Staining Of Lipid Droplets Research Articles

YAP and ECM Stiffness: Key Drivers of Adipocyte Differentiation and Lipid Accumulation.

ECM stiffness significantly influences the differentiation of adipose-derived stem cells (ADSCs), with YAP-a key transcription factor in the Hippo signaling pathway-playing a pivotal role. This study investigates the effects of ECM stiffness on ADSC differentiation and its relationship with YAP signaling. Various hydrogel concentrations were employed to simulate different levels of ECM stiffness, and their impact on ADSC differentiation was assessed through material properties, adipocyte-specific gene expression, lipid droplet staining, YAP localization, and protein levels. Our results demonstrated that increasing hydrogel stiffness enhanced adipocyte differentiation in a gradient manner. Notably, inhibiting YAP signaling further increased lipid droplet accumulation, suggesting that ECM stiffness influences adipogenesis by modulating YAP signaling and its cytoplasmic phosphorylation. This study elucidates the molecular mechanisms underlying ECM stiffness-dependent lipid deposition, highlighting YAP's regulatory role in adipogenesis. These findings provide valuable insights into the regulation of cell differentiation and have important implications for tissue engineering and obesity treatment strategies.

Complex‐mediated nucleophilic aromatic substitution with aryl nitriles to realize intramolecular flapping‐restricted D‐A AIEgens for bioimaging

AbstractDonor‐acceptor (D‐A) compounds are particularly important in optoelectronic and biological applications. However, they are normally synthesized in the presence of transition metal catalysts. Herein, we report a metal‐free method by a complex‐mediated nucleophilic aromatic substitution of aryl nitriles with amines. The method can lead to rich D‐A type aggregation‐induced emission luminogens (AIEgens) with tunable properties. They emit from deep‐blue to yellow‐green and possess high photoluminescence quantum yields up to 70.5% in the aggregate state. Interestingly, the suppression of intramolecular flapping is proved to play an indispensable role in the AIE behavior, which is different from the mechanism met in other AIEgens. Moreover, the biocompatible AIEgens possess specific staining of lipid droplets in HeLa cells and the superiority of identifying fatty liver over traditional Oil Red O staining is exhibited.

Limited Adipogenic Differentiation Potential of Human Dental Pulp Stem Cells Compared to Human Bone Marrow Stem Cells.

Bone marrow and teeth contain mesenchymal stem cells (MSCs) that could be used for cell-based regenerative therapies. MSCs from these two tissues represent heterogeneous cell populations with varying degrees of lineage commitment. Although human bone marrow stem cells (hBMSCs) and human dental pulp stem cells (hDPSCs) have been extensively studied, it is not yet fully defined if their adipogenic potential differs. Therefore, in this study, we compared the in vitro adipogenic differentiation potential of hDPSCs and hBMSCs. Both cell populations were cultured in adipogenic differentiation media, followed by specific lipid droplet staining to visualise cytodifferentiation. The in vitro differentiation assays were complemented with the expression of specific genes for adipogenesis and osteogenesis-dentinogenesis, as well as for genes involved in the Wnt and Notch signalling pathways. Our findings showed that hBMSCs formed adipocytes containing numerous and large lipid vesicles. In contrast to hBMSCs, hDPSCs did not acquire the typical adipocyte morphology and formed fewer lipid droplets of small size. Regarding the gene expression, cultured hBMSCs upregulated the expression of adipogenic-specific genes (e.g., PPARγ2, LPL, ADIPONECTIN). Furthermore, in these cells most Wnt pathway genes were downregulated, while the expression of NOTCH pathway genes (e.g., NOTCH1, NOTCH3, JAGGED1, HES5, HEY2) was upregulated. hDPSCs retained their osteogenic/dentinogenic molecular profile (e.g., RUNX2, ALP, COLIA1) and upregulated the WNT-specific genes but not the NOTCH pathway genes. Taken together, our in vitro findings demonstrate that hDPSCs are not entirely committed to the adipogenic fate, in contrast to the hBMSCs, which are more effective to fully differentiate into adipocytes.

Introducing FISCAS, a Tool for the Effective Generation of Single Cell MALDI-MSI Data.

We introduce Fluorescence Integrated Single-Cell Analysis Script (FISCAS), which combines fluorescence microscopy with MALDI-MSI to streamline single-cell analysis. FISCAS enables automated selection of tight measurement regions, thereby reducing the acquisition of off-target pixels, and makes use of established algorithms for cell segmentation and coregistration to rapidly compile single-cell spectra. MALDI-compatible staining of membranes, nuclei, and lipid droplets allows the collection of fluorescence data prior to the MALDI-MSI measurement on a timsTOF fleX MALDI-2. Usefulness of the software is demonstrated by the example of THP-1 cells during stimulated differentiation into macrophages at different time points. In this proof-of-principle study, FISCAS was used to automatically generate single-cell mass spectra along with a wide range of morphometric parameters for a total number of roughly 1300 cells collected at 24, 48, and 72 h after the onset of stimulation. Data analysis of the combined morphometric and single-cell mass spectrometry data shows significant molecular heterogeneity within the cell population at each time point, indicating an independent differentiation of each individual cell rather than a synchronized mechanism. Here, the grouping of cells based on their molecular phenotype revealed an overall clearer distinction of the different phases of differentiation into macrophages and delivered an increased number of lipid signals as possible markers compared with traditional bulk analysis. Utilizing the linkage between mass spectrometric data and fluorescence microscopy confirmed the expected positive correlation between lipid droplet staining and the overall signal for triacylglyceride (TG), demonstrating the usefulness of this multimodal approach.

Super-Resolution Microscopic Imaging of Lipid Droplets in Living Cells via Carbonized Polymer Dot-Based Polarity-Responsive Nanoprobe.

Lipid droplets (LDs) are dynamic subcellular organelles that participate in various physiological processes, and their abnormality can also lead to various diseases. Tracing the dynamics of LDs in living cells will be valuable for understanding cell physiological states. Here, we employed a structured light illumination super-resolution imaging assisted with a carbonized polymer dot (CPD)-based fluorescence nanoprobe to track the travel paths of LDs and other organelles. The CPDs we developed are highly biocompatible with living cells and exhibit a highly sensitive response to solvent polarity, allowing for high specificity in staining LDs in living cells. Aided by these nanoprobes, we successfully observed many real-time LD-involved dynamics in living cells, such as intracellular LD interactions, communications with other organelles, and dynamic behaviors under external stimuli (oxidative stress inducer). These studies deepen our comprehension of the physiological role of LDs and drive the advancement of super-resolution fluorescent probes.

Testosterone Inhibits Lipid Accumulation in Porcine Preadipocytes by Regulating ELOVL3.

Castration is commonly used to reduce stink during boar production. In porcine adipose tissue, castration reduces androgen levels resulting in metabolic disorders and excessive fat deposition. However, the underlying detailed mechanism remains unclear. In this study, we constructed porcine preadipocyte models with and without androgen by adding testosterone exogenously. The fluorescence intensity of lipid droplet (LD) staining and the fatty acid synthetase (FASN) mRNA levels were lower in the testosterone-treated cells than in the untreated control cells. In contrast, the mRNA levels of adipose triglycerides lipase (ATGL) and androgen receptor (AR) were higher than in the testosterone-treated cells than in the control cells. Subsequently, transcriptomic sequencing of porcine preadipocytes incubated with and without testosterone showed that the mRNA expression levels of very long-chain fatty acid elongase 3 (ELOVL3), a key enzyme involved in fatty acids synthesis and metabolism, were high in control cells. The siRNA-mediated knockdown of ELOVL3 reduced LD accumulation and the mRNA levels of FASN and increased the mRNA levels of ATGL. Next, we conducted dual-luciferase reporter assays using wild-type and mutant ELOVL3 promoter reporters, which showed that the ELOVL3 promoter contained an androgen response element (ARE); furthermore, its transcription was negatively regulated by AR overexpression. In conclusion, our study reveals that testosterone inhibits fat deposition in porcine preadipocytes by suppressing ELOVL3 expression. Moreover, our study provides a theoretical basis for further studies on the mechanisms of fat deposition caused by castration.

MT1G induces lipid droplet accumulation through modulation of H3K14 trimethylation accelerating clear cell renal cell carcinoma progression

BackgroundLipid droplet formation is a prominent histological feature in clear cell renal cell carcinoma (ccRCC), but the significance and mechanisms underlying lipid droplet accumulation remain unclear.MethodsExpression and clinical significance of MT1G in ccRCC were analyzed by using TCGA data, GEO data and scRNASeq data. MT1G overexpression or knockdown ccRCC cell lines were constructed and in situ ccRCC model, lung metastasis assay, metabolomics and lipid droplets staining were performed to explore the role of MT1G on lipid droplet accumulation in ccRCC.ResultsInitially, we observed low MT1G expression in ccRCC tissues, whereas high MT1G expression correlated with advanced disease stage and poorer prognosis. Elevated MT1G expression promoted ccRCC growth and metastasis both in vitro and in vivo. Mechanistically, MT1G significantly suppressed acylcarnitine levels and downstream tricarboxylic acid (TCA) cycle activity, resulting in increased fatty acid and lipid accumulation without affecting cholesterol metabolism. Notably, MT1G inhibited H3K14 trimethylation (H3K14me3) modification. Under these conditions, MT1G-mediated H3K14me3 was recruited to the CPT1B promoter through direct interaction with specific promoter regions, leading to reduced CPT1B transcription and translation.ConclusionsOur study unveils a novel mechanism of lipid droplet accumulation in ccRCC, where MT1G inhibits CPT1B expression through modulation of H3K14 trimethylation, consequently enhancing lipid droplet accumulation and promoting ccRCC progression.Graphical abstract figure Schematic diagram illustrating MT1G/H3K14me3/CPT1B-mediated lipid droplet accumulation promoted ccRCC progression via FAO inhibition.

Single‐Benzene Fluorophores: Controlling Electron Density on Amino of 2,5‐Diaminoterephthalates to Manipulate Optical/Electronic Properties for Efficient and Diversified Functions

AbstractSingle‐benzene fluorophores have attracted increasing interest owing to their simple molecular structure and unique photophysical properties. Developing novel single‐benzene fluorophores with facile synthesis, bright and tunable dual‐state emissions, and diversified functions is highly desirable. Herein, a strategy of controlling electron density on amino of 2,5‐diaminoterephthalates is proposed to modulate their dual‐state emission, acidichromism, Cu2+ detection, and lipid droplets (LDs) staining. A series of efficient dual‐state single‐benzene fluorophores based on 2,5‐diaminoterephthalate skeleton with tunable emissions in blue to red region and large Stokes shifts is facilely developed. The obtained fluorophores with various substituents on amino groups exhibit different acidichromism property. Two of fluorophores (ABB and ABM) with high electron density on amino groups exhibit high sensitivity and selectivity toward Cu2+ detection with a ratiometric and “turn‐off” fluorescence mechanism for ABM and ABB, respectively. The limit of detection for Cu2+ is determined to be 1.40 × 10−7 and 4.35 × 10−9 mol L−1 for ABB and ABM, respectively. Moreover, three of fluorophores (ABT, ABB, and ABM) with green to red emission display superior LDs‐targeting capability with high specificity, and brightness. This molecular design philosophy provides a new way of designing highly bright dual‐state fluorophores for practical applications.

NAD(P)H-quinone oxidoreductase 1 induces complicated effects on mitochondrial dysfunction and ferroptosis in an expression level-dependent manner.

NAD(P)H-quinone oxidoreductase 1 (NQO1) is an essential redox enzyme responsible for redox balance and energy metabolism. Despite of its importance, the brain contains high capacity of polyunsaturated fatty acids and maintains low levels of NQO1 expression. In this study, we examined how levels of NQO1 expression affects cell survival in response to toxic insults causing mitochondrial dysfunction and ferroptosis, and whether NQO1 has a potential as a biomarker in different stressed conditions. Following treatment with rotenone, overexpressed NQO1 in SH-SY5Y cells improved cell survival by reducing mitochondrial reductive stress via increased NAD+ supply without mitochondrial biogenesis. However, NQO1 overexpression boosted lipid peroxidation following treatment with RSL3 and erastin. A lipid droplet staining assay showed increased lipid droplets in cells overexpressing NQO1. In contrast, NQO1 knockdown protected cells against ferroptosis by increasing GPX4, xCT, and the GSH/GSSG system. Also, NQO1 knockdown showed lower iron contents and lipid droplets than non-transfectants and cells overexpressing NQO1, even though it could not attenuate cell death when exposed to rotenone. In summary, our study suggests that different NQO1 levels may have advantages and disadvantages depending on the surrounding environments. Thus, regulating NQO1 expression could be a potential supplementary tool when treating neuronal diseases.

Abstract 4436: Phenotypic heterogeneity in pancreatic ductal adenocarcinoma revealed by single-cell lipidomics

Abstract Introduction: The high chemoresistance of pancreatic ductal adenocarcinoma (PDAC) is partly attributed to the phenotypic plasticity and high adaptability of PDAC cells to the challenges of the harsh microenviroment. Phenotypic presentation of the PDAC cell is also determined by the lipid composition of the plasma membrane and lipid species that organize the rigid or more flexible lipid bilayer and determine the cellular mobility and plasticity. Lipids are also important energy and signaling molecules that contribute to the aggressiveness of the cell. Here we focus on differences in lipid profile of PDAC cells in epithelial and mesenchymal cell state using single-cell lipidomics approach (SpaceM). Methods: Lipid profile of 2D cultures of eight conventional and ten primary patient derived cell lines of different source (patient derived xenograft, ascites, primary tumor, organoid derived) was investigated. Epithelial (E) or mesenchymal (M) cell state was evaluated by gene and protein expression of EMT markers. Matrix-Assisted Laser Desorption/Ionization (MALDI)-imaging MS was used for data acquisition on positive ion mode at 25 µm step size and m/z range of 600-1000. For annotating lipids in MALDI-imaging data, we used the METASPACE cloud software. For constructing single-cell lipidomic profiles we used the SpaceM method. In total, around 150.000 cells were analyzed and 1400 lipid ions detected. Results: Gene expression analysis suggested prominent metabolism of structural lipids in the conventional epithelial cells. In UMAP analysis of single cell lipidomics data, each cell line, conventional and primary, presented a unique lipid fingerprint with however prominent clustering into the two phenotypes, epithelial and mesenchymal. We detected enrichment of alkyl-acyl phosphatidylcholines, alkyl-acyl phospatidylethanolamines, ceramides and sphingolipids in the epithelial phenotype. Mesenchymal phenotype was enriched for lipids involved in lateral diffusion and low bilayer thickness supporting the increased fluidity of the mesenchymal cell membrane. Furthermore, mesenchymal phenotype was enriched for lipid droplets and energy storing triacylglycerols, also confirmed by histological lipid droplet staining. Conclusions: Using the single cell lipid information, a successful grouping to epithelial or mesenchymal subtype could be performed regardless of sample source, suggesting a conserved lipid profile typical for the two states. Further detailed investigation of single cell lipidomics data aims to unveil the prominent epithelial-mesenchymal state lipid markers and open a niche for functional investigations of cell-state specific markers and potential cell-state targeting. Citation Format: Marija Trajkovic-Arsic, Jeany Delafiori, Mohammed Shahraz, Corinna Münch, Smiths S. Lueong, Sven T. Liffers, Doris Hellerschmied, Theodore Alexandrov, Jens T. Siveke. Phenotypic heterogeneity in pancreatic ductal adenocarcinoma revealed by single-cell lipidomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4436.

Indoleamine 2,3-dioxygenase 1-mediated iron metabolism in macrophages contributes to lipid deposition in nonalcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is a rapidly progressing chronic liver disease of global significance. However, the underlying mechanisms responsible for NASH remain unknown. Indoleamine 2,3-dioxygenase 1 (IDO1) has been recognized as essential factor in immune response and metabolic regulation. Here we aimed to investigate the functions and mechanisms of the IDO1 in macrophages on hepatic lipid deposition and iron metabolism in NASH. The effect of IDO1 in NASH was evaluated by WT and IDO1-/- mice model fed with methionine/choline-deficient (MCD) diet in vivo. Macrophages scavenger clodronate liposomes (CL) and overexpressing of IDO1 in macrophages by virus were employed as well. Lipid deposition was assessed through pathological examination and lipid droplet staining, while iron levels were measured using an iron assay kit and western blotting. Primary hepatocytes and bone marrow-derived macrophages were treated with oleic acid/palmitic acid (OA/PA) to assess IDO1 expression via Oil Red O staining and immunofluorescence staining in vitro. Pathological images demonstrated that the increase of IDO1 exacerbated lipid accumulation in the livers of mice with MCD diet, while reduction of iron accumulation was observed in the liver and the serum of MCD-fed mice. Scavenging of macrophages effectively mitigated both lipid and iron accumulation. In addition, the deficiency of IDO1 in macrophages significantly mitigated lipid accumulation and iron overload in hepatic parenchymal cells. Finally, lentivirus-mediated overexpression of IDO1 in liver macrophages exacerbated hepatic steatosis and iron deposition in NASH. Our results demonstrated that effective inhibition of IDO1 expression in macrophages in NASH alleviated hepatic parenchymal cell lipid accumulation and iron deposition, which provided new insights for the future treatment of NASH.

CRISPR/Cas genome editing revealed non-angiogenic role of VEGFA gene in porcine luteal cells: a preliminary report.

The angiogenic cytokine vascular endothelial growth factor A (VEGFA) also exerts non-angiogenic effects on endocrine functionality of porcine luteal cells critical for progesterone (P4) production. The expression dynamics of VEGFA-FLT/KDR system were investigated using RT-qPCR during luteal stages and VEGFA gene knock out (KO) porcine luteal cells were generated using CRISPR/Cas9 technology. The downstream effects of VEGFA ablation were studied using RT-qPCR, Annexin V, MTT, ELISA for P4 estimation and scratch wound assay. Bioinformatics analysis of RNA-Seq data of porcine mid-luteal stage was conducted for exploring protein-protein interaction network, KEGG pathways, transcription factors and kinase mapping for VEGFA-FLT/KDR interactomes. The VEGFA-FLT/KDR system expressed throughout the luteal stages with highest expression during mid- luteal stage. Cellular morphology, structure and oil-red-o staining for lipid droplets did not differ significantly between VEGFA KO and wild type cells, however, VEGFA KO significantly decreased (p < 0.05) viability and proliferation efficiency of edited cells on subsequent passages. Expression of apoptotic gene, CASP3 and hypoxia related gene, HIF1A were significantly (p < 0.05) upregulated in KO cells. The relative mRNA expression of VEGFA and steroidogenic genes STAR, CYP11A1 and HSD3B1 decreased significantly (p < 0.05) upon KO, which was further validated by the significant (p < 0.05) decrease in P4 output from KO cells. Bioinformatics analysis mapped VEGFA-FLT/KDR system to signalling pathways associated with steroidogenic cell functionality and survival, which complemented the findings of the study. The ablation of VEGFA gene resulted in decreased steroidogenic capability of luteal cells, which suggests that VEGFA exerts additional non-angiogenic regulatory effects in luteal cell functionality.

Electrochemical Diselenation of BODIPY Fluorophores for Bioimaging Applications and Sensitization of 1 O2.

We report a rapid, efficient, and scope-extensive approach for the late-stage electrochemical diselenation of BODIPYs. Photophysical analyses reveal red-shifted absorption - corroborated by TD-DFT and DLPNO-STEOM-CCSD computations - and color-tunable emission with large Stokes shifts in the selenium-containing derivatives compared to their precursors. In addition, due to the presence of the heavy Se atoms, competitive ISC generates triplet states which sensitize 1 O2 and display phosphorescence in PMMA films at RT and in a frozen glass matrix at 77 K. Importantly, the selenium-containing BODIPYs demonstrate the ability to selectively stain lipid droplets, exhibiting distinct fluorescence in both green and red channels. This work highlights the potential of electrochemistry as an efficient method for synthesizing unique emission-tunable fluorophores with broad-ranging applications in bioimaging and related fields.

BNIP3 in hypoxia-induced mitophagy: Novel insights and promising target for non-alcoholic fatty liver disease

BNIP3 localizes to the outer mitochondrial membrane, has been demonstrated to be extensively involved in abnormalities to mitochondrial metabolic function and dynamicsand in non-alcoholic fatty liver disease (NAFLD). However, its role in NAFLD under hypoxia remains unclear. This study aimed to investigate the expression and the role of BNIP3 in NAFLD under hypoxia, and explore its involvement in regulating NAFLD mitophagy, fatty acid β-oxidation both in vivo and in vitro. BNIP3-mediated mitophagy level was analyzed using real-time quantitative polymerase chain reaction, Western blotting, immunofluorescence and electron microscopy. The role of BNIP3 in fatty acid β-oxidation was evaluated using lipid droplet staining, triglyceride content determination, and cellular energy metabolism. The results showed that compared with the HFD-2200 m, the body weight, inflammatory liver injury, and lipid deposition were significantly reduced in the HFD-4500 m group (P < 0.05), but autophagy and mitophagy were increased, and the expression of the mitophagy receptor BNIP3 was increased (P < 0.05). Compared to the control group, BNIP3 knockdown in the hypoxia group resulted in decreased levels of CPT1, ATGL, and p-HSL in lipid-accumulating hepatocytes, lipid droplet accumulation and triglyceride content increased (P < 0.05). Moreover, the ability of lipid-accumulating hepatocytes to oxidize fatty acids was reduced by BNIP3 knockdown in the hypoxia group (P < 0.05). Therefore, it can be concluded that, in NAFLD mice under hypoxia, BNIP3-mediated mitophagy promotes fatty acid β-oxidation. This study elucidated the role of BNIP3 in promoting fatty acid β-oxidation in NAFLD under hypoxia, and suggests BNIP3 may serve as a novel potential therapeutic target for NAFLD.

Thiophene-conjugated benzothiazole derivatives as versatile skeletons for staining subcellular organelles in living cells

A series of hydroxy thiophene-conjugated benzothiazole derivatives (HTBs) was prepared to investigate their fluorescence emission behaviors and capability to selectively stain subcellular organelles for fluorescence imaging. The HTB derivatives exhibited excited-state intramolecular proton transfer (ESIPT) and intramolecular charge transfer (ICT) behaviors, according to their molecular structure. Subtle changes in the molecular structure of the HTB influenced its response to changes in the surrounding environment, such as the solvent polarity and/or pH. In vitro cellular imaging experiments revealed that HTBA (5-(benzo[d]thiazol-2-yl)-[2,2′-bithiophen]-4-ol) could selectively stain mitochondria. In contrast, HTBB-CO2Et (ethyl 5'-(benzo[d]thiazol-2-yl)-3′-hydroxy-[2,2′-bithiophene]-5-carboxylate), which contains a regioisomeric hydroxy thiophene unit with an electron-withdrawing ethyl ester group, showed the capability to stain lipid droplets (LDs) in living cells. When the hydroxyl group in HTBB-CO2Et was protected with a benzyl group, effective staining of LDs in live cells was achieved. Thus, we successfully obtained an effective fluorescent probe. Collectively, the results indicated that small structural changes resulted in substantial changes in the intracellular localization and in vitro imaging ability of the prepared compounds.

Hydroxyurea attenuates hepatic stellate cell proliferation in vitro and liver fibrogenesis in vivo.

Liver fibrosis results from excessive proliferation of, and collagen production by hepatic stellate cells (HSCs) that is caused by chronic liver injury. No drugs are available to cure liver fibrosis. Hydroxyurea is an anti-proliferative drug that is used in benign and malignant disorders. Here, we studied the effect of hydroxyurea on primary HSCs and its anti-fibrotic effect in the CCl4 mouse model of liver fibrosis. Primary rat HSCs were cultured in the absence or presence of hydroxyurea (0.1-1.0 mmol/L). CCl4 or vehicle was administered to C57BL/6/J mice for 4 weeks, with or without hydroxyurea (100 mg/kg/day) co-treatment. We used real-time cell proliferation analysis, Oil Red O (lipid droplet) staining, immunohistochemistry, Acridine Orange staining (apoptosis), Sytox green staining (necrosis), RT-qPCR, ELISA, and Western Blotting for analysis. Hydroxyurea dose-dependently suppressed lipid droplet-loss and mRNA levels of Col1α1 and Acta2 in transdifferentiating HSCs. In fully-activated HSCs, hydroxyurea dose-dependently attenuated PCNA protein levels and BrdU incorporation, but did not reverse Col1α1 and Acta2 mRNA expression. Hydroxyurea did not induce apoptosis or necrosis in HSCs or hepatocytes. Hydroxyurea suppressed accumulation of desmin-positive HSCs and hepatic collagen deposition after CCl4 treatment. CCl4 -induced regenerative hepatocyte proliferation, Col1α1 and Acta2 mRNA expression and α-SMA protein levels were not affected. This study demonstrates that hydroxyurea inhibits HSC proliferation in vitro and attenuates early development of liver fibrosis in vivo, while preserving hepatocyte regeneration after toxic insults by CCl4. Thus, hydroxyurea may have therapeutic value against liver fibrosis.

Abstract GS.103: Human Cardiomyopathy Disease Variant In Formin Homology 2 Domain Containing 3 (FHOD3) Protein Revealed Novel Early Autophagy Dysregulation And Mitochondrial Dysfunction

Introduction: The FHOD3-V1151I variant has been identified by multiple Genome Wide Association Studies to be associated with hypertrophic cardiomyopathy (HCM). FHOD3 regulates actin filament formation which is central to key cardiomyocyte processes including autophagy. The precise role of autophagy in cardiomyopathy is an emerging area of research but poorly characterized. We hypothesize that V1151I may perturb FHOD3-dependent cardiac autophagy regulation. Methods: The homologous V1151I variant was introduced into mice using CRISPR. Cardiac function was studied by echocardiography, cardiac mitochondrial function measured by Seahorse (Agilent) and oil red O staining of lipid droplets in cardiac sections. Cultured H9C2 cardiomyoblasts were transfected with FHOD3-V1151V (WT) or V1151I (variant) expression plasmids and immunoprecipitation and immunofluorescence were performed to assess protein interaction and localization. Phosphatidylinositol 3-phosphate (PI3P, Echelon) was used for the rescue experiments. Results: Heart tissue from FHOD3-V1151I variant mice (n=7) showed a 5-fold (p&lt;0.0001) increase in lipid droplet accumulation and impaired mitochondrial respiration and ATP production (p&lt;0.0001), while cardiac structure and function assessed by echocardiography was similar to V1151V WT littermate control hearts (n=7). A bioinformatics analysis predicted FHOD3 interaction with PFN4 which we confirmed by co-immunoprecipitation in addition to ATG16, a regulator of autophagic vesicle membrane biogenesis. The FHOD3-PFN4-ATG16 interaction was however lost in FHOD3-V1151I hearts. Immunofluorescence of H9C2 cells expressing FHOD3-V1151I revealed that the variant disrupts interaction of LC3 with PI3P, a hallmark of early autophagy. The disrupted LC3-PI3P interaction seen with the V1151I variant phenocopies cells treated with early autophagy inhibitors 3-MA or spautin, but not late autophagy inhibitor chloroquine. Furthermore, exogenous PI3P restored the molecular interaction and autophagy dysregulation. Conclusions: We demonstrate for the first time that early autophagy dysregulation underlies energetic defects observed in cardiomyopathy, and is reversible, thus providing a novel potential therapeutic target.

Cover Picture: Eur. J. Lipid Sci. Technol. 6/2023

European Journal of Lipid Science and TechnologyVolume 125, Issue 6 2370017 Cover PictureFree Access Cover Picture: Eur. J. Lipid Sci. Technol. 6/2023 First published: 01 June 2023 https://doi.org/10.1002/ejlt.202370017AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Graphical Abstract Simplified cholesterol backbone with images of a random spectral trace, a palm leaf (Rainer Sturm/pixelio.de), fluorescently stained lipid droplets (red) and associated proteins (green) in a T3T-L1 adipocyte (Johanna Spandl, University of Bonn, Germany), and fish (Vera/pixelio.de). Volume125, Issue6June 20232370017 RelatedInformation

Trimethylamine N-oxide Promotes Atherosclerosis by Regulating Low-Density Lipoprotein-Induced Autophagy in Vascular Smooth Muscle Cells Through PI3K/AKT/mTOR Pathway.

The research aimed to study the mechanism of how trimethylamine N-oxide (TMAO) regulates autophagy to promote atherosclerosis (AS). The AS in vitro model was constructed with vascular smooth muscle cells (VSMCs) treated with ox-LDL. The Cell Counting Kit-8 (CCK-8) trial was chosen to examine VSMCs' absorbance (OD) value. A transmission electron microscope (TEM) was selected for monitoring autophagosomes. Western blotting (WB) was adopted for examining the expression of Beclin-1, p62, LC3, α-SMA, SM22-α, OPN, PI3K, AKT, mTOR, p-PI3K, p-AKT, and p-mTOR proteins. Real-time fluorescent quantitative PCR (RT-qPCR) was accepted for testing the expression of α-SMA, SM22-α, OPN, PI3K, AKT, mTOR, Beclin-1, p62, and LC3 genes. The transwell assay was employed to examine the ability of migration in VSMCs. Oil red O staining assay was accepted to stain lipid droplets in VSMCs. TMAO noticeably promoted autophagy inhibition and the phenotypic transformation of AS. Protein expressions of p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR, and p62 of the TMAO+ox-LDL group were higher than the ox-LDL group, while Beclin-1 and LC3 were lower than the ox-LDL group. Gene expressions of PI3K, AKT, mTOR, and p62 of the TMAO+ox-LDL group were higher than the ox-LDL group, while Beclin-1 and LC3 were lower than the ox-LDL group. The intervention of LY294002 reversed the regulation of the corresponding proteins and genes. The study proved that TMAO could promote autophagy inhibition of AS via activating the PI3K/AKT/mTOR pathway. It supplied a reliable basis for improving clinical diagnostic methods and developing targeted AS drugs.

Cover Picture: Eur. J. Lipid Sci. Technol. 4/2023

European Journal of Lipid Science and TechnologyVolume 125, Issue 4 2370010 Cover PictureFree Access Cover Picture: Eur. J. Lipid Sci. Technol. 4/2023 First published: 03 April 2023 https://doi.org/10.1002/ejlt.202370010AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Graphical Abstract Simplified cholesterol backbone with images of a random spectral trace, a palm leaf (Rainer Sturm/pixelio.de), fluorescently stained lipid droplets (red) and associated proteins (green) in a T3T-L1 adipocyte (Johanna Spandl, University of Bonn, Germany), and fish (Vera/pixelio.de). Volume125, Issue4April 20232370010 RelatedInformation