The Key Role of Retina Microglia in Pathological Angiogenesis and the Immunotherapeutic Potential of the cGAS-STING Pathway

Background

Pathological angiogenesis is common in various neovascular ophthalmic diseases, such as Diabetic Retinopathy (DR) and Age-related Macular Degeneration (AMD) [1]. During these diseases, bone marrow cells, including retinal microglia and monocyte-derived macrophages, are activated, leading to neuroinflammation in the retina and choroid. These immune cells release pro-inflammatory factors and other molecules that stimulate endothelial cell proliferation and migration, forming abnormal new blood vessels [2]. Currently, non-specific anti-inflammatory therapies such as steroids are widely used to treat neovascular eye diseases, but their efficacy is limited and they carry potential risks of side effects [3, 4]. Therefore, it is important to deeply study the immune inflammation pathways in angiogenesis and identify potential specific immunotherapy targets.

The cGAS-STING pathway has recently been discovered as an important innate immune signaling mechanism in host defense. As a key molecule, cGAS mainly recognizes DNA in the cytoplasm, especially pathogen DNA from invading viruses and bacteria, and triggers downstream IRF3/IRF7 or NF-κB signaling, producing type I interferons and other inflammatory cytokines [5, 6]. In the retina, activation of the cGAS-STING pathway is associated with inflammation and degeneration [7]. Inhibiting the cGAS-STING pathway can alleviate retinal ganglion cell death caused by neuroinflammation [8]. However, the specific mechanisms and intervention strategies of this pathway in the retina are still unclear.

Research Source

This article, co-authored by Biyan Ni, Ziqi Yang, and Tian Zhou, was published in 2024 in the “Journal of Neuroinflammation” [9]. The research team is affiliated with the Zhongshan Ophthalmic Center of Sun Yat-sen University and the Guangdong Provincial Key Laboratory of Ophthalmology and Vision Science.

Detailed Research Process

RNA-Seq Data Analysis

The study first downloaded and re-analyzed an existing RNA-Seq dataset (GSE160306), which contains retinal tissue samples from healthy controls, diabetic patients, patients with Non-Proliferative Diabetic Retinopathy (NPDR), and patients with Proliferative Diabetic Retinopathy (PDR). Differential gene expression analysis was performed using DESeq2 software (v1.30.1), followed by Gene Ontology (GO) enrichment analysis and Gene Set Enrichment Analysis (GSEA) [11].

Establishment of Animal Models

The study used STING-deficient mice (STINGgt) from Jackson Laboratory and C57BL/6J mice purchased from GenPharmatech to establish laser-induced choroidal neovascularization (CNV) models and oxygen-induced retinopathy (OIR) models. Specific experimental methods included using 532 nm wavelength argon laser to induce Bruch’s membrane rupture and oxygen chamber to create OIR models [12, 13].

Cell Isolation and Single-Cell RNA Sequencing Analysis

Bone marrow cells were isolated from retinal and choroidal tissues using EasySep CD11b+ cell isolation tools after mechanical dissociation and enzymatic digestion, followed by RNA sequencing data analysis [13]. Additionally, cell culture experiments were conducted under hypoxic conditions to observe cell responses under different conditions.

Combination of Various Experimental Methods

The study employed various experimental techniques, such as quantitative PCR, Western Blot, immunofluorescence (IF) staining, Hematoxylin and Eosin (H&E) staining, and TUNEL analysis to explore the specific mechanisms and effects of the cGAS-STING pathway in microglia and macrophages [14].

Detailed Experimental Results

Significant Upregulation of the cGAS-STING Pathway in the Retina of PDR Patients

RNA-Seq data showed that the expression of cGAS and STING genes was significantly upregulated in PDR patients, closely related to the process of retinal neovascularization.

Activation of the cGAS-STING Pathway in CNV and OIR Mouse Models

Western Blot and immunofluorescence staining results indicated that the cGAS-STING signaling pathway was significantly activated during angiogenesis in CNV and OIR models, mainly concentrated in bone marrow cells (such as microglia and macrophages).

Significant Reduction of Pathological Angiogenesis in STING-deficient Mice

Using STING-deficient mouse models, the study found that mice lacking STING showed smaller areas of neovascularization and less inflammatory response in multiple models of pathological angiogenesis (such as CNV and OIR). This indicates that STING activation plays a key role in pathological angiogenesis.

Combined Treatment with VEGF and STING Inhibitors

Further research showed that combining STING inhibitors (such as c-176 and sn-011) with anti-VEGF therapy could significantly enhance anti-angiogenic effects. This finding suggests the potential of combining immunotherapy with anti-VEGF treatment to significantly improve the treatment of neovascular eye diseases.

Research Conclusions and Significance

This study provides strong evidence for the key role of the cGAS-STING pathway in pathological angiogenesis, indicating that targeting the cGAS-STING signaling axis, represented by STING inhibitors such as c-176 or sn-011, can significantly inhibit neuroinflammation and pathological angiogenesis associated with microglia and macrophage necroptosis. Additionally, combining anti-VEGF therapy offers a better approach to treating neovascular ophthalmic diseases.

Highlights and Innovations

1. Discovery of Key Pathological Mechanisms: This study systematically reveals for the first time the key role of the cGAS-STING pathway in retinal microglia.
2. Innovative Treatment Strategy: Proposes a new method of combining STING inhibitors and anti-VEGF immunotherapy, which is expected to improve the treatment of neovascular eye diseases.
3. Rigorous Experimental Design: The combination of multiple models and experimental methods makes the research conclusions highly credible and promising for application.

Other Valuable Information

The study also demonstrated the regulatory mechanisms of STING at different molecular levels (such as genes and proteins) and potential feedback loops, suggesting that further exploration of these pathways’ fine regulatory mechanisms could provide new targets for anti-inflammatory therapy.

Conclusion

This study indicates that by targeting the cGAS-STING pathway, we can effectively inhibit neuroinflammation and pathological angiogenesis mediated by microglia. This provides a new approach for the immunotherapy of neovascular ophthalmic diseases and has broad clinical application prospects.