Evaluated therapeutic efficacy and mechanism of EV30, a novel pterostilbene derivative, in suppressing CNV suggest that EV30 represents a potential therapeutic candidate for CNV by suppressing pathological angiogenesis and modulating inflammation-associated signaling pathways.
Abstract
Wet age-related macular degeneration (wAMD) is a leading cause of irreversible vision loss characterized by pathological choroidal neovascularization (CNV). While anti-VEGF therapies are the standard of care, limitations such as treatment resistance and side effects necessitate novel therapeutic agents. This study evaluates the therapeutic efficacy and mechanism of EV30, a novel pterostilbene derivative, in suppressing CNV. EV30 was synthesized based on the pterostilbene template. In vitro, the effects of EV30 on human umbilical vein endothelial cells (HUVECs) proliferation, migration, and tube formation were assessed using Cell Counting Kit-8 (CCK-8), scratch wound, and tube formation assays, respectively. Mechanistic pathways were investigated via Western blotting and RT-qPCR. In vivo, a laser-induced CNV mouse model was treated with intravitreal EV30. Efficacy was evaluated utilizing fundus photography, fluorescein angiography (FFA), optical coherence tomography (OCT), and choroidal flat mounts (IB4 staining). Finally, biosafety was assessed through histology (H&E), electroretinography (ERG), and blood analysis. EV30 demonstrated potent anti-angiogenic properties in vitro, significantly inhibiting HUVEC proliferation, migration, and tube formation in a dose- and time-dependent manner. EV30 reduced vascular endothelial growth factor A (VEGFA) expression and modulated the phosphorylation status of proteins associated with the mTOR/NF-κB/p38 MAPK signaling pathway. In the laser-induced CNV model, EV30 effectively reduced lesion area and vascular leakage comparable to bevacizumab. Furthermore, ERG analysis revealed that EV30 partially preserved retinal electrophysiological function, as indicated by improved scotopic a-wave amplitudes, suggesting functional protection of the retina in the CNV model. EV30 exerted anti-angiogenic effects and was associated with modulation of mTOR/NF-κB/p38 MAPK signaling activity. Together, these findings suggest that EV30 represents a potential therapeutic candidate for CNV by suppressing pathological angiogenesis and modulating inflammation-associated signaling pathways.
The chemokine receptor CXCR4 has been recognized as a pivotal mediator of cytokine-driven angiogenesis. Wet age-related macular degeneration (wet-AMD) is characterized by choroidal neovascularization (CNV) leading to vision loss. However, the current anti-VEGF therapy remains limited by recurrent CNV in refractory wet-AMD. Herein we developed small-molecule inhibitors targeting CXCR4-mediated neovascularization in wet-AMD. Employing computational molecular docking simulations for candidate screening, BPRCX807 exhibited superior binding affinity as a CXCR4 antagonist relative to other compounds tested. A laser-induced wet-AMD murine model was established, demonstrating subretinal CNV and upregulation of proteins implicated in CXCR4 signaling and angiogenesis. Retinal structural alterations and CNV-progression were monitored via real-time fluorescein angiography and optical coherence tomography (OCT), revealing that BPRCX807 treatment significantly attenuated angiogenesis compared to conventional anti-VEGF therapy. Retinal safety evaluation further indicated that BPRCX807 administration did not induce detectable retinal toxicity or structural abnormalities. Ex vivo choroid sprouting assays corroborated the reduction of vascular leakage and CNV-formation by BPRCX807. The visual-functional electroretinographic evaluations indicated that, while anti-VEGF agents produced modest functional improvements, BPRCX807 achieved superior restoration of retinal visual function. Notably, bioinformatic analyses supported that inhibition of CXCR4 signaling effectively suppressed VEGF-driven and cytokine-mediated angiogenic pathways. Finally, biomolecular simulations combined with predictive signaling-pathway analyses suggested that BPRCX807 modulates multiple VEGF-regulatory signaling cascades, thereby enhancing its inhibitory effects on VEGF expression and downstream angiogenesis. Collectively, these computational and experimental data demonstrate that BPRCX807 disrupts CXCR4 receptor interactions and suppresses angiogenic factor expression via inhibition of the CXCR4/p-AKT signaling pathway, representing a promising therapeutic approach for wet-AMD.
Shih-Jie Chou, Chia-Hao Wang, P. Tsai et al.· European Journal of Pharmaco...· 0 citations
Age-related macular degeneration (AMD), characterized by pathologic choroidal neovascularization (CNV), is a leading cause of vision loss in the elderly. Vascular endothelial growth factor A (VEGFa) antagonists can prevent acute vision loss, but high treatment burden and loss of efficacy with chronic therapy highlight the need to explore alternative mechanisms. Recently, microRNA-34a (miR-34a) has emerged as a key regulator in aging and age-related diseases, but its role in neovascular AMD is unclear. In an injury-induced murine CNV model, we found miR-34a promoted pathological angiogenesis, without altering expression of Vegfa or its receptor Kdr, the canonical regulators of CNV. Mechanistically, miR-34a directly targets and inhibits the transcription factor KLF2, thereby upregulating the proangiogenic factors CXCR4 and CXCL12. Finally, we show miR-34a exacerbates CNV in aged mice and is expressed in CNV lesions excised from wet AMD patients. These findings establish a causal link between the age-related miR-34a and neovascularization in AMD.
Jason J. Colasanti, A. Santeford, Joseph B. Lin et al.· Proceedings of the National...· 0 citations
Neovascular age-related macular degeneration (nAMD) is a major cause of blindness and is characterized by pathologic angiogenesis, specifically choroidal neovascularization (CNV). Mononuclear phagocytes (MPs), including infiltrating systemic monocyte-derived macrophages and retinal microglia, play critical roles in promoting CNV. The cGAS/STING pathway is increasingly implicated in multiple neuronal and systemic diseases and recently in ocular neovascularization. Given its roles across multiple cell types and the absence of MP-targeted therapies, we investigated the MP-specific role of cGAS/STING and a strategy for its selective targeting. In the laser-induced CNV mouse model, cGAS/STING was predominantly expressed in MPs. To enable selective targeting, we used a hydroxyl dendrimer (HD) previously shown to target MPs. HD conjugated to Cy3 selectively localized to MPs in laser CNV. HD conjugated to the STING inhibitor SN-011 (HD-SN-011) effectively inhibited cGAS/STING activation in cultured MPs. In the laser-CNV model, HD-SN-011 significantly reduced CNV leakage and lesion size, both important clinical endpoints in nAMD. RiboTag profiling confirmed selective suppression of cGAS/STING signaling and inflammatory gene expression in MPs. Together, our results implicate the specific importance of MP cGAS/STING signaling in CNV and provide proof of concept for specific modulation of STING in MPs as a therapy for nAMD.
Le Shi, D. Cherukaraveedu, Hongkwan Cho et al.· JCI Insight· 0 citations
Purpose
To evaluate the therapeutic potential of nicotinamide mononucleotide (NMN) for modulating the neurovascular inflammatory microenvironment and blunting tissue remodeling in neovascular age-related macular degeneration (nAMD).
Methods
A laser-induced choroidal neovascularization (CNV) model was established in C57BL/6J mice, and CNV lesion size was quantified on RPE/choroid flat mounts. Immunostaining evaluated myeloid cell accumulation and fibrosis-associated remodeling. Quantitative RT-PCR and Western blotting assessed inflammatory/angiogenic gene expression and signaling activation in the retina and RPE/choroid. In vitro, lipopolysaccharide (LPS)-stimulated bEnd.3 and primary mouse RPE cells and TGF-β-stimulated THP-1-derived macrophage cells were used to model inflammatory and profibrotic responses.
Results
NMN treatment significantly reduced CNV size in the laser-induced CNV model. This was accompanied by decreased myeloid cell accumulation within CNV lesions. NMN attenuated inflammatory and angiogenesis-related gene expression in the RPE/choroid and neural retina and reduced downstream signaling activation. In vitro, NMN suppressed LPS-induced inflammatory and proangiogenic responses in primary RPE cells and bEnd.3 endothelial cells and inhibited NF-κB activation. NMN further attenuated tissue remodeling, as shown by reduced collagen I-positive area under prolonged and delayed dosing regimens, together with decreased F4/80-positive area and α-smooth muscle actin-positive area within CNV lesions. In TGF-β-induced THP-1-derived macrophage cells, NMN suppressed profibrotic responses.
Conclusions
Our findings indicate that NMN reduces inflammatory signaling and alleviates the inflammatory microenvironment in CNV, accompanied by decreased angiogenesis-related gene expression and fibrosis-related remodeling. By attenuating inflammatory activation and tissue remodeling processes, NMN warrants further evaluation as an adjunctive approach to limit CNV progression and late-stage tissue remodeling in nAMD.
Jue Wang, Hideto Osada, Steve Chen et al.· Investigative Ophthalmology...· 0 citations
Wet age-related macular degeneration (wAMD), characterized by choroidal neovascularization (CNV), faces a clinical challenge of diminishing efficacy during long-term anti-VEGF monotherapy. An AI-assisted analytical framework pinpointed impaired retinal pigment epithelium (RPE) autophagy and a pro-inflammatory microenvironment driven by retinal microglia-recruited monocytes as co-conspirators in CNV progression. To tackle these dual culprits, we developed an intravitreal injectable hydrogel (Rab&BCL-M@G) that co-encapsulates ranibizumab (Rab) and a small-sized baicalin-loaded microemulsion (BCL-M), enabling controlled and sustained co-delivery of both agents to the retina for over 14 days. Guided by the AI-identified targets, we demonstrated that baicalin promoted dysfunctional mitochondria clearance via the AKT2-PGC-1α-mediated autophagic flux and suppressed monocyte recruitment by disrupting microglial CCL4 signaling. This two-pronged action ameliorated inflammation and angiogenesis, synergizing with Rab. In a laser-induced wAMD mouse model, a single intravitreal injection of Rab&BCL-M@G sustainably reduced CNV area, promoted repair, restored autophagy, and diminished microglial infiltration. Crucially, subsequent wet-lab validation confirmed a positive pathological correlation among defective autophagy, inflammation, and angiogenesis, thereby closing the loop from computational prediction to experimental verification and explaining suboptimal long-term performance after Rab monotherapy. This study proposes a promising synergistic strategy, advancing wAMD therapy through precision delivery and AI-informed mechanism discovery.
Xi Jiang, Fengqi Yang, Hui Gong et al.· Small· 0 citations
Pathological choroidal neovascularization underlies vision loss in neovascular age-related macular degeneration (nAMD), yet the molecular regulators coordinating vascular and immune components remain incompletely defined. Here, we investigated the role of the endolysosomal cation channel, two-pore channel 2 (TPC2) in choroidal angiogenesis. Loss of TPC2 in mice markedly reduced ex vivo choroidal sprouting, while pharmacological activation enhanced vascular growth. Mechanistically, Tpc2-deficiency led to downregulation of multiple microglia-derived pro-angiogenic factors and impaired the ability of the microglial secretome to stimulate neovascularization. In choroidal vascular cells, TPC2 loss attenuated NF-κB/MAPK signaling pathways. Tpc2-deficiency is also associated with lysosomal secretion of cathepsins, especially CTSD, resulting in decreased extracellular proteolytic activity and impaired paracrine regulation of angiogenesis. Extending these findings to human cells, TPC2 knockout in iPSC-derived endothelial cells impaired migration, tube formation, and CTSD activity in the secretome, mirroring the murine phenotype. Together, these results establish TPC2 as one of the regulators of lysosome-mediated choroidal angiogenesis, highlighting its potential as a therapeutic target in nAMD.
Yi Lu, Alice Reschigna, Franz Kynast et al.· Angiogenesis· 0 citations
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