Free Radical Biology and Medicine

Reactive oxygen species (ROS) are central signaling molecules in many biological processes by inducing oxidative modifications of protein cysteine residues, including S-glutathionylation. Increasing evidence supports that ROS contribute to cancer progression via promoting cancer cell migration, invasion, and metastasis. Nevertheless, the protein targets of S-glutathionylation that regulate cancer cell motility remain ill-defined. In this study, we report on the redox regulation of ARHGEF7, a guanine nucleotide exchange factor highly expressed in metastatic cancer cells, that plays a major role in regulating cell migration. Our data demonstrates that ARHGEF7 is selectively glutathionylated at the highly conserved C312 residue in its PH domain, which is implicated in regulating its enzymatic activity. Breast cancer cell lines showed increased cell migration and invasion upon glutathionylation of ARHGEF7 at C312 in response to both oxidative stress and epidermal growth factor (EGF). Mechanistically, upon C312 glutathionylation, ARHGEF7 exhibited significantly enhanced binding to Rac1 and increased Rac1 recruitment to the cell membrane and lamellipodia. ARHGEF7 S-glutathionylation also increased its enzymatic rate of GDP-GTP nucleotide exchange, resulting in Rac1 activation. Consequently, ARHGEF7 C312 S-glutathionylation induced Rac1-PAK1 activation and their downstream pathways, including LIMK1 and MEK1, thereby enhancing migration and invasion. Our data reveal a new redox player in cell migration, with its potential implications for ROS-induced cancer progression.

Rhodiola rosea is a traditional medicinal plant often classified as an adaptogen, with reported effects in supporting the bodys response to physical, environmental, and emotional stressors. The present study investigated the antioxidant properties of Rhodiola rosea extract and its major chemical constituents to provide insight into their potential mechanisms of action. Through in vitro biochemical assays, we demonstrated that Rhodiola rosea extract has the capacity to reduce hydrogen peroxide (H2O2) levels. Among its primary chemical components, rosavin significantly decreased H2O2, whereas salidroside had no effect. Neither compound affected superoxide levels. Structural analysis revealed that the intact phenylpropanoid glycoside architecture of rosavin is required for activity, as its individual components, arabinose and rosin, showed no inhibitory effect. Further investigation demonstrated that rosavin attenuates H2O2-mediated oxidation of thiol groups, supporting a role in cellular redox regulation. In cultured human cells, rosavin mitigated reductions in cell viability induced by exposure to H2O2, indicating cytoprotective effects under oxidative stress conditions. Finally, in an in vivo model, administration of SARS-CoV-2 spike protein increased circulating levels of H2O2, which were subsequently reduced following rosavin treatment. Collectively, these findings identify rosavin as a structurally dependent antioxidant component of Rhodiola rosea that modulates H2O2-associated oxidative stress and supports further investigation of phenylpropanoid glycosides as adaptogens.

ObjectiveN-acetylcysteine (NAC) is a clinically available antioxidant with potential applications in trauma-induced hypermetabolic states, including burn injury and crush syndrome. However, its effects on heat-stressed skeletal muscle cells remain incompletely characterized. This study conducted a secondary analysis of a publicly available dataset to quantify NACs protective effects against heat-stress-induced cellular damage. MethodsWe re-analyzed a publicly available dataset (Lu J, 2024, Mendeley Data, doi:10.17632/wffrtcgbnx.1) containing 21 observations across three conditions: Control (n=3), Heat Stress only (HS, n=3), and HS with NAC at five doses (0.5-8.0 mM, n=3 per dose). The primary outcome was the protective ratio [(HS+NAC - HS) / (Control - HS)], where 1.0 indicates complete protection. Statistical analyses included one-way ANOVA, post-hoc t-tests with Bonferroni correction, Cohens d effect sizes, and bootstrap confidence intervals. ResultsHeat stress significantly reduced cell viability by 56.3% (Control: 100.0 {+/-} 12.2 vs HS: 43.7 {+/-} 5.1; t(4)=7.37, p=0.002, Cohens d=6.02). NAC demonstrated a biphasic dose-response with maximal protection at 2.0 mM (66.7 {+/-} 14.4), yielding a protective ratio of 0.409 (95% CI: 0.146-0.675), representing 40.9% protection against heat stress damage. The comparison between HS and HS+NAC (2.0 mM) showed a large effect size (Cohens d = 2.12) but did not reach statistical significance (p = 0.060) due to the small sample size. One-way ANOVA confirmed overall group differences (F(2,18)=32.39, p<0.001, 2=0.783). ConclusionsNAC provides partial protection against heat stress-induced skeletal muscle cell damage at 2.0 mM, with a large effect size suggesting clinical relevance despite limited statistical power. These preliminary findings support further investigation of NAC as an adjunct therapy in trauma-induced hypermetabolic states. All analysis code is provided for reproducibility.

Mitochondrial morphology and function are critical determinants of neuronal function and survival, with disruptions in mitochondrial dynamics often preceding the overt neuronal dysfunction seen in neurodegenerative diseases such as Alzheimers disease, Huntingtons disease and Parkinsons disease. The kynurenine pathway accounts for 95% of dietary tryptophan catabolism and many of the metabolites are neuroactive, including redox-active 3-hydroxykynurenine (3-HK). 3-HK is present under normal physiological conditions in the central nervous system (CNS) and is elevated during inflammation. While supraphysiological levels of 3-HK have been associated with neurotoxicity, the effects of physiological concentrations on neuronal cells, and specifically their mitochondria, remain poorly understood. Here we assessed viability, ATP levels and redox status to determine cellular health and function in neuronal cells exposed to physiological levels of 3-HK, alongside confocal imaging and transcriptomic profiling, finding significant alterations in mitochondrial function and morphology. Interestingly, a biphasic influence of 3-HK on mitochondrial morphology was observed, with an elongated network as well as decreased surface area and volume being observed only at the lowest concentration of 3-HK, reflecting normal physiological levels. At the highest 3-HK concentration tested, reflecting an inflammatory situation, an increased number of mitochondria were present, accompanied by increased activation of caspase-3/7 and enhanced production of mitochondrial superoxide. These results highlight a previously unknown role for 3-HK in regulating mitochondrial function and structure, possibly through altered fission and fusion events, suggesting that subtle changes in kynurenine pathway metabolism may contribute to early mitochondrial dysfunction in neurological disease.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a mitochondrial lipid storage myopathy characterized by impaired fatty acid {beta}-oxidation, mitochondrial dysfunction, and progressive neuromuscular and cardiac disease. MADD is most commonly caused by pathogenic variants in electron transfer flavoprotein dehydrogenase (ETFDH), which encodes electron transfer flavoprotein-ubiquinone oxidoreductase (Etf-QO), a critical redox enzyme that transfers electrons from acyl-CoA dehydrogenases to the mitochondrial electron transport chain. Defective Etf-QO activity disrupts electron flow, promotes reactive oxygen species (ROS) production, and impairs cellular energy metabolism, linking abnormal lipid oxidation to oxidative stress-mediated tissue damage. To investigate the role of redox imbalance in MADD pathogenesis, we generated CRISPR/Cas9 knock-in Drosophila melanogaster models carrying patient-relevant Etf-QO missense mutations (L127R, S296C, and L399F; corresponding to human L138R, S307C, and L409F) within conserved FAD- and ubiquinone-binding domains. Mutant flies developed progressive locomotor impairment, reduced muscle performance, and marked lipid droplet accumulation in skeletal muscle, cardiac tissue, and fat bodies, indicating systemic defects in mitochondrial lipid utilization. Cardiac analyses demonstrated reduced fractional shortening, prolonged heart period, and increased arrhythmia index, consistent with metabolic cardiomyopathy associated with mitochondrial oxidative stress. In vivo respirometry revealed significantly decreased oxygen consumption, reflecting impaired oxidative phosphorylation. At the molecular level, mutant flies exhibited elevated ROS levels and ATP depletion, accompanied by increased expression of AMPK, PGC-1, and Tfam, suggesting activation of energy stress signaling and compensatory mitochondrial biogenesis. Importantly, endurance exercise significantly improved locomotor and cardiac function while reducing lipid accumulation and oxidative stress. Together, these findings establish a redox-centered in vivo model of MADD and identify oxidative stress as a major driver of disease pathology and a potential therapeutic target.

Objective Fatty Acid esters of Hydroxy-Fatty Acids (FAHFAs) are anti-diabetic and anti-inflammatory lipokines produced mainly by adipose tissue (AT). As exercise training enhances FAHFA levels, we investigated the impact of acute exercise (AE) and exercise-mimicking conditions on circulating and adipocyte FAHFA levels. Methods Clinical trial (NCT05572905) in 60 women, grouped by BMI (lean vs. obese) and age (young vs. older), was combined with in vitro experiments on human adipocytes. Following baseline characterization (body composition, VO2max, insulin sensitivity, AT/plasma FAHFAs), women underwent a cross-over AE and control interventions with repeated blood sampling for FAHFA analysis. Results In AT, lean and older women exhibited higher FAHFA levels than obese and young women, respectively; older women also showed a shift toward higher levels of 13/12-carbon-branched FAHFAs. Circulating FAHFA levels were similar across all groups and were not positively associated with insulin sensitivity, VO2max or FAHFA levels in AT. Although AE increased circulating free fatty acids (FFA), plasma FAHFAs dropped in response to both AE and control interventions. In adipocytes, FAHFAs were unaffected by glucocorticoids but increased in response to lipolysis together with gene expression related to FFA oxidation (FAO). Nevertheless, blocking mitochondrial FAO partially mimicked the lipolytic effect, while peroxisomal inhibition synergistically boosted FAHFA lipolysis-driven production despite having no effect alone. Conclusions While adiposity and aging modulate FAHFA levels in AT, circulating levels remain stable and unaffected by AE, challenging subcutaneous AT as their primary systemic source. In vitro, FAHFA synthesis is driven by high FFA availability but limited by competing peroxisomal FAO.

Catechinopyranocyanidins (Cpcs) which consist of diastereomers A and B are pigments derived from adzuki beans and are compounds in which the catechin and cyanidin skeletons are condensed to a pyrano ring. While catechins and anthocyanidins possess high antioxidant capacity, the physiological functions of Cpcs remains unclear. In this study, the antioxidant capacity of Cpcs was evaluated by in vitro antioxidant assays and by assessing their cytoprotective activity against oxidative stress in normal human dermal fibroblasts (NHDFs). Antioxidant capacity based on the hydrogen atom transfer (HAT) mechanism, as assessed by the ORAC assay revealed that Cpcs exhibit 14.1 mol TE/mol (Trolox equivalent antioxidant capacity: TEAC). Meanwhile, capacity based on the single electron transfer (SET) mechanism, as assessed by the DPPH, ABTS and CUPRAC assays revealed, they exhibit 2.1-3.6 mol TE/mol. Since TEAC value of Cpcs demonstrated by the HAT based mechanism higher than its SET based oxidative capacity suggesting that the antioxidant capacity of Cpcs is driven by the HAT mechanism. In cell culture experiments, Cpcs ameliorate cell toxicity in rotenone-induced injury model, suggesting to cytoprotective activity against mitochondrial dysfunction-dependent apoptosis. These results reveal novel physiological functions of Cpcs which may serve as a design guideline for elucidating in vivo dynamics based on antioxidant mechanisms.

Rhodoquinone (RQ) is a recently discovered component of the mammalian electron transport chain (ETC) with a high degree of tissue-specificity. Currently, a lack of pure analytical standards limits efforts to precisely quantify its levels using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and interrogate its biochemical functions within mammalian ETC complexes. Here, rhodoquinone-9 (RQ-9) and rhodoquinone-10 (RQ-10), and their isomeric by-products isorhodoquinone-9 (isoRQ-9) and isorhodoquinone-10 (isoRQ-10), were synthesized from ubiquinone-9 and ubiquinone-10 starting materials. Isomers were separated and purified by flash chromatography and structurally confirmed with nuclear magnetic resonance (NMR) spectroscopy. The chromatographic and fragmentation patterns of both the oxidized and reduced forms of these electron carriers were further characterized by LC-MS/MS, establishing signatures for their confident identification in lipidomics studies. LC-MS/MS analysis of murine kidney tissue with RQ-9 analytical standard spike-in corroborate the identity of the endogenous murine RQ-9 and enable absolute quantification of its levels. Thus, we synthesized and purified RQ-9 and RQ-10 analytical standards that will enable absolute quantification in mammalian tissues and in vitro reconstitution studies on RQ-9 and RQ-10 in the mammalian ETC.

BackgroundCalcific aortic valve disease (CAVD) is the most common valvular heart disease in developed countries, yet no pharmacological therapy is available to slow or halt its progression. CAVD is driven by progressive calcification of aortic valve leaflets, in which myeloid cells play a central role. While macrophages have been implicated in CAVD pathogenesis, the contribution of their precursors, monocytes, remains poorly understood. We hypothesized that circulating monocytes acquire a pro-calcific and pro-inflammatory phenotype contributing to valve remodelling and CAVD progression. MethodsWe profiled circulating CD14+ monocytes from healthy volunteers (Vol), patients with CAVD, and without CAVD (NCAVD). Peripheral blood mononuclear cells (PBMCs) were isolated, and monocyte subpopulations were phenotyped by flow cytometry. Transcriptome profiling by RNA sequencing identified disease-associated gene signatures, which were validated by RT-qPCR. The CD14+ monocyte secretome was analysed using multiplex assays. Functional ability of CAVD-derived CD14+ monocytes to induce myofibroblastic transdifferentiation (MT) and osteoblastic differentiation (OD) of human valvular interstitial cells (VICS) was evaluated by immunocytochemistry and quantitative o-cresolphthalein complexone assays. ResultsIn PBMCs, CAVD monocytes displayed a subpopulation shift, with an increased proportion of CD14CD16- classical monocytes and a reduced CD14CD16 non-classical monocyte levels. In CD14+ monocytes, transcriptomic analysis revealed upregulation of inflammation-related (PDK4) and calcification-related (ATP2B1) genes, alongside downregulation of immunomodulatory genes (DDR1, IKBKE). Secretome analysis showed reduced production of immunomodulatory and anti-osteoblastogenic cytokines (IL-4, CCL3) while promoting gene expression of factors promoting MT and OD in VICS. These alterations were associated with a marked monocyte-induced increase in SMA and OPN expression in VICS and a two-fold increase in calcification. ConclusionWe demonstrate for the first time that circulating monocytes from patients with CAVD exhibit enhanced pro-inflammatory and pro-calcific properties that may contribute to CAVD progression. Additionally, we identify dysregulated gene sets within these monocytes that represent potential novel therapeutic targets for CAVD.

Background and AimsSteatotic liver disease (SLD) is characterized by excessive lipid accumulation in hepatocytes, and alcohol consumption may modify the disease course, but the evidence is inclusive. This systematic review and meta-analysis aimed to holistically evaluate the impact of mild, moderate, and high levels of alcohol consumption on hepatic and extrahepatic outcomes in SLD. MethodsWe systematically searched EMBASE, MEDLINE, Web of Science, and the Cochrane Central Register of Controlled Trials for relevant studies. The study outcomes included liver related events, malignancy, mortality and cardiovascular disease among adults with SLD who consumed alcohol. ResultsOf 2228 records identified, twenty-six studies comprising 466611 adults with SLD were included. High alcohol consumption was associated with an increased risk of liver-related events compared with abstinence (2.97, 95% CI 1.61-5.50; p<0.001), and a similar association was observed among alcohol drinkers overall (HR 1.93, 95% CI 1.60-2.33; p<0.001). Moderate alcohol consumption was associated with a higher incidence of malignancy (HR 1.41, 95% CI 1.13-1.78; p=0.677). In contrast, mild alcohol consumption was associated with lower all-cause mortality compared with abstinence (HR 0.88, 95% CI 0.78-0.98; p=0.001). No association was observed between alcohol consumption and cardiovascular disease incidence or hepatocellular carcinoma ConclusionsAlcohol intake may increase the risk of liver-related complications and cancer risk in individuals with SLD. Mild alcohol consumption was associated with lower all-cause mortality, and alcohol intake showed no association with cardiovascular disease incidence. Further studies are needed to clarify the dose-dependent effects of alcohol on hepatic and extrahepatic outcomes in SLD.

O2-tolerance is a desirable property for [FeFe] hydrogenases, which are highly efficient H2-producing catalysts. While most such enzymes are highly sensitive to aerobic environments, a small number of explored representatives exhibit exceptional stability and even H2-producing activity under oxygenic conditions. However, the genetic signatures of the O2-tolerance in this class of enzymes remain largely unknown. To address this knowledge gap, we explored a close homologue of a well-characterized O2-tolerant [FeFe] hydrogenase from Clostridium beijerinckii (CbHydA1) - a hydrogenase from Terrisporobacter glycolicus (TgHydA1). Our investigation indeed confirms that TgHydA1 can transition to the O2-stable Hinact state, a hallmark of O2 tolerance. The surprising outcome is that despite the high amino acid similarity, TgHydA1 shows a substantially higher propensity to remain in the Hinact state than CbHydA1. Using protein film electrochemical experiments, we demonstrate that the root of this behavior lies in roughly tenfold slower reactivation rates than those of CbHydA1 at any applied potential. This degree and direction of variation in reactivation kinetics have not been observed before for any other O2-tolerant [FeFe] hydrogenases or their variants to date, uncovering a yet-to-be-explored facet of reactivity alteration available to these enzymes. Overall, the results presented here highlight the importance of a holistic analysis of [FeFe] hydrogenase sequences in the context of their interaction with O2 that encompasses the protein environment and properties of the auxiliary metallocofactors.

Punicalagin, an ellagic acid polyphenol from pomegranate, has been proposed as an antagonist of protein disulfide isomerase (PDI) and endoplasmic reticulum resident protein 57 (ERp57), thiol oxidoreductases that regulate protein folding and extracellular thrombotic signaling. Here, biochemical oxidase and reductase assays on PDI show that punicalagin inhibits both activities with micromolar potency, thereby extending earlier work that described only disulfide reductase inhibition. In parallel, thiol labeling of catalytic cysteines revealed no change in the redox state, supporting a noncovalent, allosteric of inhibition. Molecular docking and molecular dynamics simulations showed that punicalagin binds stably and preferentially to defined sites on the Nterminal domains of PDI through extensive hydrogen bonding and van der Waals contacts, which is an alternative binding mode to previously reported C-terminal binding. Finally, artificial intelligence-driven network analysis identified PDI as a high-confidence target of punicalagin and related galloylated polyphenols, alongside additional signaling proteins. Together, these findings provide further mechanistic framework for punicalagin-mediated antagonism of PDI and highlight galloylated polyphenols as promising scaffolds for protein disulfide isomerase-targeted therapeutics. HighlightsO_LIPunicalagin, a galloylated polyphenol, antagonizes not only the reductase activity but also the oxidase activity of protein disulfide isomerase C_LIO_LIProtein disulfide isomerase inhibition by punicalagin is through N-terminal binding C_LIO_LIPunicalagin inhibits conformationally rather than catalytic cysteine modification C_LIO_LIArtificial intelligence network analysis reveals pathway inhibition by punicalagin C_LI

BackgroundThe ketogenic diet is being explored as an adjuvant intervention in breast cancer because it lowers circulating glucose and elevates ketone bodies such as {beta}-hydroxybutyrate (BHB), but how individual ER+ breast cancer subtypes adapt to these conditions remains poorly characterized. We examined metabolic responses to BHB supplementation under glucose restriction in two ER+ breast cancer cell lines, asking whether metabolic adaptation patterns differ between models. MethodsMCF-7 and T47D cells were cultured under high glucose, glucose-restricted (5% of standard), or glucose-restricted with 10 mM BHB conditions and profiled by comprehensive two-dimensional gas chromatography-mass spectrometry (GCxGC-MS). Pairwise Welchs t-tests with Benjamini-Hochberg false discovery rate (FDR) correction were applied to identify treatment-responsive metabolites. Targeted assays quantified intracellular glycine, SHMT1 protein, and total branched-chain amino acid (BCAA) concentrations across a BHB dose range (2.5-15 mM). Patient tumor transcriptomic data from TCGA (n=1,084) and paired tumor-normal samples from GSE58135 (n=20) were analyzed for genes involved in one-carbon, ketone body, and BCAA metabolism. ResultsMCF-7 and T47D cells exhibited markedly divergent metabolic responses to BHB. In MCF-7 cells, BHB supplementation produced a broad pattern-level metabolic shift: 75% of detected metabolites trended upward when BHB was added to glucose-restricted cultures (C vs. B comparison), with 1,4-butanediol reaching nominal significance (FC=2.35, p=0.016) and a 4.1-fold trend increase in lactic acid (p=0.11), although no individual metabolite survived FDR correction. T47D cells showed essentially no metabolic response to BHB at the global level. Targeted assays detected an elevation in glycine at 5 mM BHB in both cell lines that did not follow a monotonic dose response and was not accompanied by changes in SHMT1 protein expression. Total BCAA levels were elevated by BHB in T47D cells but remained unchanged in MCF-7 cells. In paired patient samples, OXCT1 (log2FC = -1.41), SHMT1 (log2FC = -1.31), and ACAT1 (log2FC = -1.07) were significantly downregulated in ER+ tumors relative to matched normal tissue (adjusted p < 0.001 for all three). ConclusionsER+ breast cancer cell lines show heterogeneous metabolic responses to BHB supplementation under glucose restriction. The broad pattern of metabolite elevation in MCF-7 but not T47D cells suggests that capacity to utilize ketone bodies as metabolic substrate varies between ER+ models. The downregulation of OXCT1, ACAT1, and SHMT1 in ER+ tumors compared to normal tissue identifies these enzymes as candidate biomarkers that may help stratify which patients are likely to benefit from ketogenic interventions. Findings related to individual metabolites should be regarded as exploratory and require validation in larger, adequately powered cohorts.

Age-related macular degeneration (AMD) is a progressive complex eye disease and one of the leading causes of blindness. AMD progression is marked by molecular changes in the retinal pigmented epithelium (RPE) which include increased reactive oxygen species (ROS) accumulation, mitochondrial dysfunction - eventually leading to dysfunctional RPE. Mitophagy regulator, Pink1, is reduced in the RPE of AMD patients and Pink1 loss leads to a shift from mitochondrial respiration to glycolysis. Serine is a non-essential amino acid which is de novo synthesized from glycolytic intermediate 3-PG via the rate limiting enzyme PHGDH. Serine is tightly integrated into anabolic processes like glutathione (GSH) cycling, maintaining NADH/NADPH pools leading to changes in AMPK signaling. Here, we show that Pink1 loss leads to a reduction in PHGDH and serine levels in the RPE leading to impaired mitochondrial structure and function, increased ROS mediated damage, increased inflammation, and hampered retinal function. Serine supplementation rescued ROS accumulation, balanced GSH abundance, and increased retinal function. Overall, our study highlights the potential of dietary serine in ROS management in AMD.

Aortic dissection is life-threatening due to continued loss of medial integrity that may culminate in secondary rupture within hours to days. While pre-existing defects or hemodynamic loads compound structural deterioration of the aorta, pathological progression from symptomatic dissection channel to lethal transmural tear is poorly understood. We examined the structure of referent and acutely dissected ascending aortas by microscopy. Elastic, collagen, and cellular components of non-dissected media were intricately interconnected. Medial damage in dissection lesions was traced from ingress to central to peripheral areas. Entry tears broke cleanly through successive laminae leading to cavernous false lumens in which medial structure was destroyed. Nearby laminae with widening between flanking elastic lamellae (termed minor delaminations) were filled with blood and showed severe medial damage. Farther laminae without delamination but containing red blood cells (termed blood extravasation) displayed moderate medial damage. More distant, non-delaminated laminae with accumulation of albumin but not red blood cells (termed plasma extravasation) exhibited mild medial damage. Varying medial hemorrhage with scattered sloughing of laminae was observed along the entire false lumen. We conclude that hydraulic fracturing of residual dissected media by pressurized blood via communications from the false lumen contributes to further structural weakening of the aortic wall.

Glycyrrhiza uralensis is a widely used medicinal plant present in more than 70% of Kampo formulations in Japan owing to its diverse pharmacological activities, including immunomodulatory, antitumor, and antioxidant effects. Isoliquiritigenin (ILG), a major chalcone constituent of G. uralensis, exhibits anti-inflammatory activity; however, its molecular mechanism remains unclear. Here, we employed an activity-based protein profiling approach to identify the molecular targets of ILG. Given that the ,{beta}-unsaturated carbonyl moiety of ILG can covalently react with reactive cysteine residues via nucleophilic addition, we used an iodoacetamide-based probe to globally profile cysteine-reactive proteomes. The comparative analysis between ILG- and vehicle-treated RAW 264.7 macrophages identified cysteine 65 (Cys65) of lipocalin-type prostaglandin D2 synthase (L-PGDS) as a potential covalent target. ILG treatment did not alter L-PGDS expression levels, indicating that reduced probe labeling reflects direct covalent competition rather than changes in expression. Consistently, ILG significantly suppressed prostaglandin D2 (PGD2) production, comparable to the selective L-PGDS inhibitor AT-56. Although both ILG and AT-56 reduced interleukin-6 expression, ILG exerted a stronger inhibitory effect. Our results demonstrate that covalent inhibition of L-PGDS and subsequent suppression of PGD2 production represent a key mechanism underlying the anti-inflammatory activity of ILG.

BackgroundCurrent microphysiological models do not support long-term investigations into the chronic effects of vascular risk factors and the development of vascular diseases. Prolonged culture frequently leads to cellular senescence and loss of functional integrity, resulting in variability and inconsistency in modeling chronic vascular responses. Here we aimed to develop and sustain a long-term multicellular human vascular avatar, addressing the critical need for long-term disease modeling and drug testing. MethodsTo identify the optimal media for longevity, cell identity and function were assessed by morphology, qPCR, beta-gal staining, ELISA, bulk RNA-seq and single cell RNA-seq analysis. After optimizing the culture media, iPSCs-derived ECs and VSMCs from unaffected and Hutchinson-Gilford Progeria Syndrome (HGPS) donors were grown in Gravitational Lumen Patterning (GLP) Vessel- Chips for 1-6 months to generate a long-lived vascular avatar for the study of vascular aging. ResultsGuided by quantitative morphological analyses and bulk RNAseq profiling, we generated a novel optimized culture media VSL (VEGF, SB431542 as a TGF-{beta} inhibitor, low fetal bovine serum) that enhances the long-term health of vascular endothelial cells (ECs). Furthermore, we modified the VSL formulation (mVSL) by modulating 8Br-cAMP, FGF, PDGF, and a cell viability enhancer HMH1015 levels to enhance EC-VSMC (vascular smooth muscle cell) crosstalk and support long-term cellular viability. Subsequently, we maintained and characterized a human vascular avatar with a three-dimensional extracellular matrix environment and 3D vascular architecture for over 180 days. Finally, we demonstrated that this long-lived human vascular avatar enabled modeling vascular aging using iPSC-derived vascular cells from patients with Hutchinson-Gilford Progeria Syndrome (HGPS). ConclusionsWe have successfully engineered and maintained a human vascular avatar for over 180 days. The vascular avatar provides a robust platform for modeling disease-associated vascular aging and for evaluating therapeutic strategies targeting chronic vascular disorders.

The tight regulation of iron homeostasis is of great importance for cellular health. An increase in intracellular iron levels results in the formation of free radicals, which damages macromolecules and membranes, eventually resulting in cell death by Ferroptosis. Recently, we showed that patients with mutations in VPS41 display a severe neurodegenerative phenotype with iron deposition in the brain. VPS41 is well known as subunit of the HOPS complex required for fusion of late endosomes and autophagosomes with lysosomes. However, VPS41 has also been identified as inhibitor of Ferroptosis and regulator of redox homeostasis. How VPS41 exerts these functions and if these are dependent on the HOPS complex is unknown. Here we show that depletion of VPS41 results in increased intracellular iron levels, ROS formation and mitochondrial fission. Our findings indicate an important role for VPS41 in the regulation of iron homeostasis and mitochondrial fission and suggest Ferroptosis as a possible cause for neurodegeneration in VPS41 patients.

Sarcoidosis is a multisystem disorder that primarily affects the lungs and is characterizedby granulomatous inflammation. However, much of the underlying disease mechanisms remain poorly understood. Extracellular vesicles (EVs) are small membrane-bound particles released by all cells and carry various cargos including metabolites. They are involved in intercellular communication that can be dysregulated in diseases.This study characterizes the metabolic cargo of EVs isolated from bronchoalveolar lavage fluid (BALF), using liquid chromatography-mass spectrometry (LC-MS)-based metabolomic analysis, in patients with sarcoidosis (n=37), compared to healthy controls (n=10). Additionally, the sarcoidosis signature was compared to another pulmonary disorder, anti-synthetase syndrome (ASyS, n=10). Arachidonic acid (AA) results were verified by ELISA. A total of 1202 metabolites were detected, with 111 annotated ones further analyzed. EVs from sarcoidosis patients showed distinct metabolomic profiles compared to both ASyS patients and healthy controls, with 38 annotated metabolites differentially expressed in any of the groups. In both annotated and non-annotated data, sarcoidosis patients clustered separately from ASyS patients and healthy individuals. Furthermore, sarcoidosis patients clustered in 3 subgroups, whereof one was similar to ASyS patients and one stood out as showing higher cell counts in BALF. Higher AA levels were found in sarcoidosis patient EVs by LC-MS, and AA results were verified by ELISA. Our data show that BALF EV metabolites are disease-dependent and support the notion thatsarcoidosis patients should be further subgrouped for better diagnosis and treatment.

Interleukin-6 (IL-6), produced by skeletal muscle and extramuscular tissues, regulates skeletal muscle function through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. However, the interaction between intrinsic (locally produced) IL-6 and extrinsic (circulating) IL-6 in skeletal muscle remains unclear. We investigated whether and how intrinsic expression of IL-6 in cultured primary human myoblasts influences their response to extrinsic stimulation with recombinant human IL-6 (rhIL-6). Using gene silencing, we found that suppression of intrinsic IL-6 enhanced rhIL-6-induced phosphorylation of STAT1 and STAT3. Silencing STAT3 also increased rhIL-6-induced STAT1 phosphorylation, but silencing STAT1 had no effect on STAT3 phosphorylation. Pretreatment of myoblasts with neutralising anti-IL-6 antibodies increased phosphorylation of STAT1 and STAT3 induced by 50 ng/mL rhIL-6, whereas pretreatment with 5 ng/mL rhIL-6 reduced this response. Despite increased JAK/STAT signalling, IL-6 silencing decreased glucose and oleic acid uptake and oxidation under both basal and rhIL-6-stimulated conditions. Collectively, our results imply that intrinsic IL-6 restrains activation of the JAK/STAT pathway by extrinsic IL-6, but acts synergistically with it to promote myoblast energy metabolism.