Dual anti-angiogenic and anti-inflammatory action of tRNA-Cys-5-0007 in ocular vascular disease.

BACKGROUND: Intravitreal injections of angiogenesis inhibitors have proved efficacious in the majority of patients with ocular angiogenesis. However, one-fourth of all treated patients fail to derive benefits from intravitreal injections. tRNA-derived small RNA (tsRNA) emerges as a crucial class of non-coding RNA molecules, orchestrating key roles in the progression of human diseases by modulating multiple targets. Through our prior sequencing analyses and bioinformatics predictions, tRNA-Cys-5-0007 has shown as a potential regulator of ocular angiogenesis. This study endeavors to elucidate the precise role of tRNA-Cys-5-0007 in the context of ocular angiogenesis. METHODS: Quantitative reverse transcription PCR (qRT-PCR) assays were employed to detect tRNA-Cys-5-0007expression. EdU assays, sprouting assays, transwell assays, and Matrigel assays were conducted to elucidate the involvement of tRNA-Cys-5-0007 in endothelial angiogenic effects. STZ-induced diabetic model, OIR model, and laser-induced CNV model were utilized to replicate the pivotal features of ocular vascular diseases and evaluate the influence of tRNA-Cys-5-0007 on ocular angiogenesis and inflammatory responses. Bioinformatics analysis, luciferase activity assays, RNA pull-down assays, and in vitro studies were employed to elucidate the anti-angiogenic mechanism of tRNA-Cys-5-0007. Exosomal formulation was employed to enhance the synergistic anti-angiogenic and anti-inflammatory efficacy of tRNA-Cys-5-0007. RESULTS: tRNA-Cys-5-0007 expression was down-regulated under angiogenic conditions. Conversely, tRNA-Cys-5-0007 overexpression exhibited anti-angiogenic effects in retinal endothelial cells, as evidenced by reduced proliferation, sprouting, migration, and tube formation abilities. In diabetic, laser-induced CNV, and OIR models, tRNA-Cys-5-0007 overexpression led to decreased ocular vessel leakage, inhibited angiogenesis, and reduced ocular inflammation. Mechanistically, these effects were attributed to the targeting of vascular endothelial growth factor A (VEGFA) and TGF-ß1 by tRNA-Cys-5-0007. The utilization of an exosomal formulation further potentiated the synergistic anti-angiogenic and anti-inflammatory efficacy of tRNA-Cys-5-0007. CONCLUSIONS: Concurrent targeting of tRNA-Cys-5-0007 for anti-angiogenic and anti-inflammatory therapy holds promise for enhancing the effectiveness of current anti-angiogenic therapy.

Novel CCR3-targeted cyclic peptides as potential therapeutic agents for age-related macular degeneration via inhibiting angiogenesis and reducing retinal photoreceptor damage.

The prolonged intravitreal administration of anti-vascular endothelial growth factor (VEGF) drugs is prone to inducing aberrant retinal vascular development and causing damage to retinal neurons. Hence, we have taken an alternative approach by designing and synthesizing a series of cyclic peptides targeting CC motif chemokine receptor 3 (CCR3). Based on the binding mode of the N-terminal region in CCR3 protein to CCL11, we used computer-aided identification of key amino acid sequence, conformational restriction through different cyclization methods, designed and synthesized a series of target cyclic peptides, and screened the preferred compound IB-2 through affinity. IB-2 exhibits excellent anti-angiogenic activity in HRECs. The apoptosis level of 661W cells demonstrated a significant decrease with the escalating concentration of IB-2. This suggests that IB-2 may have a protective effect on photoreceptor cells. In vivo experiments have shown that IB-2 significantly reduces retinal vascular leakage and choroidal neovascularization (CNV) area in a laser-induced mouse model of CNV. These findings indicate the potential of IB-2 as a safe and effective therapeutic agent for AMD, warranting further development.

Metrnl inhibits choroidal neovascularization by attenuating the choroidal inflammation via inactivating the UCHL-1/NF-κB signaling pathway.

Objective: Choroidal neovascularization (CNV) represents the predominant form of advanced wet Age-related Macular Degeneration (wAMD). Macrophages play a pivotal role in the pathological progression of CNV. Meteorin-like (Metrnl), a novel cytokine known for its anti-inflammatory properties in macrophages, is the focus of our investigation into its mechanism of action and its potential to impede CNV progression. Methods: Cell viability was evaluated through CCK-8 and EdU assays following Metrnl treatment. Expression levels of inflammatory cytokines and proteins were assessed using quantitative reverse-transcription polymerase chain reaction(qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and western blot techniques. Protein-protein interactions were identified through protein mass spectrometry and co-immunoprecipitation (Co-IP). Additionally, in vivo and in vitro neovascularization models were employed to evaluate angiogenesis. Results: Our results revealed downregulated Metrnl levels in the choroid-sclera complex of CNV mice, the aqueous humor of wAMD patients, and activated macrophages. Metrnl overexpression demonstrated a reduction in pro-inflammatory cytokine production, influenced endothelial cell function, and suppressed angiogenesis in choroid explants and CNV models. Through protein mass spectrometry and Co-IP, we confirmed Metrnl binds to UCHL-1 to modulate the NF-κB signaling pathway. This interaction inhibited the transcription and expression of pro-inflammatory cytokines, ultimately suppressing angiogenesis. Conclusion: In summary, our findings indicate that Metrnl down-regulates macrophage pro-inflammatory cytokine secretion via the UCHL-1/NF-κB signaling pathway. This mechanism alleviates the inflammatory microenvironment and effectively inhibits choroidal neovascularization.

The landscape of angiogenesis and inflammatory factors in eyes with myopic choroidal neovascularization before and after anti-VEGF injection.

INTRODUCTION: To investigate the levels of angiogenesis and inflammatory cytokines in individuals with myopic choroidal neovascularization (mCNV) and the changes in these factors following intravitreal anti-VEGF injection. METHODS: Aqueous humor samples were gathered from eyes with mCNV, those with single macular bleeding (SMB) without mCNV in highly myopic eyes, and those with age-related cataracts. Using a multiplex bead immunoassay, we analyzed 28 angiogenesis and inflammatory factors in the aqueous humor. Furthermore, clinical data were documented for correlation analysis. RESULTS: In this study, the levels of vascular endothelial growth factor A (VEGF-A), interleukin 8 (IL-8), and fibroblast growth factors 1 (FGF-1) were significantly elevated in mCNV compared to SMB eyes (p < 0.05). Their odds ratios for mCNV occurrence were 1.05, 3.45, and 2.64, respectively. Hepatocyte growth factor (HGF) and VEGF-C were notably higher in mCNV than in cataract patients (p < 0.05), and VEGF-C correlated to the degree of myopic atrophic maculopathy (p = 0.024). Axial length exhibited a negative correlation with VEGF-A and positive correlations with VEGF-C, HGF, and MCP-1 (p < 0.01). Following anti-VEGF treatment, a reduction in VEGF-A, endothelin-1, and FGF-2 was noted in mCNV patients (p < 0.05), but MCP-1 levels increased. CONCLUSION: Our findings highlight the predominant role of angiogenesis and inflammation factors in mCNV pathogenesis. VEGF-C's correlation with axial length and atrophy suggests its involvement in the process of myopic atrophic maculopathy.

Novel therapeutic perspectives for wet age-related macular degeneration: RGD-modified liposomes loaded with 2-deoxy-D-glucose as a promising nanomedicine.

Choroidal neovascularization (CNV), characterized as a prominent feature of wet age-related macular degeneration (AMD), is a primary contributor to visual impairment and severe vision loss globally, while the prevailing treatments are often unsatisfactory. The development of conventional treatment strategies has largely been based on the understanding that the angiogenic switch of endothelial cells is dictated by angiogenic growth factors alone. Even though treatments targeting vascular endothelial growth factor (VEGF), like Ranibizumab, are widely administered, more than half of the patients still exhibit inadequate or null responses, emphasizing the imperative need for solutions to this problem. Here, aiming to explore therapeutic strategies from a novel perspective of endothelial cell metabolism, a biocompatible nanomedicine delivery system is constructed by loading RGD peptide-modified liposomes with 2-deoxy-D-glucose (RGD@LP-2-DG). RGD@LP-2-DG displayed good targeting performance towards endothelial cells and excellent in vitro and in vivo inhibitory effects on neovascularization were demonstrated. Moreover, our mechanistic studies revealed that 2-DG interfered with N-glycosylation, leading to the inhibition of vascular endothelial growth factor receptor 2 (VEGFR2) and its downstream signaling. Notably, the remarkable inhibitory effect on neovascularization and biocompatibility of RGD@LP-2-DG render it a highly promising and clinically translatable therapeutic candidate for the treatment of wet AMD and other angiogenic diseases, particularly in patients who are unresponsive to currently available treatments.

Microglial repopulation restricts ocular inflammation and choroidal neovascularization in mice.

Introduction: Age-related macular degeneration (AMD) is a prevalent, chronic and progressive retinal degenerative disease characterized by an inflammatory response mediated by activated microglia accumulating in the retina. In this study, we demonstrate the therapeutically effects and the underlying mechanisms of microglial repopulation in the laser-induced choroidal neovascularization (CNV) model of exudative AMD. Methods: The CSF1R inhibitor PLX3397 was used to establish a treatment paradigm for microglial repopulation in the retina. Neovascular leakage and neovascular area were examined by fundus fluorescein angiography (FFA) and immunostaining of whole-mount RPE-choroid-sclera complexes in CNV mice receiving PLX3397. Altered cellular senescence was measured by beta-galactosidase (SA-ß-gal) activity and p16INK4a expression. The effect and mechanisms of repopulated microglia on leukocyte infiltration and the inflammatory response in CNV lesions were analyzed. Results: We showed that ten days of the CSF1R inhibitor PLX3397 treatment followed by 11 days of drug withdrawal was sufficient to stimulate rapid repopulation of the retina with new microglia. Microglial repopulation attenuated pathological choroid neovascularization and dampened cellular senescence in CNV lesions. Repopulating microglia exhibited lower levels of activation markers, enhanced phagocytic function and produced fewer cytokines involved in the immune response, thereby ameliorating leukocyte infiltration and attenuating the inflammatory response in CNV lesions. Discussion: The microglial repopulation described herein are therefore a promising strategy for restricting inflammation and choroidal neovascularization, which are important players in the pathophysiology of AMD.

Mast cells in human choroid and their role in age-related macular degeneration (AMD).

The role of mast cells in physiologic and pathological processes extends far beyond the allergy processes: they are involved in wound healing, chronic inflammation, and tumor growth. This short article emphasizes the role played by mast cells in age-related macular degeneration (AMD). Mast cells can induce angiogenesis and are present around Bruch's membrane during the early and late stages of choroidal neovascularization in AMD. Proteolytic enzymes released by mast cells lead to thinning of the choroid in AMD as well as degradation of vascular basement membranes and Bruch's membrane, which in turn could result in retinal pigment epithelial death and choriocapillaris degeneration in geographical atrophy and exudative AMD.

Effects of slit lamp-delivered retinal laser photobiomodulation in a rat model of choroidal neovascularization.

Neovascular age-related macular degeneration, also known as exudative or wet age-related macular degeneration, is the leading cause of blindness in the developed world. Photobiomodulation has the potential to target the up-stream hypoxic and pro-inflammatory drivers of choroidal neovascularization. This study investigated whether photobiomodulation attenuates characteristic pathological features of choroidal neovascularization in a rodent model. Experimental choroidal neovascularization was induced in Brown Norway rats with laser photocoagulation. A custom-designed, slit-lamp-mounted, 670 nm laser was used to administer retinal photobiomodulation every 3 days, beginning 6 days prior to choroidal neovascularization induction and continuing until the animals were killed 14 days later. The effect of photobiomodulation on the size of choroidal neovascular membranes was determined using isolectin-B4 immunohistochemistry and spectral domain-optical coherence tomography. Vascular leakage was determined with fluorescein angiography. The effect of treatment on levels of vascular endothelial growth factor expression was quantified with enzyme-linked immunosorbent assay. Treatment with photobiomodulation was associated with choroidal neovascular membranes that were smaller, had less fluorescein leakage, and a diminished presence of inflammatory cells as compared to sham eyes. These effects were not associated with a statistically significant difference in the level of vascular endothelial growth factor when compared to sham eyes. The data shown herein indicate that photobiomodulation attenuates pathological features of choroidal neovascularization in a rodent model by mechanisms that may be independent of vascular endothelial growth factor.

Role of NLRP3 Inflammasomes in Monocyte and Microglial Recruitments in Choroidal Neovascularization.

Although the pathogenesis of choroidal neovascularization (CNV) is largely unknown in age-related macular degeneration (AMD), inflammasomes may contribute to CNV development and progression. To understand the role NLRP3 inflammasomes in CNV, we used Ccr2RFPCx3cr1GFP dual-reporter mice and immunostaining techniques to confirm localization of NLRP3 inflammasomes in the laser-induced CNV (LCNV) lesions. Confocal microscopy was used to image and quantify LCNV volumes. MCC950 was used as NLRP3 inhibitor. ELISA and quantitative RT-PCR were used to confirm the activation of NLRP3 by monitoring the expression of IL-1ß protein and mRNA in choroidal tissues from LCNV mice. In addition, NLRP3 (-/-) LCNV mice were used to investigate whether NLRP3 inflammasomes contribute to the development of LCNV lesions. We observed that red fluorescent protein (RFP)-positive monocyte-derived macrophages and GFP-positive microglia-derived macrophages, in addition to other cell types, were localized in LCNV lesions at day 7 post-laser injury. In addition, NLRP3 inflammasomes are associated with LCNV lesions. Inhibition of NLRP3 inflammasomes, using MCC950, caused an increased Ccr2RFP-positive macrophages, Cx3cr1GFP-positive microglia, and other cells, resulting in an increase in total lesion size. NLRP3 (-/-) LCNV mice showed significantly increased lesion size compared with age-matched controls. Inhibition of NLRP3 resulted in decreased IL-1ß mRNA and protein expression in the choroidal tissues, suggesting that increased lesion size may not be directly related to IL-1ß.

Exploring the Impact of Saccharin on Neovascular Age-Related Macular Degeneration: A Comprehensive Study in Patients and Mice.

Purpose: We aimed to determine the impact of artificial sweeteners (AS), especially saccharin, on the progression and treatment efficacy of patients with neovascular age-related macular degeneration (nAMD) under anti-vascular endothelial growth factor (anti-VEGF-A) treatment. Methods: In a cross-sectional study involving 46 patients with nAMD undergoing intravitreal anti-VEGF therapy, 6 AS metabolites were detected in peripheral blood using liquid chromatography - tandem mass spectrometry (LC-MS/MS). Disease features were statistically tested against these metabolite levels. Additionally, a murine choroidal neovascularization (CNV) model, induced by laser, was used to evaluate the effects of orally administered saccharin, assessing both imaging outcomes and gene expression patterns. Polymerase chain reaction (PCR) methods were used to evaluate functional expression of sweet taste receptors in a retinal pigment epithelium (RPE) cell line. Results: Saccharin levels in blood were significantly higher in patients with well-controlled CNV activity (P = 0.004) and those without subretinal hyper-reflective material (P = 0.015). In the murine model, saccharin-treated mice exhibited fewer leaking laser scars, lesser occurrence of bleeding, smaller fibrotic areas (P < 0.05), and a 40% decrease in mononuclear phagocyte accumulation (P = 0.06). Gene analysis indicated downregulation of inflammatory and VEGFR-1 response genes in the treated animals. Human RPE cells expressed taste receptor type 1 member 3 (TAS1R3) mRNA and reacted to saccharin stimulation with changes in mRNA expression. Conclusions: Saccharin appears to play a protective role in patients with nAMD undergoing intravitreal anti-VEGF treatment, aiding in better pathological lesion control and scar reduction. The murine study supports this observation, proposing saccharin's potential in mitigating pathological VEGFR-1-induced immune responses potentially via the RPE sensing saccharin in the blood stream.

miR-199a-5p modulates choroidal neovascularization by regulating Wnt7b/Wnt/ß-catenin signaling pathway.

Choroidal neovascularization (CNV) can be seen in many fundus diseases, and lead to fundus exudation, bleeding, or vision loss. miRNAs are vital regulator in CNV. miR-199a-5p has been proved to be involved in regulating vascular formation of endothelial cells, but its role in CNV remains unclear. This study aims to study the role of miR-199a-5p in CNV. Laser irradiation was used to induce CNV model. The lesion area of CNV was calculated by high-resolution angiography with fluorescein isothiocyanate-dextran. Wnt family member 7b (Wnt7b), ß-catenin, and Wnt pathway proteins was measured by western blot. Immunofluorescence was performed to test Wnt7b, ß-catenin, CD31, and p-p65. miR-199a-5p and Wnt7b mRNA were tested by reverse transcription real-time polymerase chain reaction. Cell count kit-8, wound healing, Transwell, tube formation, and flow cytometry were used to detect the function of miR-199a-5p and Wnt7b on human retinal microvascular endothelial cells (HRMEC). TargetScan database and dual-luciferase reporter assay verified the interaction between miR-199a-5p and Wnt7b. The results revealed that Wnt7b increased in CNV rats. Knocking down Wnt7b repressed cell proliferation, migration, invasion, and angiogenesis, and accelerated cell apoptosis of HRMEC. Dual-luciferase reporter assay verified that miR-199a-5p targeted Wnt7b. Overexpression of miR-199a-5p inhibited the angiogenesis of HRMEC and promoted cell apoptosis by inhibiting Wbt7b. In vivo experiment found that Wnt7b rescued the promotion of miR-199a-5p inhibition on CNV lesion of rats. In addition, Wnt7b positively regulated Wnt/ß-catenin signaling pathway and promoted the angiogenesis of HRMEC. In conclusion, overexpression of miR-199a-5p inhibited the angiogenesis of HRMEC by regulating Wnt7b/Wnt/ß-catenin signaling pathway, which may serve as a promising therapy target of CNV.

Sphingosine-1-phosphate receptor 1/5 selective agonist alleviates ocular vascular pathologies.

Ocular abnormal angiogenesis and edema are featured in several ocular diseases. S1P signaling via S1P1 likely is part of the negative feedback mechanism necessary to maintain vascular health. In this study, we conducted pharmacological experiments to determine whether ASP4058, a sphingosine 1-phosphate receptor 1/5 (S1P1/5) agonist, is useful in abnormal vascular pathology in the eye. First, human retinal microvascular endothelial cells (HRMECs) were examined using vascular endothelial growth factor (VEGF)-induced cell proliferation and hyperpermeability. ASP4058 showed high affinity and inhibited VEGF-induced proliferation and hyperpermeability of HRMECs. Furthermore, S1P1 expression and localization changes were examined in the murine laser-induced choroidal neovascularization (CNV) model, a mouse model of exudative age-related macular degeneration, and the efficacy of ASP4058 was verified. In the CNV model mice, S1P1 tended to decrease in expression immediately after laser irradiation and colocalized with endothelial cells and Müller glial cells. Oral administration of ASP4058 also suppressed vascular hyperpermeability and CNV, and the effect was comparable to that of the intravitreal administration of aflibercept, an anti-VEGF drug. Next, efficacy was also examined in a retinal vein occlusion (RVO) model in which retinal vascular permeability was increased. ASP4058 dose-dependently suppressed the intraretinal edema. In addition, it suppressed the expansion of the perfusion area observed in the RVO model. ASP4058 also suppressed the production of VEGF in the eye. Collectively, ASP4058 can be a potential therapeutic agent that normalizes abnormal vascular pathology, such as age-related macular degeneration and RVO, through its direct action on endothelial cells.

Therapeutic targeting of telomerase ameliorates experimental choroidal neovascularization.

Choroidal neovascularization (CNV) is the principal driver of blindness in neovascular age-related macular degeneration (nvAMD). Increased activity of telomerase, has been associated with endothelial cell proliferation, survival, migration, and invasion in the context of tumor angiogenesis. Expanding on this knowledge, we investigated the role of telomerase in the development of CNV in mouse model. We observed increased gene expression and activity of telomerase in mouse CNV. Genetic deficiency of the telomerase components, telomerase reverse transcriptase (Tert) and telomerase RNA component (Terc) suppressed laser-induced CNV in mice. Similarly, a small molecule inhibitor of TERT (BIBR 1532), and antisense oligonucleotides (ASOs) targeting Tert and Terc reduced CNV growth. Bone marrow chimera studies suggested that telomerase activity in non-bone marrow-derived cells is crucial for the development of CNV. Comparison of BIBR 1532 with VEGF neutralizing therapeutic strategy in mouse revealed a comparable level of angiosuppressive activity. However, when BIBR and anti-VEGF antibodies were administered as a combination at sub-therapeutic doses, a statistically significant suppression of CNV was observed. These findings underscore the potential benefits of combining sub-therapeutic doses of BIBR and anti-VEGF antibodies for developing newer therapeutic strategies for NV-AMD. Telomerase inhibition with BIBR 1532 suppressed induction of multiple cytokines and growth factors critical for neovascularization. In conclusion, our study identifies telomerase as a promising therapeutic target for treating neovascular disease of the eye and thus provides a proof of principle for further exploration of telomerase inhibition as a novel treatment strategy for nvAMD.

Single-cell profiling transcriptomic reveals cellular heterogeneity and cellular crosstalk in choroidal neovascularization model.

Choroidal neovascularization (CNV) is a hallmark of neovascular age-related macular degeneration (nAMD) and a major contributor to vision loss in nAMD cases. However, the identification of specific cell types associated with nAMD remains challenging. Herein, we performed single-cell sequencing to comprehensively explore the cellular diversity and understand the foundational components of the retinal pigment epithelium (RPE)/choroid complex. We unveiled 10 distinct cell types within the RPE/choroid complex. Notably, we observed significant heterogeneity within endothelial cells (ECs), fibroblasts, and macrophages, underscoring the intricate nature of the cellular composition in the RPE/choroid complex. Within the EC category, four distinct clusters were identified and EC cluster 0 was tightly associated with choroidal neovascularization. We identified five clusters of fibroblasts actively involved in the pathogenesis of nAMD, influencing fibrotic responses, angiogenic effects, and photoreceptor function. Additionally, three clusters of macrophages were identified, suggesting their potential roles in regulating the progression of nAMD through immunomodulation and inflammation regulation. Through CellChat analysis, we constructed a complex cell-cell communication network, revealing the role of EC clusters in interacting with fibroblasts and macrophages in the context of nAMD. These interactions were found to govern angiogenic effects, fibrotic responses, and inflammatory processes. In summary, this study reveals noteworthy cellular heterogeneity in the RPE/choroid complex and provides valuable insights into the pathogenesis of CNV. These findings will open up potential avenues for deep understanding and targeted therapeutic interventions in nAMD.

Progression of retinal choroidal neovascularization by latent human cytomegalovirus infection and immunological signaling among neonatal patients admitted to tertiary care hospital.

Choroidal neovascularization (CNV) is a serious condition that affects the retina, causing partial or complete blindness in people of different ages. While CNV is a common occurrence in various chorioretinopathies, research on its occurrence in neonates is limited. Human cytomegalovirus (HCMV) is a significant health threat to neonates, with a strong association with retinal angiogenesis. However, there has been limited investigation into HCMV-associated CNV progression. In this article, we extensively studied the expression of different inflammatory cytokines and chemokines during latent HCMV-associated retinal neovascularization. Our research found that HCMV-induced CNV progression was significantly prominent in the presence of AT2R-dependent angiogenesis (p < 0.001), whereas in the absence of HCMV, AT1R-dependent CCL-5-mediated angiogenesis was documented. We also observed significant increases in CCL-19, CCL-21 chemokine responses, followed by CCR-7 chemokine receptor activation (p < 0.001) in HCMV-induced CNV patients compared to HCMV non-induced CNV groups. Furthermore, significant changes in predictive chemokine markers of HCMV-induced CNV were positively correlated with HCMV viremia. These immunological alterations ultimately lead to the switching of NFκB canonical and noncanonical pathways, respectively, in HCMV-induced neonatal CNV and HCMV non-induced CNV. This clinical observation presents a novel hypothesis that ocular HCMV latency poses a noteworthy risk factor for the progression of retinal neovascularization through a distinctive immunological signaling pathway. The current study represents the first of its kind to report on this association, which may have significant implications for the clinical management of patients with ocular HCMV.

5-Aza-2'-Deoxycytidine Ameliorates Choroidal Neovascularization by Inhibiting the Wnt/ß-Catenin Signaling Pathway.

Purpose: Choroidal neovascularization (CNV) can constitute the final pathology of many ocular diseases and result in severe vision loss. Studies have demonstrated that DNA methylation is critical in retinal development, aging, and disorders. The current work investigated the effects and underlying mechanism of 5-Aza-2'-deoxycytidine (5-aza-dC), a suppressor of DNA methylation, in the pathological progression of CNV. Methods: The DNA methylation profiles of retinal pigment epithelial (RPE)/choroidal complexes in normal and laser-induced CNV mice were assessed by Arraystar Mouse RefSeq Promoter Arrays. The CNV area and blood flow density and intensity were observed by optical coherence tomography angiography, and fluorescence leakage was examined by fundus fluorescein angiography in CNV mice with systemic administration of 5-aza-dC. The effects of 5-aza-dC on the biological functions of bEnd.3 cells were estimated by related assays. Notum gene promoter methylation was measured using bisulfite sequencing PCR. Methyltransferases and Wnt signaling-related genes were detected in animal and cell culture experiments by real-time PCR and immunoblot. Results: Methyltransferases were upregulated, but Notum (a secretion inhibitor of Wnt signaling) was downregulated in the RPE/choroidal complexes of mice with experimental CNV. Intraperitoneal injection of 5-aza-dC inactivated the Wnt pathway and ameliorated the lesion area and the intensity and density of blood flow, as well as the degree of leakage in CNV. In vitro, vascular endothelial growth factor A (VEGFA) stimulation promoted methyltransferases expression and suppressed Notum expression, consequently activating Wnt signaling, whereas exogenous 5-aza-dC reversed VEGFA-induced hyperpermeability, proliferation, migration, and tube formation in bEnd.3 cells via demethylation of Notum promoter. Conclusions: We observed that 5-aza-dC attenuates the growth of CNV by inhibiting the Wnt signaling pathway via promoter demethylation of the Wnt antagonist Notum. These findings provide a theoretical basis for methylation-based treatment with the Notum gene as a potential target for CNV treatment.

Topical Ophthalmic Liposomes Dual-Modified with Penetratin and Hyaluronic Acid for the Noninvasive Treatment of Neovascular Age-Related Macular Degeneration.

Introduction: Since intrinsic ocular barrier limits the intraocular penetration of therapeutic protein through eye drops, repeated intravitreal injections of anti-vascular endothelial growth factor (anti-VEGF) agents are the standard therapy for neovascular age-related macular degeneration (nAMD), which are highly invasive and may cause particular ocular complications, leading to poor patient compliance. Methods: Using Penetratin (Pen) as the ocular penetration enhancer and hyaluronic acid (HA) as the retina-targeting ligand, a dual-modified ophthalmic liposome (Penetratin hyaluronic acid-liposome/Conbercept, PenHA-Lip/Conb) eye drop was designed to non-invasively penetrate the ocular barrier and deliver anti-VEGF therapeutic agents to the targeted intraocular tissue. Results: PenHA-Lip effectively penetrates the ocular barrier and targets the retinal pigment epithelium via corneal and non-corneal pathways. After a single topical administration of conbercept-loaded PenHA-Lip (PenHA-Lip/Conb), the intraocular concentration of conbercept peaked at 18.74 ± 1.09 ng/mL at 4 h, which is 11.55-fold higher than unmodified conbercept. In a laser-induced choroidal neovascularization (CNV) mouse model, PenHA-Lip/Conb eye drops three times daily for seven days inhibited CNV formation and progression without any significant tissue toxicity and achieved an equivalent effect to a single intravitreal conbercept injection. Conclusion: PenHA-Lip efficiently and safely delivered conbercept to the posterior eye segment and may be a promising noninvasive therapeutic option for nAMD.

Human lens epithelial-secreted exosomes attenuate ocular angiogenesis via inhibiting microglial activation.

The lens is an avascular tissue, where epithelial cells (LECs) are the primary living cells. The role of LECs-derived exosomes (LEC-exos) is largely unknown. In our study, we determined the anti-angiogenic role of LEC-exos, manifested as regressed retinal neovascularization (NV) using the oxygen-induced retinopathy (OIR), and reduced choroidal NV size and pathological vascular leakage using the laser-induced choroidal neovascularization (laser-induced CNV). Furthermore, the activation and accumulation of microglia were also restricted by LEC-exos. Based on Luminex multiplex assays, the expressions of chemokines such as SCYB16/CXCL16, MCP-1/CCL2, I-TAC/CXCL11, and MIP 3beta/CCL19 were decreased after treatment with LEC-exos. Transwell assays showed that LEC-exos restricted the migration of the mouse microglia cell line (BV2 cells). After incubation with LEC-exos-treated BV2 cells, human umbilical vein endothelial cells (hUVECs) were collected for further evaluation using tube formation, Transwell assays, and 5-ethynyl-2'-deoxyuridine (EDU) assays. Using in vitro experiments, the pro-angiogenic effect of microglia was restricted by LEC-exos. Hence, it was investigated that LEC-exos attenuated ocular NV, which might attribute to the inhibition of microglial activation and accumulation.

A Sustainable Retinal Drug Co-Delivery for Boosting Therapeutic Efficacy in wAMD: Unveiling Multifaceted Evidence and Synergistic Mechanisms.

Sustainable retinal codelivery poses significant challenges technically, although it is imperative for synergistic treatment of wet age-related macular degeneration (wAMD). Here, a microemulsion-doped hydrogel (Bor/PT-M@TRG) is engineered as an intravitreal depot composing of temperature-responsive hydrogel (TRG) and borneol-decorated paeoniflorin (PF) & tetramethylpyrazine (TMP)-coloaded microemulsions (Bor/PT-M). Bor/PT-M@TRG, functioning as the "ammunition depot", resides in the vitreous and continuously releases Bor/PT-M as the therapeutic "bullet", enabling deep penetration into the retina for 21 days. A single intravitreal injection of Bor/PT-M@TRG yields substantial reductions in choroidal neovascularization (CNV, a hallmark feature of wAMD) progression and mitigates oxidative stress-induced damage in vivo. Combinational PF&TMP regulates the "reactive oxygen species/nuclear factor erythroid-2-related factor 2/heme oxygenase-1" pathway and blocks the "hypoxia inducible factor-1α/vascular endothelial growth factor" signaling in retina, synergistically cutting off the loop of CNV formation. Utilizing fluorescence resonance energy transfer and liquid chromatography-mass spectrometry techniques, they present compelling multifaceted evidence of sustainable retinal codelivery spanning formulations, ARPE-19 cells, in vivo eye balls, and ex vivo section/retina-choroid complex cell levels. Such codelivery approach is elucidated as the key driving force behind the exceptional therapeutic outcomes of Bor/PT-M@TRG. These findings highlight the significance of sustainable retinal drug codelivery and rational combination for effective treatment of wAMD.

Emerging Co-Assembled and Sustained Released Natural Medicinal Nanoparticles for Multitarget Therapy of Choroidal Neovascularization.

Age-related macular degeneration (AMD) disease has become a worldwide senile disease, and frequent intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) is the mainstream treatment in the clinic, which is associated with sight-threatening complications. Herein, nintedanib, an inhibitor of angiogenesis, and lutein, a potent antioxidant, can co-assemble into nanoparticles through multiple noncovalent interactions. Interestingly, the co-assembled lutein/nintedanib nanoparticles (L/N NPs) exhibit significantly improved stability and achieve long-term sustained release of two drugs for at least two months in mice. Interestingly, in rabbit eyeball with a more complete barrier system, the L/N NPs still successfully distribute in the retina and choroid for a month. In the laser-induced mouse choroidal neovascularization model, the L/N NPs after a minimally invasive subconjunctival administration can successfully inhibit angiogenesis and achieve comparable and even better therapeutic results to that of standard intravitreal injection of anti-VEGF. Therefore, the subconjunctival injection of L/N NPs with long-term sustained drug release behavior represents a promising and innovative strategy for AMD treatment. Such minimally invasive administration together with the ability to effectively inhibit angiogenesis reduce inflammation and counteract oxidative stress and holds great potential for improving patient outcomes and quality of life in those suffering from this debilitating eye condition.