A feedback loop driven by H3K9 lactylation and HDAC2 in endothelial cells regulates VEGF-induced angiogenesis.

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.

A de novo missense mutation in MPP2 confers an increased risk of Vogt-Koyanagi-Harada disease as shown by trio-based whole-exome sequencing.

Vogt-Koyanagi-Harada (VKH) disease is a leading cause of blindness in young and middle-aged people. However, the etiology of VKH disease remains unclear. Here, we performed the first trio-based whole-exome sequencing study, which enrolled 25 VKH patients and 50 controls, followed by a study of 2081 VKH patients from a Han Chinese population to uncover detrimental mutations. A total of 15 de novo mutations in VKH patients were identified, with one of the most important being the membrane palmitoylated protein 2 (MPP2) p.K315N (MPP2-N315) mutation. The MPP2-N315 mutation was highly deleterious according to bioinformatic predictions. Additionally, this mutation appears rare, being absent from the 1000 Genome Project and Genome Aggregation Database, and it is highly conserved in 10 species, including humans and mice. Subsequent studies showed that pathological phenotypes and retinal vascular leakage were aggravated in MPP2-N315 mutation knock-in or MPP2-N315 adeno-associated virus-treated mice with experimental autoimmune uveitis (EAU). In vitro, we used clustered regularly interspaced short palindromic repeats (CRISPR‒Cas9) gene editing technology to delete intrinsic MPP2 before overexpressing wild-type MPP2 or MPP2-N315. Levels of cytokines, such as IL-1ß, IL-17E, and vascular endothelial growth factor A, were increased, and barrier function was destroyed in the MPP2-N315 mutant ARPE19 cells. Mechanistically, the MPP2-N315 mutation had a stronger ability to directly bind to ANXA2 than MPP2-K315, as shown by LC‒MS/MS and Co-IP, and resulted in activation of the ERK3/IL-17E pathway. Overall, our results demonstrated that the MPP2-K315N mutation may increase susceptibility to VKH disease.

YY1 lactylation in microglia promotes angiogenesis through transcription activation-mediated upregulation of FGF2.

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.

Fibroblast growth factor 7 alleviates myocardial infarction by improving oxidative stress via PI3Kα/AKT-mediated regulation of Nrf2 and HXK2.

Acute myocardial infarction (MI) triggers oxidative stress, which worsen cardiac function, eventually leads to remodeling and heart failure. Unfortunately, effective therapeutic approaches are lacking. Fibroblast growth factor 7 (FGF7) is proved with respect to its proliferative effects and high expression level during embryonic heart development. However, the regulatory role of FGF7 in cardiovascular disease, especially MI, remains unclear. FGF7 expression was significantly decreased in a mouse model at 7 days after MI. Further experiments suggested that FGF7 alleviated MI-induced cell apoptosis and improved cardiac function. Mechanistic studies revealed that FGF7 attenuated MI by inhibiting oxidative stress. Overexpression of FGF7 actives nuclear factor erythroid 2-related factor 2 (Nrf2) and scavenging of reactive oxygen species (ROS), and thereby improved oxidative stress, mainly controlled by the phosphatidylinositol-3-kinase α (PI3Kα)/AKT signaling pathway. The effects of FGF7 were partly abrogated in Nrf2 deficiency mice. In addition, overexpression of FGF7 promoted hexokinase2 (HXK2) and mitochondrial membrane translocation and suppressed mitochondrial superoxide production to decrease oxidative stress. The role of HXK2 in FGF7-mediated improvement of mitochondrial superoxide production and protection against MI was verified using a HXK2 inhibitor (3-BrPA) and a HXKII VDAC binding domain (HXK2VBD) peptide, which competitively inhibits localization of HXK2 on mitochondria. Furthermore, inhibition of PI3Kα/AKT signaling abolished regulation of Nrf2 and HXK2 by FGF7 upon MI. Together, these results indicate that the cardio protection of FGF7 under MI injury is mostly attributable to its role in maintaining redox homeostasis via Nrf2 and HXK2, which is mediated by PI3Kα/AKT signaling.

LGALS3BP in Microglia Promotes Retinal Angiogenesis Through PI3K/AKT Pathway During Hypoxia.

Purpose: Retinal microglia promote angiogenesis and vasculopathy in oxygen-induced retinopathy (OIR); however, its specific molecular mechanism in the formation of retinal angiogenesis remains unclear. The lectin galactoside-binding soluble 3 binding protein (LGALS3BP), a member of the scavenger receptor cysteine-rich (SRCR) domain protein family, is involved in tumor neovascularization, and we therefore hypothesized that LGALS3BP plays an active role in microglia-induced angiogenesis. Methods: The expression of LGALS3BP in microglia was detected by immunofluorescence, RT-qPCR, and western blotting. Functional assays of human umbilical vein endothelial cells (HUVECs) such as migration, proliferation, and tube formation were measured by Transwell, EdU, and Matrigel assays. Angiogenesis-related factors and PI3K/AKT levels were detected by western blotting. The relationship between LGALS3BP and PI3K or HIF-1α was investigated by immunoprecipitation. Results: Our results showed that the expression of LGALS3BP was significantly increased in microglia surrounding neovascularization of the OIR mice and was also upregulated in human microglial clone 3 (HMC3) cells after hypoxia. Moreover, HUVECs co-cultured with hypoxic HMC3 cells showed increased migration, proliferation, and tube formation, as well as levels of angiogenesis-related factor. However, the proangiogenic ability and angiogenesis-related factor expression of HMC3 cells was suppressed after silencing LGALS3BP. LGALS3BP induces the upregulation of angiogenesis-related factors through the PI3K/AKT pathway and then promotes angiogenesis in microglia. Conclusions: Collectively, our findings suggest that LGALS3BP in microglia plays an important role in angiogenesis, suggesting a potential therapeutic target of LGALS3BP for angiogenesis.

Baicalein Acts against Candida albicans by Targeting Eno1 and Inhibiting Glycolysis.

Baicalein (BE) is a promising antifungal small-molecule compound with an extended antifungal spectrum, good synergy with fluconazole, and low toxicity, but its target protein and antifungal mechanism remain elusive. In this study, we found that BE can function against Candida albicans by disrupting glycolysis through targeting Eno1 and inhibiting its function. Eno1 acts as a key therapeutic target of the drug, as BE had no antifungal activity against the eno1 null mutant in a Galleria mellonella model of C. albicans infection. To investigate the mechanism of action, we solved the crystal structure of C. albicans Eno1(CaEno1) and then compared the difference between this structure and that of Eno1 from humans. The predicted primary binding site of BE on CaEno1 is between amino acids D261 and W274, with D263, S269, and K273 playing critical roles in the interaction with BE. Both positions S269 and K273 have different residues in the human Eno1 (hEno1). This finding suggests that BE may bind selectively to CaEno1, which would limit the potential for side effects in humans. Our findings demonstrate that Eno1 is a target protein of BE and thus may serve as a novel target for the development of antifungal therapeutics acting through the inhibition of glycolysis. IMPORTANCE Baicalein (BE) is a promising antifungal agent which has been well characterized, but its target protein is still undiscovered. The protein Eno1 plays a crucial role in the survival of Candida albicans. However, there are few antifungal agents which inhibit the functions of Eno1. Here, we found that BE can function against Candida albicans by disrupting glycolysis through targeting Eno1 and inhibiting its function. We further solved the crystal structure of C. albicans Eno1(CaEno1) and predicted that the primary binding site of BE on CaEno1 is between amino acids D261 and W274, with D263, S269, and K273 playing critical roles in the interaction with BE. Our findings will be helpful to get specific small-molecule inhibitors of CaEno1 and open the way for the development of new antifungal therapeutics targeted at inhibiting glycolysis.

Multifactorial Mechanisms of Tolerance to Ketoconazole in Candida albicans.

Candida albicans is a prevalent opportunistic human fungal pathogen for which treatment is limited to only four main classes of antifungal drugs, with the azole and echinocandin classes being used most frequently. Drug tolerance, the ability of some cells to grow slowly in supra-MIC drug concentrations, decreases the number of available treatment options. Here, we investigated factors affecting tolerance and resistance to ketoconazole in C. albicans. We found both temperature and the composition of growth medium significantly affected tolerance with little effect on resistance. In deletion analysis of known efflux pump genes, CDR1 was partially required for azole tolerance, while CDR2 and MDR1 were dispensable. Tolerance also required Hsp90 and calcineurin components; CRZ1, which encodes a transcription factor downstream of calcineurin, was required only partially. Deletion of VMA11, which encodes a vacuolar ATPase subunit, and concanamycin A, a V-ATPase inhibitor, abolished tolerance, indicating the importance of vacuolar energy transactions in tolerance. Thus, tolerance to ketoconazole is regulated by multiple factors, including physiological and genetic mechanisms. IMPORTANCE Due to the ever-expanding range of invasive medical procedures and treatments, invasive fungal infections now pose a serious global threat to many people living in an immunocompromised status. Like humans, fungi are eukaryotic, which significantly limits the number of unique antifungal targets; the current arsenal of antifungal agents is limited to just three frontline drug classes. Additional treatment complexities result from the development of drug tolerance and resistance, which further narrows therapeutic options; however, the difference between tolerance and resistance remains largely unknown. This study demonstrates that tolerance and resistance are regulated by multiple genetic and physiological factors. It is prudent to note that some factors affect tolerance only, while other factors affect both tolerance and resistance. The complex underlying mechanisms of these drug responses are highlighted by the fact that there are both shared and distinct mechanisms that regulate tolerance and resistance.

The protective effects of fibroblast growth factor 10 against hepatic ischemia-reperfusion injury in mice.

Hepatic ischemia-reperfusion injury (IRI) is a major complication of liver surgery and transplantation. IRI leads to hepatic parenchymal cell death, resulting in liver failure, and lacks effective therapeutic approaches. Fibroblast growth factor 10 (FGF10) is a paracrine factor which is well-characterized with respect to its pro-proliferative effects during embryonic liver development and liver regeneration, but its role in hepatic IRI remains unknown. In this study, we investigated the role of FGF10 in liver IRI and identified signaling pathways regulated by FGF10. In a mouse model of warm liver IRI, FGF10 was highly expressed during the reperfusion phase. In vitro experiments demonstrated that FGF10 was primarily secreted by hepatic stellate cells and acted on hepatocytes. The role of FGF10 in liver IRI was further examined using adeno-associated virus-mediated gene silencing and overexpression. Overexpression of FGF10 alleviated liver dysfunction, reduced necrosis and inflammation, and protected hepatocytes from apoptosis in the early acute injury phase of IRI. Furthermore, in the late phase of IRI, FGF10 overexpression also promoted hepatocyte proliferation. Meanwhile, gene silencing of FGF10 had the opposite effect. Further studies revealed that overexpression of FGF10 activated nuclear factor-erythroid 2-related factor 2 (NRF2) and decreased oxidative stress, mainly through activation of the phosphatidylinositol-3-kinase/AKT pathway, and the protective effects of FGF10 overexpression were largely abrogated in NRF2 knockout mice. These results demonstrate the protective effects of FGF10 in liver IRI, and reveal the important role of NRF2 in FGF10-mediated hepatic protection during IRI.