or CD11c. RESULTS: The CCR7 level was significantly increased by 9.6-fold in pterygia compared with control conjunctivae (p = 0.008). The higher the expression of CCR7 was, the more blood vessels appeared in pterygia (r = 0.437, p = 0.002) and the more general ocular redness was (r = 0.51, p < 0.001) in pterygium patients. CCR7 was significantly associated with pterygium extent (r = 0.286, p = 0.048). In addition, we found that CCR7 colocalized with CD11b, CD11c or MHC II in dendritic cells, and immunofluorescence staining showed that CCR7-CCL21 is a potential chemokine axis in pterygium. CONCLUSIONS: This work verified that CCR7 impacts the extent of primary pterygia invading the cornea and inflammation at the ocular surface, which may provide a possibility for a further in-depth understanding of the immunological mechanism in pterygia.

Treatment With Melatonin After Corneal Graft Attenuates Rejection.

Background: Immunologic graft rejection is the main complication of corneal transplants. This study aimed to investigate the effect of melatonin (MT) on the rejection of corneal transplantation. Methods: Corneal allografts were performed by grafting corneas from BALB/C mice to C57BL/6 hosts. MT (50 mg/kg) was intraperitoneally injected into the hosts every day from the day of transplantation. The survival of grafts was observed by slit lamp biomicroscopy, and inflammatory cell infiltration was detected by hematoxylin and eosin staining and immunohistochemistry. The balance of Teff and Treg immune cells in draining lymph nodes (DLNs) was detected by flow cytometry. The levels of cytokines related to the grafts and DLNs were detected using real-time fluorescence quantitative PCR. Additionally, we used the mouse macrophage line RAW264.7 to study the effect of MT on the activation of NLRP3 inflammatory body. Results: MT treatment improved the graft survival rate, reduced inflammatory cell infiltration in the graft, decreased the percentage of Th1/Th17 cells in the DLNs, and increased the percentage of Treg cells. Melatonin inhibited the activation of the NLRP3 inflammasome, thereby reducing the expression of IL-1ß and other related proinflammatory cytokines such as MCP-1, MIP-1, NLRP3, ASC, TNF-a and VEGF-A (all p < 0.05). Conclusion: Our study demonstrates that MT promotes the survival of mouse corneal grafts by inhibiting NLRP3-mediated immune regulation, reducing immune cell activation and cell migration, and inhibiting the production of inflammatory-related cytokines. Treatment with MT might provide a potential clinical therapeutic target for corneal transplantation.

Females with Type 2 Diabetes Mellitus Are Prone to Diabetic Retinopathy: A Twelve-Province Cross-Sectional Study in China.

AIMS: To investigate the distribution of diabetic retinopathy (DR) by sex in patients with type 2 diabetes mellitus (T2DM) in a twelve-province cross-sectional study in China. METHODS: Patients with T2DM, whose ages were ≥18 years, were recruited from 76 cities/counties in 12 provinces in mainland China between January 2015 and December 2018. All participants received a standardized interview, eye examinations, and digital fundus photography. The presence and severity of DR were diagnosed and classified by retina specialists according to the DR domestic typing method. RESULTS: A total of 12,766 participants (5963 males and 6803 females) were eligible for this study. The total prevalence of DR was 30.1%. Females exhibited a significantly higher prevalence of DR than males (31.1% vs. 29.0%, P = 0.011). A multivariate logistic regression analysis confirmed that female sex was an independent predictor for a higher prevalence of DR after adjusting for age, the duration of diabetes, economic status, and the presence of hypertension (OR: 1.096, 95% CI: 1.013-1.186, P = 0.023). Even after stratification by the diabetic duration, age, and economic status, female sex was still independently associated with the presence of DR in patients whose T2DM history was more than 10 years, whose ages were over 60 years, or who were in a relatively intermediate economic area. CONCLUSION: Females had a higher prevalence of DR than males in T2DM patients with a diabetic history of more than 10 years, ages over 60 years, or a relatively intermediate economic status.

Inhibition of retinal ganglion cell apoptosis: regulation of mitochondrial function by PACAP.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an endogenous peptide with neuroprotective effects on retinal neurons, but the precise mechanism underlying these effects remains unknown. Considering the abundance of mitochondria in retinal ganglion cells (RGCs), we postulate that the protective effect of PACAP is associated with the regulation of mitochondrial function. RGC-5 cells were subjected to serum deprivation for 48 hours to induce apoptosis in the presence or absence of 100 nM PACAP. As revealed with the Cell Counting Kit-8 assay, PACAP at different concentrations significantly increased the viability of RGC-5 cells. PACAP also inhibited the excessive generation of reactive oxygen species in RGC-5 cells subjected to serum deprivation. We also showed by flow cytometry that PACAP inhibited serum deprivation-induced apoptosis in RGC-5 cells. The proportions of apoptotic cells and cells with mitochondria depolarization were significantly decreased with PACAP treatment. Western blot assays demonstrated that PACAP increased the levels of Bcl-2 and inhibited the compensatory increase of PAC1. Together, these data indicate protective effects of PACAP against serum deprivation-induced apoptosis in RGCs, and that the mechanism of this action is associated with maintaining mitochondrial function.

Intertrochanteric fracture: Association between the coronal position of the lag screw and stress distribution.

BACKGROUND/PURPOSE: The best position of the lag screw in the femoral head for the fixation of intertrochanteric fracture is controversial. Traditional view suggests that it should be positioned in the central axis of the femoral neck with a tip-apex distance (TAD) of <25 mm, but the mechanical properties have not been reported yet. Herein, we aimed to investigate internal fixation with the lag screw placed in different positions on the femoral coronal plane by performing a finite element analysis and to identify a reasonable lag screw position after the internal stress distributions at the femoral head. METHODS: A three-dimensional finite element model of a healthy male's femur was set up, on which the intertrochanteric fracture model with proximal femoral nail antirotation (PFN-A) was based. Nine modalities of the model were established in accordance with different lag screw positions in the femoral head. Three-dimensional finite element calculations were conducted, and the distribution trends of characteristic high-stress concentration points were observed. RESULTS: The area of high-stress concentration was distributed from the top of the femoral head to the medial cortex of the trochanteric region. Four characteristic high-stress concentration points were observed, and the following trends indicated that the lower the position of the lag screw, the greater its length. CONCLUSIONS: A longer and lower lag screw may make the fixation sustain greater stress, reduce bone tissue stress correspondingly in intertrochanteric fractures fixated with PFN-A, and sustain greater stress and more cyclic load at the same bone density.

The Blockade of Vascular Endothelial Growth Factor C Effectively Inhibits Corneal Lymphangiogenesis and Promotes Allograft Survival.

PURPOSE: To explore the inhibitory effects of antivascular endothelial growth factor C (VEGF-C) therapy on corneal lymphangiogenesis and allograft rejection in rats. METHODS: Fischer 344 rat corneas were transplanted into Lewis rat eyes. After corneal transplantation, Lewis rats (the recipients) were randomly and equally divided into 2 groups: anti-VEGF-C treatment (group A) and control (group B). Corneal hemangiogenesis and lymphangiogenesis were characterized using whole-mount immunofluorescence, and the immune rejection of the grafts was examined using a slit lamp and evaluated by scoring the rejection index (RI). In addition, the expression of VEGF-C was examined by immunohistochemistry and real-time polymerase chain reaction. The association of corneal lymphangiogenesis and hemangiogenesis with VEGF-C in transplanted corneas was also characterized. RESULTS: VEGF-C expression was markedly downregulated after anti-VEGF-C therapy. The outgrowth of corneal lymphangiogenesis dramatically decreased in group A. There was a significant relationship between VEGF-C reduction and the decrease in the lymphatic vessel area (r=0.55, P<0.05), whereas the relationship between the reduction of VEGF-C and the decrease in blood vessel area was not significant (r=0.11, P>0.05). In addition, the RI scores were significantly lower in group A compared with group B at 7, 10, and 14 days after transplantation. The graft survival time in group A rats (20.33±1.37 days) was significantly longer than that in group B rats (12.83±1.47 days; P<0.05). CONCLUSIONS: The results suggested that VEGF-C blockade had a significant role in preventing corneal lymphangiogenesis in corneal beds, which resulted in higher allograft survival rates.

[Coverslip assisted primary tissue culture for human lens epithelial cells in vitro].

PURPOSE: To set up an easy procedure of tissue culture for human lens epithelial cells in vitro and to observe the biological characteristics. METHODS: Capsules from embryo of 20 weeks, eye bank of Zhongshan Ophthalmic Centre and patients with cataract were spread on culture utensil. 10 microL of 10% DMEM medium was added and a piece of coverslip was lay to prevent crimp. Then the capsules were cultured under 37 degrees C after adding enough medium. Capsules from the same source were cultured by traditional tissue culture method. Expressions of 13 crystallin between primary tissue culture cells and SRA01/04 cell line were compared by western blotting. RESULTS: With coverslip assisted, the cells could be observed proliferated and migrated from the edge of embryo capsule 2 days later, and for capsules from eye bank and age-related cataract patients, the interval time was 3 to 4 days. By traditional tissue culture method, the interval time of embryo capsule was 3 to 4 days, and for capsules from eye bank and age-related cataract patients, the interval time was the same. And capsules floated sometimes. CONCLUSIONS: By coverslip assisted primary tissue culture human lens epithelial cells could grow faster and easier, and the method is worthy to be spread in research of lens diseases.