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Specialized retinal endothelial cells modulates blood-retina- barrier in diabetic retinopathy

ABSTRACT: Endothelial cells (EC) play essential roles in retinal vascular homeostasis. This study aimed to characterize retinal EC heterogeneity and functional diversity using single-cell RNA sequencing (scRNA-seq). Systematic analysis of cellular compositions and cell-cell interaction networks identified a unique EC cluster with high inflammatory gene expression in the diabetic retina; sphingolipid metabolism in this cluster is a prominent aspect correlated with changes in retinal function. Among the sphingolipid-related genes, alkaline ceramidase 2 (ACER2) showed the most significant increase. We collected plasma and vitreous samples from patients with non-proliferative diabetic retinopathy (NPDR) and PDR for mass spectrometry analysis. Metabolomic profiling revealed that the ceramide levels were significantly elevated in NPDR and further increased in PDR compared with control patients. In vitro analyses showed that the ACER2 overexpression retarded endothelial barrier breakdown induced by ceramide, while silencing of ACER2 further disrupted the injury. Moreover, intravitreal injection of the recombinant ACER2 adeno-associated virus rescued diabetes-induced vessel leakiness, inflammatory response, and neurovascular disease in diabetic mouse models. Together, this study revealed a new diabetes-specific retinal EC population and a negative feedback regulation pathway that reduces ceramide content and endothelial dysfunction by upregulating ACER2 expression. These findings provide insights into cell-type targeted interventions for diabetic retinopathy.

ORGANISM(S): Mus musculus

PROVIDER: GSE243100 | GEO | 2024/01/01

REPOSITORIES: GEO

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Project description:Diabetic retinopathy (DR), a leading cause of irreversible vision loss in the working-age population, is an inflammatory, neuro-vascular complication of diabetes with poorly understood mechanism. N6-methyladenosine (m6A) modification plays crucial roles in biological and pathological events, while its role in DR remain elusive. Herein, we identified the pathological involvement of m6A demethylase FTO in DR. FTO expression was elevated in proliferative membranes of DR patients, endothelial cells (EC) under diabetic stress, and retinal vessels of diabetic murine models. FTO overexpression in EC promoted EC cycle progression and tip cell formation to facilitate angiogenesis in vitro, in mice and in zebrafish. FTO also regulated EC-pericyte crosstalk to trigger diabetes-induced microvascular leakage, and mediated EC-microglia crosstalk to induce retinal inflammation and subsequent neurodegeneration in vivo and in vitro. Mechanistically, FTO regulated EC features depending on its demethylation activity via modulating CDK2 mRNA stability with YTHDF2 as the reader. Moreover, FTO up-regulation in EC under diabetic conditions was driven by lactic acid mediated histone lactylation. FB23-2, which inhibits FTO’s m6A demethylase activity, suppressed diabetes associated endothelial phenotypes in vitro and in vivo. Noteworthy, we developed a novel macrophage membrane coated and PLGA-Dil based nanoplatform encapsulating FB23-2 for systemic administration, and confirmed its targeting and therapeutic efficacy in mice. Collectively, our study demonstrated an FTO-mediated regulatory network that coordinates EC biology and retinal homeostasis in DR, providing a promising nanotherapeutic approach for DR treatment. Diabetic retinopathy (DR), a leading cause of irreversible vision loss in the working-age population, is an inflammatory, neuro-vascular complication of diabetes with poorly understood mechanism. N6-methyladenosine (m6A) modification plays crucial roles in biological and pathological events, while its role in DR remain elusive. Herein, we identified the pathological involvement of m6A demethylase FTO in DR. FTO expression was elevated in proliferative membranes of DR patients, endothelial cells (EC) under diabetic stress, and retinal vessels of diabetic murine models. FTO overexpression in EC promoted EC cycle progression and tip cell formation to facilitate angiogenesis in vitro, in mice and in zebrafish. FTO also regulated EC-pericyte crosstalk to trigger diabetes-induced microvascular leakage, and mediated EC-microglia crosstalk to induce retinal inflammation and subsequent neurodegeneration in vivo and in vitro. Mechanistically, FTO regulated EC features depending on its demethylation activity via modulating CDK2 mRNA stability with YTHDF2 as the reader. Moreover, FTO up-regulation in EC under diabetic conditions was driven by lactic acid mediated histone lactylation. FB23-2, which inhibits FTO’s m6A demethylase activity, suppressed diabetes associated endothelial phenotypes in vitro and in vivo. Noteworthy, we developed a novel macrophage membrane coated and PLGA-Dil based nanoplatform encapsulating FB23-2 for systemic administration, and confirmed its targeting and therapeutic efficacy in mice. Collectively, our study demonstrated an FTO-mediated regulatory network that coordinates EC biology and retinal homeostasis in DR, providing a promising nanotherapeutic approach for DR treatment.

Project description:Background: Proliferative diabetic retinopathy (PDR) is hallmarked by the formation of retinal neovascularization (RNV) membranes, which can lead to a tractional retinal detachment, the primary reason for severe vision loss in end-stage disease. The aim of this study was to characterize the molecular and cellular features of RNV in order to unravel potential novel drug treatments for PDR. Methods: A total of 42 patients undergoing vitrectomy for PDR, macular pucker or macular hole (control patients) were included in this study. The surgically removed RNV and epiretinal membranes were analyzed by RNA sequencing, single-cell based Imaging Mass Cytometry and conventional immunohistochemistry. Since macrophages were found to be abundant in RNV tissue, vitreal macrophages, also known as hyalocytes, were isolated from the vitreous of patients with PDR by flow cytometry, cultivated and characterized by immunhistochemistry. A bioinformatical drug repurposing approach was applied, in order to identify novel drug options for end-stage diabetic retinopathy disease. Results: The in-depth transcriptional and single-cell protein analysis of diabetic RNV tissue samples revealed an accumulation of endothelial cells, macrophages and myofibroblasts as well as an abundance of secreted ECM proteins such as SPARC, FN1 and several types of collagen in RNV tissue. The immunohistochemical staining of cultivated vitreal hyalocytes from patients with PDR showed that hyalocytes express α-SMA (alpha-smooth muscle actin), a classic myofibroblast marker. According to our drug repurposing analysis, imatinib emerged as a potential drug option for future treatment of PDR. Conclusion: This study delivers the first in-depth transcriptional and single-cell proteomic characterization of RNV tissue samples. Our data suggest an important role of hyalocyte-to-myofibroblast transdifferentiation in the pathogenesis of diabetic vitreoretinal disease and suggests their modulation as a novel possible clinical approach.

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Specialized retinal endothelial cells modulates blood-retina- barrier in diabetic retinopathy

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