Hetian Lei

Aims/hypothesis The 309G SNP in the second promoter of the gene encoding mouse double minute 2 (MDM2) has been implicated in multiple human diseases. The aims of this study were to determine whether MDM2 SNP309G is associated with proliferative diabetic retinopathy (PDR), and whether it contributes to pathological angiogenesis. Methods Sanger DNA sequencing was used to determine the MDM2 SNP309 status in peripheral blood and fibrovascular membranes (FVMs) from individuals with PDR, as well as in epiretinal membranes from individuals with proliferative vitreoretinopathy (PVR). An ELISA was used to quantify the levels of the oxidative DNA damage biomarker 8-oxo-2′-deoxyguanosine in vitreous humour samples from individuals with PDR or PVR. Prime editing was employed to introduce MDM2 SNP309G into primary human retinal microvascular endothelial cells (HRECs), which were then assessed for in vitro angiogenic activities, including proliferation, migration and tube formation. A mouse model of oxygen-induced retinopathy (OIR) was used to evaluate pathological retinal neovascularisation in humanised mice carrying MDM2 SNP309T or SNP309G. Quantitative RT-PCR and western blot analyses were performed to assess gene and protein expression related to MDM2-mediated signalling pathways. Results An association between MDM2 SNP309G and PDR was identified. Among 110 individuals with PDR, 60.1% harboured MDM2 SNP309G in their FVMs, and 20.9% exhibited a T→G substitution at position 309 in FVMs compared with matched blood samples. The vitreous humour from individuals with PDR contained significantly higher levels of 8-oxo-2′-deoxyguanosine (7.8±1.2-fold) compared with PVR control participants. Chronic exposure of primary HRECs to vitreous humour containing a high concentration of d-glucose suppressed expression of 8-oxoguanine DNA glycosylase, promoted conversion of MDM2 SNP309T to G (36.1%), and increased MDM2 protein levels. Prime editing-mediated conversion of MDM2 SNP309T to G in HRECs (51.6%) further enhanced high-glucose-induced MDM2 expression and angiogenic responses in vitro. In vivo, humanised C57BL/6J mice carrying MDM2 SNP309G exhibited increased retinal levels of MDM2, hypoxia-inducible factor-1α (HIF-1α), phosphorylated vascular endothelial growth factor receptor 2 (VEGFR2), phosphorylated Erk1/2 and phosphorylated specificity protein 1 (Sp1), together with elevated vascular endothelial growth factor (VEGF) in the vitreous humour, and exacerbated pathological retinal angiogenesis in the OIR model, compared with mice harbouring MDM2 SNP309T. Conclusions/interpretation These findings suggest that MDM2 SNP309G drives a positive feedback loop involving MDM2 and VEGF signalling in vascular endothelial cells, thereby promoting pathological angiogenesis. Targeting the second promoter of MDM2 may represent a novel therapeutic strategy for preventing aberrant angiogenesis in PDR. Graphical Abstract

Purpose: Low-dose 650 nm red light has been found to slow myopia progression, although the underlying mechanisms remain unclear. This study aimed to investigate its antioxidative effects and molecular pathways. Methods: An oxidative stress model was established in ARPE-19 cells by treatment with hydrogen peroxide (H₂O₂) at concentrations of 0, 0.5, and 0.75 mM. The cells were then irradiated with red light for 9 minutes, twice daily for 2 days, with an equivalent-power white light group serving as a control. Following irradiation, oxidative stress was quantified using a DCFH-DA assay, and DNA damage was assessed by γ-H2AX immunofluorescence. For the in vivo study, mice received ocular irradiation with red light (9 minutes/session, twice daily for 5 days) using a white light-exposed group as a control. Changes in axial length were measured post irradiation using anterior segment optical coherence tomography. Subsequently, retinal pigment epithelial (RPE) cells were isolated from the mice for RNA sequencing to analyze differential mRNA expression. Quantitative real-time PCR (qPCR) and Western blotting were employed to validate the expression of specific genes associated with ocular diseases. Results: At a concentration of 0.75 mM hydrogen peroxide, red light irradiation significantly reduces oxidative stress levels compared to the control group. RNA sequencing data revealed that there were 274 genes upregulated and 225 genes downregulated in RPE cells from mouse eyes illuminated with the 650 nm red light. The gene encoding aldehyde dehydrogenase 3A1 (ALDH3A1), which is significantly upregulated after red light irradiation, plays an important role in protecting ocular structures from oxidative damage. qPCR and Western blot analyses confirmed that ALDH3A1 was heightened in RPE cells from mouse eyes in vivo and in cultured human RPE cells in vitro illuminated by the 650 nm red light. Conclusions: ALDH3A1 may play a part in myopia improvement upon 650 nm red light illumination.

Introduction: Retinopathy of prematurity (ROP) is one of the leading causes of babies’ visual impairment and blindness. There is no effective prevention and treatment of ROP so far, and the shared genetic and developmental similarities among the brain, kidneys and retina may offer novel potential therapeutic approaches to ROP. Objectives: The aim of this study is to explore a correlation of ROP patients and the renal, eye tissue of the mouse model of oxygen-induced retinopathy (OIR). Methods and analysis: We used renal and vitreous untargeted/targeted metabolomics in OIR to conduct our study. Network association analysis and machine learning were performed with the above results and previous studies: retinal-targeted metabolomics of OIR and human blood-targeted metabolomics of ROP. Results: OIR results in retinal neovascularisation and renal injury. Nine canonical signalling pathways were enriched, which are involved in the initiation and progression of pathologic retinal neovascularisation. Arginine biosynthesis emerged as a common pathway across renal, vitreous, retinal and blood metabolomics, suggesting its potential as a predictive biomarker and therapeutic target for ROP and neonatal kidney injury. Conclusion: The presence of renal injury-related indicators may assist in diagnosing retinal neovascular diseases such as ROP. Arginine biosynthesis is the best common pathway of kidney-untargeted OIR metabolomics, vitreous- and retina-targeted OIR metabolomics and blood-targeted metabolomics of ROP, indicating that arginine biosynthesis might be the common pathway of ROP and neonatal kidney injury.

Abnormal blood vessel growth in the eye is a leading cause of vision loss globally, particularly in diseases like diabetic retinopathy where the vitreous plays a crucial but poorly understood role in disease progression. While we know the vitreous can stimulate blood vessel growth, the specific molecular mechanisms remain unclear. Here we show that a protein called G0S2 (G0/G1 switch gene 2) serves as a key regulator of blood vessel growth in response to normal vitreous. Through comprehensive gene analysis, we discovered that G0S2 levels increase significantly when blood vessel cells are exposed to normal vitreous. The importance of G0S2 is highlighted by our finding that uveal melanoma patients with higher G0S2 levels had poorer survival rates. When we removed G0S2 from blood vessel cells, they no longer responded to vitreous stimulation, confirming its critical role. Notably, we identified an existing drug that can target G0S2, potentially offering a new therapeutic approach. This discovery of G0S2’s role and its potential therapeutic targeting opens new avenues for treating eye diseases characterized by abnormal blood vessel growth, while also providing a valuable biomarker for predicting disease progression in eye cancer patients.

Platelet-derived growth factor receptor β (PDGFRβ) is the principal PDGFR isoform in retinal pigment epithelial (RPE) cells from the epiretinal membranes of patients with proliferative vitreoretinopathy (PVR). Ras-related C3 Botulinum toxin substrate 1 (Rac1), a member of the Rho family, is a crucial factor in the cell migration and contraction processes that are inherent to the pathogenesis of PVR. The mutants Rac1T17N and Rac1Q61L can block and promote Rac1 activation, respectively. The major objective of this research was to ascertain whether PDGFRβ mediates vitreous-induced Rac1 activation and whether Rac1T17N could be leveraged for the prevention of PVR pathogenesis in a rabbit model. A pull-down assay was used to examine GTP Rac1 levels, which are indicative of Rac1 activation, and western blotting was used to assess cellular protein expression. A CCK8 assay, a wound healing assay, a transwell invasion assay and a collagen contraction assay were employed to analyze cell proliferation, migration, invasion and contraction capacity, respectively. A PVR model was created by injecting platelet-rich plasma and human retinal pigment epithelial cells (ARPE-19) into the vitreous cavities of rabbits, and this model was used to evaluate the severity of PVR impacted by intravitreally injected ARPE-19 cells transduced with adeno-associated virus (AAV)1-Rac1T17N or Rac1Q61L. PVR grade was evaluated by a double-blinded investigator according to the Fastenberg classification; in addition, ultrasound and histological analyses were performed to assess PVR severity. Vitreous-induced GTP Rac1 is mediated by PDGFRβ. There was a significant decrease in vitreous-induced GTP Rac1 in ARPE-19 cells transduced with AAV1-Rac1T17N compared with those transduced with AAV1-GFP. In addition, the suppression of GTP Rac1 production in human RPE cells by transduction with AAV1-Rac1T17N inhibited vitreous-induced proliferation, migration, invasion, and contractility. Importantly, the results of the animal experiments indicated that although there was a significant increase in PVR potential in rabbits intravitreally injected with ARPE-19 cells infected with AAV1-Rac1Q61L, there was a significant decrease in PVR potential in rabbits intravitreally injected with ARPE-19 cells infected with AAV1-Rac1T17N (P < 0.01). AAV1-Rac1T17N has great potential for PVR therapy.

Introduction: Tuberculosis (TB) is a serious infectious disease that endangers human health, and TB becomes more difficult in eradiation due to its multidrug resistance (MDR). The objective of this research was to identify novel targets for treating TB.Methods: A 2-fold serial dilution method was used to determine minimal inhibitory concentrations (MIC) of compound M6 against Mycobacterium smegmatis (M. smegmatis). Compound M6 was subjected to reverse molecular docking with seven Mycobacterium tuberculosis proteins, and the best binding protein with the highest LibDock score was evaluated. The target protein with the highest score was purified through prokaryotic expression. Isolated target proteins were investigated for the enzyme activities and for the kinetic effect of compound M6 by absorbance detection. Subsequently, the CRISPR/Cas9 technology was employed to inhibit target gene expression for detecting MIC changes. Finally, potential targets were evaluated for the effect of the compound M6 in bacteria.Results: The MIC values of compound M6 against M. smegmatis were 32 μg/mL. The results from reverse molecular docking show that IspD has the highest LibDock score of 142.50, followed by Rv0674, IspF, and Dxr, with docking scores of 110.762, 71.6955, and 57.7446, respectively. IspD is a key enzyme in the 2-C-methyl-D-erythritol 4-phosphate pathway of MTB. The aKi and Ki values of M6 for the substrate MEP are 609.58 μM and 81.33 μM. For CTP, the aKi and Ki values are 657.89 μM and 40.07 μM. With tetracycline inducing CRISPR/Cas9 to suppress the expression of IspD, the MIC value of M6 against IspD went down significantly from 32 μg/mL to 4 μg/mL.Conclusion: IspD is a novel target of the compound M6 for treating TB.

Vascular endothelial growth factor receptor (VEGFR)-2 is a key switch for angiogenesis, which is observed in various human diseases. In this study, a novel system for advanced prime editing (PE), termed PE6h, is developed, consisting of dual lentiviral vectors: (1) a clustered regularly interspaced palindromic repeat-associated protein 9 (H840A) nickase fused with reverse transcriptase and an enhanced PE guide RNA and (2) a dominant negative (DN) MutL homolog 1 gene with nicking guide RNA. PE6h was used to edit VEGFR2 (c.18315T>A, 50.8%) to generate a premature stop codon (TAG from AAG), resulting in the production of DN-VEGFR2 (787 aa) in human retinal microvascular endothelial cells (HRECs). DN-VEGFR2 impeded VEGF-induced phosphorylation of VEGFR2, Akt, and extracellular signal-regulated kinase-1/2 and tube formation in PE6h-edited HRECs in vitro . Overall, our results highlight the potential of PE6h to inhibit angiogenesis in vivo .