Pitavastatin Calcium Confers Fungicidal Properties to Fluconazole by Inhibiting Ubiquinone Biosynthesis and Generating Reactive Oxygen Species.

Fluconazole (FLC) is extensively employed for the prophylaxis and treatment of invasive fungal infections (IFIs). However, the fungistatic nature of FLC renders pathogenic fungi capable of developing tolerance towards it. Consequently, converting FLC into a fungicidal agent using adjuvants assumes significance to circumvent FLC resistance and the perpetuation of fungal infections. This drug repurposing study has successfully identified pitavastatin calcium (PIT) as a promising adjuvant for enhancing the fungicidal activity of FLC from a comprehensive library of 2372 FDA-approved drugs. PIT could render FLC fungicidal even at concentrations as low as 1 µM. The median lethal dose (LD50) of PIT was determined to be 103.6 mg/kg. We have discovered that PIT achieves its synergistic effect by inhibiting the activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, thereby impeding ubiquinone biosynthesis, inducing reactive oxygen species (ROS) generation, triggering apoptosis, and disrupting Golgi function. We employed a Candida albicans strain that demonstrated a notable tolerance to FLC to infect mice and found that PIT effectively augmented the antifungal efficacy of FLC against IFIs. This study is an illustrative example of how FDA-approved drugs can effectively eliminate fungal tolerance to FLC.

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.

Icariin alleviates oxygen-induced retinopathy by targeting microglia hexokinase 2.

Retinopathy of prematurity (ROP) is a retinal disease-causing retinal neovascularization that can lead to blindness. Oxygen-induced retinopathy (OIR) is a widely used ROP animal model. Icariin (ICA) has anti-oxidative and anti-inflammation properties; however, whether ICA has a regulatory effect on OIR remains unclear. In this study, ICA alleviated pathological neovascularization, microglial activation and blood-retina barrier (BRB) damage in vivo. Further results indicated that endothelial cell tube formation, migration and proliferation were restored by ICA treatment in vitro. Proteomic microarrays and molecular mimicry revealed that ICA can directly bind to hexokinase 2 (HK2) and decrease HK2 protein expression in vivo and in vitro. In addition, ICA inhibited the AKT/mTOR/HIF1α pathway activation. The effects of ICA on pathological neovascularization, microglial activation and BRB damage disappeared after HK2 overexpression in vivo. Similarly, the endothelial cell function was revised after HK2 overexpression. HK2 overexpression reversed ICA-induced AKT/mTOR/HIF1α pathway inhibition in vivo and in vitro. Therefore, ICA prevented pathological angiogenesis in OIR in an HK2-dependent manner, implicating ICA as a potential therapeutic agent for ROP.

Natural Polymers - Based Hydrogel Dressings for Wound Healing.

SIGNIFICANCE: Acute wounds such as severe burns and chronic wounds like diabetic ulcers present a significant threat to human health. Wound dressings made from natural polymers offer inherent properties that effectively enhance wound healing outcomes and reduce healing time. RECENT ADVANCES: Numerous innovative hydrogels are being developed and translated to the clinic to successfully treat various wound types. This underscores the substantial potential of hydrogels in the future wound care market. Economically, annual sales of wound care products are projected to reach $15-22 billion by 2024. CRITICAL ISSUES: While chitosan-, cellulose-, and collagen-based hydrogel dressings are currently commercially available, scaling up and manufacturing hydrogels for commercial products remains a challenging process. Additionally, ensuring the sterility and stability of the chemical or biological components comprising the hydrogel are critical considerations. FUTURE DIRECTIONS: In light of the persistent increase in wound fatalities and the resulting economic and social impacts, as well as the importance of educating the public about dietary health and disease, there should be increased investment in new wound care dressings, particularly hydrogels derived from natural products. With numerous researchers dedicated to advancing preclinical hydrogels, the future holds promise for more innovative and more personalized hydrogel wound dressings.

YY1 Lactylation Aggravates Autoimmune Uveitis by Enhancing Microglial Functions via Inflammatory Genes.

Activated microglia in the retina are essential for the development of autoimmune uveitis. Yin-Yang 1 (YY1) is an important transcription factor that participates in multiple inflammatory and immune-mediated diseases. Here, an increased YY1 lactylation in retinal microglia within in the experimental autoimmune uveitis (EAU) group is observed. YY1 lactylation contributed to boosting microglial activation and promoting their proliferation and migration abilities. Inhibition of lactylation suppressed microglial activation and attenuated inflammation in EAU. Mechanistically, cleavage under targets & tagmentation ï¼ˆCUT&Tag） analysis revealed that YY1 lactylation promoted microglial activation by regulating the transcription of a set of inflammatory genes, including STAT3, CCL5, IRF1, IDO1, and SEMA4D. In addition, p300 is identified as the writer of YY1 lactylation. Inhibition of p300 decreased YY1 lactylation and suppressed microglial inflammation in vivo and in vitro. Collectively, the results showed that YY1 lactylation promoted microglial dysfunction in autoimmune uveitis by upregulating inflammatory cytokine secretion and boosting cell migration and proliferation. Therapeutic effects can be achieved by targeting the lactate/p300/YY1 lactylation/inflammatory genes axis.

OR11H1 Missense Variant Confers the Susceptibility to Vogt-Koyanagi-Harada Disease by Mediating Gadd45g Expression.

Vogt-Koyanagi-Harada (VKH) disease is a severe autoimmune disease. Herein, whole-exome sequencing (WES) study are performed on 2,573 controls and 229 VKH patients with follow-up next-generation sequencing (NGS) in a collection of 2,380 controls and 2,278 VKH patients. A rare c.188T>C (p Val63Ala) variant in the olfactory receptor 11H1 (OR11H1) gene is found to be significantly associated with VKH disease (rs71235604, Pcombined = 7.83 × 10-30 , odds ratio = 3.12). Functional study showes that OR11H1-A63 significantly increased inflammatory factors production and exacerbated barrier function damage. Further studies using RNA-sequencing find that OR11H1-A63 markedly increased growth arrest and DNA-damage-inducible gamma (GADD45G) expression. Moreover, OR11H1-A63 activates the MAPK and NF-κB pathways, and accelerates inflammatory cascades. In addition, inhibiting GADD45G alleviates inflammatory factor secretion, likely due to the regulatory effect of GADD45G on the MAPK and NF-κB pathways. Collectively, this study suggests that the OR11H1-A63 missense mutation may increase susceptibility to VKH disease in a GADD45G-dependent manner.

Unexpected Inhibitory Effect of Octenidine Dihydrochloride on Candida albicans Filamentation by Impairing Ergosterol Biosynthesis and Disrupting Cell Membrane Integrity.

Candida albicans filamentation plays a significant role in developing both mucosal and invasive candidiasis, making it a crucial virulence factor. Consequently, exploring and identifying inhibitors that impede fungal hyphal formation presents an intriguing approach toward antifungal strategies. In line with this anti-filamentation strategy, we conducted a comprehensive screening of a library of FDA-approved drugs to identify compounds that possess inhibitory properties against hyphal growth. The compound octenidine dihydrochloride (OCT) exhibits potent inhibition of hyphal growth in C. albicans across different hyphae-inducing media at concentrations below or equal to 3.125 µM. This remarkable inhibitory effect extends to biofilm formation and the disruption of mature biofilm. The mechanism underlying OCT's inhibition of hyphal growth is likely attributed to its capacity to impede ergosterol biosynthesis and induce the generation of reactive oxygen species (ROS), compromising the integrity of the cell membrane. Furthermore, it has been observed that OCT demonstrates protective attributes against invasive candidiasis in Galleria mellonella larvae through its proficient eradication of C. albicans colonization in infected G. mellonella larvae by impeding hyphal formation. Although additional investigation is required to mitigate the toxicity of OCT in mammals, it possesses considerable promise as a potent filamentation inhibitor against invasive candidiasis.

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.

Global lactylome reveals lactylation-dependent mechanisms underlying TH17 differentiation in experimental autoimmune uveitis.

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 TH17 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 Lys164 promoted TH17 differentiation by directly modulating the expression of TH17-related genes, including Runx1, Tlr4, interleukin-2 (IL-2), and IL-4. Delactylation of Ikzf1 at Lys164 impaired TH17 differentiation. These findings exemplify how glycolysis regulates the site specificity of protein lactylation to promote TH17 differentiation and implicate Ikzf1 lactylation as a potential therapeutic target for autoimmune diseases.

FTO-mediated m6A modification alleviates autoimmune uveitis by regulating microglia phenotypes via the GPC4/TLR4/NF-κB signaling axis.

Uveitis, a vision-threatening inflammatory disease worldwide, is closely related to resident microglia. Retinal microglia are the main immune effector cells with strong plasticity, but their role in uveitis remains unclear. N6-methyladenosine (m6A) modification has been proven to be involved in the immune response. Therefore, we in this work aimed to identify the potentially crucial m6A regulators of microglia in uveitis. Through the single-cell sequencing (scRNA-seq) analysis and experimental verification, we found a significant decrease in the expression of fat mass and obesity-associated protein (FTO) in retinal microglia of uveitis mice and human microglia clone 3 (HMC3) cells with inflammation. Additionally, FTO knockdown was found to aggravate the secretion of inflammatory factors and the mobility/chemotaxis of microglia. Mechanistically, the RNA-seq data and rescue experiments showed that glypican 4 (GPC4) was the target of FTO, which regulated microglial inflammation mediated by the TLR4/NF-κB pathway. Moreover, RNA stability assays indicated that GPC4 upregulation was mainly regulated by the downregulation of the m6A "reader" YTH domain family protein 3 (YTHDF3). Finally, the FTO inhibitor FB23-2 further exacerbated experimental autoimmune uveitis (EAU) inflammation by promoting the GPC4/TLR4/NF-κB signaling axis, and this could be attenuated by the TLR4 inhibitor TAK-242. Collectively, a decreased FTO could facilitate microglial inflammation in EAU, suggesting that the restoration or activation of FTO function may be a potential therapeutic strategy for uveitis.

Apigenin Alleviates Autoimmune Uveitis by Inhibiting Microglia M1 Pro-Inflammatory Polarization.

Purpose: Apigenin is a natural small molecule compound widely present in various vegetables and fruits. Recently, Apigenin was reported to inhibit lipopolysaccharide (LPS)-simulated microglial proinflammatory activation. Considering the important role of microglia in retinal disorders, we wonder whether Apigenin could exert a therapeutic effect on experimental autoimmune uveitis (EAU) through reprogramming retinal microglia to a beneficial subtype. Methods: EAU was induced in C57BL/6J mice by immunization with interphotoreceptor retinoid-binding protein (IRBP)651-670, followed by intraperitoneal administration of Apigenin. Disease severity was assessed based on clinical and pathological scores. In vivo, Western blotting was used to quantify protein levels of classical inflammatory factors, microglial M1/M2 markers and the tight junction protein of the blood-retinal-barrier (BRB). Immunofluorescence was used to determine the Apigenin's efficacy on microglial phenotype. In vitro, Apigenin was added in LPS and IFN-γ stimulated human microglial cell line. Western blotting and Transwell assays were used to analyze the phenotype of microglia. Results: In vivo, we found that Apigenin significantly reduced the clinical and pathological scores of EAU. The protein levels of inflammatory cytokines were significantly decreased in retina, and BRB disruption was ameliorated after Apigenin treatment. Meanwhile, Apigenin inhibited microglia M1 transition in EAU mice retina. In vitro functional studies showed that Apigenin decreased LPS and IFN-γ-induced microglial inflammatory factor production and M1-activation via the TLR4/MyD88 pathway. Conclusions: Apigenin can ameliorate retinal inflammation in IRBP induced autoimmune uveitis through inhibiting microglia M1 pro-inflammatory polarization via TLR4/MyD88 pathway.

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 18 alleviates stress-induced pathological cardiac hypertrophy in male mice.

Fibroblast growth factor-18 (FGF18) has diverse organ development and damage repair roles. However, its role in cardiac homeostasis following hypertrophic stimulation remains unknown. Here we investigate the regulation and function of the FGF18 in pressure overload (PO)-induced pathological cardiac hypertrophy. FGF18 heterozygous (Fgf18+/-) and inducible cardiomyocyte-specific FGF18 knockout (Fgf18-CKO) male mice exposed to transverse aortic constriction (TAC) demonstrate exacerbated pathological cardiac hypertrophy with increased oxidative stress, cardiomyocyte death, fibrosis, and dysfunction. In contrast, cardiac-specific overexpression of FGF18 alleviates hypertrophy, decreased oxidative stress, attenuates cardiomyocyte apoptosis, and ameliorates fibrosis and cardiac function. Tyrosine-protein kinase FYN (FYN), the downstream factor of FGF18, was identified by bioinformatics analysis, LC-MS/MS and experiment validation. Mechanistic studies indicate that FGF18/FGFR3 promote FYN activity and expression and negatively regulate NADPH oxidase 4 (NOX4), thereby inhibiting reactive oxygen species (ROS) generation and alleviating pathological cardiac hypertrophy. This study uncovered the previously unknown cardioprotective effect of FGF18 mediated by the maintenance of redox homeostasis through the FYN/NOX4 signaling axis in male mice, suggesting a promising therapeutic target for the treatment of cardiac hypertrophy.

iPSC-based model of Vogt-Koyanagi-Harada disease for phenotype recapitulation and drug screening.

Vogt-Koyanagi-Harada (VKH) disease, a major blinding eye disease, is characterized by an autoimmune response against melanocytes in multiple organs throughout the body. Currently, the aetiology and pathogenesis of VKH disease are unclear, and the treatment strategy needs to be further optimized. The retinal pigment epithelium (RPE), a monolayer of pigmented cells of the fundus, is essential for maintaining normal visual function and is involved in both the acute and chronic stages of VKH disease. Therefore, the functions of the RPE may play a critical role in the aetiology and treatment of VKH disease. Herein, we established a human induced pluripotent stem cell (hiPSC) RPE model of VKH disease by reprogramming peripheral blood mononuclear cells (PBMCs) into iPSCs and then differentiating them into RPE cells. Patient-derived RPE cells exhibited barrier disruption, impaired phagocytosis, and depigmentation compared with those from normal controls, which was consistent with the features of VKH disease. Furthermore, a small molecular compound targeting EGR2 was found to rescue the barrier and phagocytic functions of the hiPSC-RPE cells through high-throughput virtual screening and functional studies, suggesting a promising strategy for the treatment of VKH disease.

A Small Molecule Inhibitor of Erg251 Makes Fluconazole Fungicidal by Inhibiting the Synthesis of the 14α-Methylsterols.

Fluconazole (FLC) is widely used to prevent and treat invasive fungal infections. However, FLC is a fungistatic agent, allowing clinical FLC-susceptible isolates to tolerate FLC. Making FLC fungicidal in combination with adjuvants is a promising strategy to avoid FLC resistance and eliminate the persistence and recurrence of fungal infections. Here, we identify a new small molecule compound, CZ66, that can make FLC fungicidal. The mechanism of action of CZ66 is targeting the C-4 sterol methyl oxidase, encoded by the ERG251 gene, resulting in decreased content of sterols with the 14α-methyl group and ultimately eliminating FLC tolerance of Candida albicans. CZ66 most likely interacts with Erg251 through residues Glu195, Gly206, and Arg241. Establishing Erg251 as a synergistic lethal target protein of FLC should direct research to identify specific small molecule inhibitors of 14α-methylsterol synthesis and open the way to abolishing fungal FLC tolerance. IMPORTANCE Fluconazole (FLC) tolerance increases the frequency of acquired FLC resistance, and a high FLC tolerance level is associated with persistent candidemia. Multiple functional proteins, such as calcineurin, heat shock protein 90 (Hsp90), and ADP ribosylation factor, are essential for the survival of C. albicans exposed to FLC, but how these factors increase the fungicidal activity of FLC remains to be determined. In this study, we found that 14α-methylsterols replace ergosterol to allow C. albicans to survive FLC, but Erg251 inactivated by CZ66 results in loss of 14α-methylsterol synthesis and cell death of C. albicans treated with FLC. Establishing Erg251 as a synergistic lethal target protein of FLC should direct research to identify specific small molecule inhibitors of 14α-methylsterol synthesis and open the way to abolishing fungal FLC tolerance.

SOX2-positive retinal stem cells are identified in adult human pars plicata by single-cell transcriptomic analyses.

Stem cell therapy is a promising strategy to rescue visual impairment caused by retinal degeneration. Previous studies have proposed controversial theories about whether in situ retinal stem cells (RSCs) are present in adult human eye tissue. Single-cell RNA sequencing (scRNA-seq) has emerged as one of the most powerful tools to reveal the heterogeneity of tissue cells. By using scRNA-seq, we explored the cell heterogeneity of different subregions of adult human eyes, including pars plicata, pars plana, retinal pigment epithelium (RPE), iris, and neural retina (NR). We identified one subpopulation expressing SRY-box transcription factor 2 (SOX2) as RSCs, which were present in the pars plicata of the adult human eye. Further analysis showed the identified subpopulation of RSCs expressed specific markers aquaporin 1 (AQP1) and tetraspanin 12 (TSPAN12). We, therefore, isolated this subpopulation using these two markers by flow sorting and found that the isolated RSCs could proliferate and differentiate into some retinal cell types, including photoreceptors, neurons, RPE cells, microglia, astrocytes, horizontal cells, bipolar cells, and ganglion cells; whereas, AQP1- TSPAN12- cells did not have this differentiation potential. In conclusion, our results showed that SOX2-positive RSCs are present in the pars plicata and may be valuable for treating human retinal diseases due to their proliferation and differentiation potential.

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.

Fibroblast growth factor 20 attenuates pathological cardiac hypertrophy by activating the SIRT1 signaling pathway.

Cardiac hypertrophy occurs initially in response to an increased cardiac load as a compensatory mechanism to maintain cardiac output. However, sustained pathological hypertrophy can develop into heart failure and cause sudden death. Fibroblast growth factor 20 (FGF20) is a member of the fibroblast growth factor family, which involved in apoptosis, aging, inflammation, and autophagy. The precise function of FGF20 in pathological cardiac hypertrophy is unclear. In this study, we demonstrated that FGF20 was significantly decreased in response to hypertrophic stimulation. In contrast, overexpression of FGF20 protected against pressure overload-induced cardiac hypertrophy. Mechanistically, we found that FGF20 upregulates SIRT1 expression, causing deacetylation of FOXO1; this effect promotes the transcription of downstream antioxidant genes, thus inhibits oxidative stress. In content, the anti-hypertrophic effect of FGF20 was largely counteracted in SIRT1-knockout mice, accompanied by an increase in oxidative stress. In summary, our findings reveal a previously unknown protective effect of FGF20 on pathological cardiac hypertrophy by reducing oxidative stress through activation of the SIRT1 signaling pathway. FGF20 is a potential novel molecular target for preventing and treating pressure overload-induced myocardial injury.