Background: Ocular neovascularization is a leading cause of blindness. Retinal microglia have been implicated in hypoxia-induced angiogenesis and vasculopathy, but the underlying mechanisms are not entirely clear. Lactylation is a novel lactate-derived posttranslational modification that plays key roles in multiple cellular processes. Since hypoxia in ischemic retinopathy is a precipitating factor for retinal neovascularization, lactylation is very likely to be involved in this process. The present study aimed to explore the role of lactylation in retinal neovascularization and identify new therapeutic targets for retinal neovascular diseases. Results: Microglial depletion by the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX3397 suppresses retinal neovascularization in oxygen-induced retinopathy. Hypoxia increased lactylation in microglia and accelerates FGF2 expression, promoting retinal neovascularization. We identify 77 sites of 67 proteins with increased lactylation in the context of increased lactate under hypoxia. Our results show that the nonhistone protein Yin Yang-1 (YY1), a transcription factor, is lactylated at lysine 183 (K183), which is regulated by p300. Hyperlactylated YY1 directly enhances FGF2 transcription and promotes angiogenesis. YY1 mutation at K183 eliminates these effects. Overexpression of p300 increases YY1 lactylation and enhances angiogenesis in vitro and administration of the p300 inhibitor A485 greatly suppresses vascularization in vivo and in vitro. Conclusions: Our results suggest that YY1 lactylation in microglia plays an important role in retinal neovascularization by upregulating FGF2 expression. Targeting the lactate/p300/YY1 lactylation/FGF2 axis may provide new therapeutic targets for proliferative retinopathies.

Background: Vascular endothelial growth factor (VEGF) is one of the most powerful proangiogenic factors and plays an important role in multiple diseases. Increased glycolytic rates and lactate accumulation are associated with pathological angiogenesis.Results: Here, we show that a feedback loop between H3K9 lactylation (H3K9la) and histone deacetylase 2 (HDAC2) in endothelial cells drives VEGF-induced angiogenesis. We find that the H3K9la levels are upregulated in endothelial cells in response to VEGF stimulation. Pharmacological inhibition of glycolysis decreases H3K9 lactylation and attenuates neovascularization. CUT& Tag analysis reveals that H3K9la is enriched at the promoters of a set of angiogenic genes and promotes their transcription. Interestingly, we find that hyperlactylation of H3K9 inhibits expression of the lactylation eraser HDAC2, whereas overexpression of HDAC2 decreases H3K9 lactylation and suppresses angiogenesis.Conclusions: Collectively, our study illustrates that H3K9la is important for VEGF-induced angiogenesis, and interruption of the H3K9la/HDAC2 feedback loop may represent a novel therapeutic method for treating pathological neovascularization.

Dysregulation of CD4 ⁺ T cell differentiation is linked to autoimmune diseases. Metabolic reprogramming from oxidative phosphorylation to glycolysis and accumulation of lactate are involved in this process. However, the underlying mechanisms remain unclear. Our study showed that lactate-derived lactylation regulated CD4 ⁺ T cell differentiation. Lactylation levels in CD4 ⁺ T cells increased with the progression of experimental autoimmune uveitis (EAU). Inhibition of lactylation suppressed T _H 17 differentiation and attenuated EAU inflammation. The global lactylome revealed the landscape of lactylated sites and proteins in the CD4 ⁺ T cells of normal and EAU mice. Specifically, hyperlactylation of Ikzf1 at Lys ¹⁶⁴ promoted T _H 17 differentiation by directly modulating the expression of T _H 17-related genes, including Runx1, Tlr4, interleukin-2 (IL-2), and IL-4. Delactylation of Ikzf1 at Lys ¹⁶⁴ impaired T _H 17 differentiation. These findings exemplify how glycolysis regulates the site specificity of protein lactylation to promote T _H 17 differentiation and implicate Ikzf1 lactylation as a potential therapeutic target for autoimmune diseases.

Uveitis causes blindness and critical visual impairment in people of all ages, and retinal microglia participate in uveitis progression. Unfortunately, effective treatment is deficient. Icariin (ICA) is a bioactive monomer derived from Epimedium. However, the role of ICA in uveitis remains elusive. Our study indicated that ICA alleviated intraocular inflammation in vivo. Further results showed the proinflammatory M1 microglia could be transferred to anti-inflammatory M2 microglia by ICA in the retina and HMC3 cells. However, the direct pharmacological target of ICA is unknown, to this end, proteome microarrays and molecular simulations were used to identify the molecular targets of ICA. Data showed that ICA binds to peroxiredoxin-3 (PRDX3), increasing PRDX3 protein expression in both a time- and a concentration-dependent manner and promoting the subsequent elimination of H₂O₂. In addition, GPX4/SLC7A11/ACSL4 pathways were activated accompanied by PRDX3 activation. Functional tests demonstrated that ICA-derived protection is afforded through targeting PRDX3. First, ICA-shifted microglial M1/M2 phenotypic polarization was no longer detected by blocking PRDX3 both in vivo and in vitro. Next, ICA-activated GPX4/SLC7A11/ACSL4 pathways and downregulated H₂O₂ production were also reversed via inhibiting PRDX3 both in vivo and in vitro. Finally, ICA-elicited positive effects on intraocular inflammation were eliminated in PRDX3-deficient retina from experimental autoimmune uveitis (EAU) mice. Taking together, ICA-derived PRDX3 activation has therapeutic potential for uveitis, which might be associated with modulating microglial M1/M2 phenotypic polarization.

Galectin-3, an attractive molecule of innate immunity, has been reported to be involved in the neuroinflammatory diseases. However, the role of Galectin-3 in autoimmune uveitis is still unclear. The purpose of this study was to investigate the effect and mechanism of Galectin-3 on microglial activation and inflammation of experimental autoimmune uveitis (EAU)_. We immunized female C57BL/6 J mice with IRBP_651-670 to induce EAU and the specific inhibitor was intravitreally injected in EAU mice. Disease severity was evaluated by clinical and histopathological scores. Immunofluorescence, western blot, qRT-PCR analysis and immunoprecipitation were used to detect the functional phenotypes and mechanisms on microglia after Galectin-3 inhibition. Our results showed that the expression of Galectin-3 was conspicuously increased in microglia of EAU retinas. The specific inhibitor of Galectin-3, TD139 was found to ameliorate the clinical and histological manifestations of EAU mice. In addition, TD139 reduced the expression of proinflammatory factors in vivo and vitro, which are related to the severity of uveitis. In mechanism, TD139 down-regulated the expression of TLR4 and MyD88, and then inhibited the activation of NF-κB p65 in microglia. In conclusion, Galectin-3 may play important roles in a variety of immune related diseases including autoimmune uveitis. Additionally, the inhibition of Galectin-3 may attenuate the microglial activation and inflammatory response through TLR4/MyD88/NF-κB pathway, highlighting a potential therapeutic target of Galectin-3 for autoimmune uveitis.