Intracellular Cholesterol Transport Research Articles

Transcriptional landscape of a hypoxia response identifies cell-specific pathways for adaptation.

How the HIF-1 (Hypoxia-Inducible) transcription factor drives and coordinates distinct responses to low oxygen across diverse cell types is poorly understood. We present a multi-tissue single-cell gene-expression atlas of the hypoxia response of the nematode Caenorhabditis elegans . This atlas highlights how cell-type-specific HIF-1 responses overlap and diverge among and within neuronal, intestinal, and muscle tissues. Using the atlas to guide functional analyses of candidate muscle-specific HIF-1 effectors, we discovered that HIF-1 activation drives downregulation of the tspo-1 ( TSPO, Translocator Protein) gene in vulval muscle cells to modulate a hypoxia-driven change in locomotion caused by contraction of body-wall muscle cells. We further showed that in human cardiomyocytes HIF-1 activation decreases levels of TSPO and thereby alters intracellular cholesterol transport and the mitochondrial network. We suggest that TSPO-1 is an evolutionarily conserved mediator of HIF-1-dependent modulation of muscle and conclude that our gene-expression atlas can help reveal how HIF-1 drives cell-specific adaptations to hypoxia.

Associations of lipids and lipid-modifying drug target genes with atrial fibrillation risk based on genomic data

BackgroundThe causal associations of lipids and the drug target genes with atrial fibrillation (AF) risk remain obscure. We aimed to investigate the causal associations using genetic evidence.MethodsMendelian randomization (MR) analyses were conducted using summary-level genome-wide association studies (GWASs) in European and East Asian populations. Lipid profiles (low-density lipoprotein cholesterol, triglyceride, and lipoprotein[a]) and lipid-modifying drug target genes (3-hydroxy-3-methylglutaryl-CoA reductase, proprotein convertase subtilisin/kexin type 9, NPC1-like intracellular cholesterol transporter 1, apolipoprotein C3, angiopoietin-like 3, and lipoprotein[a]) were used as exposures. AF was used as an outcome. The inverse variance weighted method was applied as the primary method. Summary-data-based Mendelian randomization analyses were performed for further validation using expression quantitative trait loci data. Mediation analyses were conducted to explore the indirect effect of coronary heart disease.ResultsIn the European population, MR analyses demonstrated that elevated levels of lipoprotein(a) increased AF risk. Moreover, analyses focusing on drug targets revealed that the genetically proxied target gene LPA, which simulates the effects of drug intervention by reducing lipoprotein(a), exhibited an association with AF risk. This association was validated in independent datasets. There were no consistent and significant associations observed for other traits when analyzed in different datasets. This finding was also corroborated by Summary-data-based Mendelian randomization analyses between LPA and AF. Mediation analyses revealed that coronary heart disease plays a mediating role in this association. However, in the East Asian population, no statistically significant evidence was observed to support these associations.ConclusionsThis study provided genetic evidence that Lp(a) may be a causal factor for AF and that LPA may represent a promising pharmacological target for preventing AF in the European population.

NPC Intracellular Cholesterol Transporter 1 Regulates Ovarian Maturation and Molting in Female Macrobrachium nipponense.

NPC intracellular cholesterol transporter 1 (NPC1) plays an important role in sterol metabolism and transport processes and has been studied in many vertebrates and some insects, but rarely in crustaceans. In this study, we characterized NPC1 from Macrobrachium nipponense (Mn-NPC1) and evaluated its functions. Its total cDNA length was 4283 bp, encoding for 1344 amino acids. It contained three conserved domains typical of the NPC family (NPC1_N, SSD, and PTC). In contrast to its role in insects, Mn-NPC1 was mainly expressed in the adult female hepatopancreas, with moderate expression in the ovary and heart. No expression was found in the embryo (stages CS-ZS) and only weak expression in the larval stages from hatching to the post-larval stage (L1-PL15). Mn-NPC1 expression was positively correlated with ovarian maturation. In situ hybridization showed that it was mainly located in the cytoplasmic membrane and nucleus of oocytes. A 25-day RNA interference experiment was employed to illustrate the Mn-NPC1 function in ovary maturation. Experimental knockdown of Mn-NPC1 using dsRNA resulted in a marked reduction in the gonadosomatic index and ecdysone content of M. nipponense females. The experimental group showed a significant delay in ovarian maturation and a reduction in the frequency of molting. These results expand our understanding of NPC1 in crustaceans and of the regulatory mechanism of ovarian maturation in M. nipponense.

Presenilin Deficiency Results in Cellular Cholesterol Accumulation by Impairment of Protein Glycosylation and NPC1 Function.

Presenilin proteins (PS1 and PS2) represent the catalytic subunit of γ-secretase and play a critical role in the generation of the amyloid β (Aβ) peptide and the pathogenesis of Alzheimer disease (AD). However, PS proteins also exert multiple functions beyond Aβ generation. In this study, we examine the individual roles of PS1 and PS2 in cellular cholesterol metabolism. Deletion of PS1 or PS2 in mouse models led to cholesterol accumulation in cerebral neurons. Cholesterol accumulation was also observed in the lysosomes of embryonic fibroblasts from Psen1-knockout (PS1-KO) and Psen2-KO (PS2-KO) mice and was associated with decreased expression of the Niemann-Pick type C1 (NPC1) protein involved in intracellular cholesterol transport in late endosomal/lysosomal compartments. Mass spectrometry and complementary biochemical analyses also revealed abnormal N-glycosylation of NPC1 and several other membrane proteins in PS1-KO and PS2-KO cells. Interestingly, pharmacological inhibition of N-glycosylation resulted in intracellular cholesterol accumulation prominently in lysosomes and decreased NPC1, thereby resembling the changes in PS1-KO and PS2-KO cells. In turn, treatment of PS1-KO and PS2-KO mouse embryonic fibroblasts (MEFs) with the chaperone inducer arimoclomol partially normalized NPC1 expression and rescued lysosomal cholesterol accumulation. Additionally, the intracellular cholesterol accumulation in PS1-KO and PS2-KO MEFs was prevented by overexpression of NPC1. Collectively, these data indicate that a loss of PS function results in impaired protein N-glycosylation, which eventually causes decreased expression of NPC1 and intracellular cholesterol accumulation. This mechanism could contribute to the neurodegeneration observed in PS KO mice and potentially to the pathogenesis of AD.

Abstract 2071: Identification Of Genetic Loci Influencing Lethal Atherothrombosis In Mice

Introduction: Atherothrombosis leading to myocardial infarction and stroke is a major cause of mortality. Ruptured atherosclerotic plaques release prothrombotic molecules such as tissue factor, which initiates thrombosis. Both genetic and environmental factors contribute to atherothrombosis. Combined complete deficiency of the Scavenger Receptor Class B type 1 (Scarb1) and Apolipoprotein E (ApoE) genes (dKO) on a mixed C57BL/6J (B6) and 129S1/SvImJ (129S1) background leads to an atherothrombotic phenotype and early death before 8 weeks of age. However, the dKO phenotype is dramatically different if the Scarb1 and ApoE deficient mice are backcrossed extensively to the B6 mouse strain, with no dKOs surviving to the weaning age. Hypothesis/Goals: We hypothesized that 129S1 genetic variants reduce atherothrombosis severity in dKO mice and used mouse genetics to identify these loci. Methods: To identify potential genetic factors, we produced and analyzed Scarb1 and ApoE dKO mice from 6 backcross/intercross generations with progressively more B6 genomic contribution. Weights were taken for 38 dKO mice and littermates from 3 weeks old until death of the dKO mice at 8 weeks of age. dKO mice from F1-F3 generations were divided into 3 distinct phenotypes based on their weights relative to their littermates. dKO mouse DNA was subjected to genome wide marker genotyping using the GigaMUGA platform. Quantitative trait loci analysis (QTL) was performed to identify loci responsible for the phenotypic weight differences. Results: dKO weights fell into 3 categories: indistinguishable from littermates, 14%-39% less than littermates, and greater than 39% less than littermates. QTL analyses identified significant interactions of Chr 12 with Chrs 1, 2, 3, and 14. Niemann Pick type C2 (Npc2), an intracellular cholesterol transporter, is within the interaction region on Chr 12 and is a prime candidate dKO modifier. Conclusion: We used weight data as a representative of overall health to categorize dKO phenotypes relative to non dKO littermates. We identified 6 significant genetic loci, with Npc2 as a candidate modifier gene for the Chromosome 12 locus. Identification of atherothrombosis modifier genes will provide novel therapeutic targets for this complex disease.

Endogenous Protein-Protein Interaction Network of the NPC Cholesterol Transporter 1 in the Cerebral Cortex.

NPC intracellular cholesterol transporter 1 (NPC1) is a multipass, transmembrane glycoprotein mostly recognized for its key role in facilitating cholesterol efflux. Mutations in the NPC1 gene result in Niemann-Pick disease, type C (NPC), a fatal, lysosomal storage disease. Due to the progressively expanding implications of NPC1-related disorders, we investigated endogenous NPC1 protein-protein interactions in the mouse cortex and human-derived iPSCs neuronal models of the disease through coimmunoprecipitation-coupled with LC-MS based proteomics. The current study investigated protein-protein interactions specific to the wild-type and the most prevalent NPC1 mutation (NPC1I1061T) while filtering out any protein interactor identified in the Npc1-/- mouse model. Additionally, the results were matched across the two species to map the parallel interactome of wild-type and mutant NPC1I1061T. Most of the identified wild-type NPC1 interactors were related to cytoskeleton organization, synaptic vesicle activity, and translation. We found many putative NPC1 interactors not previously reported, including two SCAR/WAVE complex proteins that regulate ARP 2/3 complex actin nucleation and multiple membrane proteins important for neuronal activity at synapse. Moreover, we identified proteins important in trafficking specific to wild-type and mutant NPC1I1061T. Together, the findings are essential for a comprehensive understanding of NPC1 biological functions in addition to its classical role in sterol efflux.

Sterol O-Acyltransferase 1 (SOAT1): A Genetic Modifier of Niemann-Pick Disease, Type C1.

Niemann-Pick disease type C1 (NPC1) is a lysosomal disorder due to impaired intracellular cholesterol transport out of the endolysosomal compartment.. Marked heterogeneity has been observed in individuals with the same NPC1 genotype, thus suggesting a significant effect of modifier genes. Prior work demonstrated that decreased SOAT1 activity decreased disease severity in an NPC1 mouse model. Thus, we hypothesized that a polymorphism associated with decreased SOAT1 expression might influence the NPC1 phenotype. Phenotyping and genomic sequencing of 117 individuals with NPC1 was performed as part of a Natural History trial. Phenotyping included determination of disease severity and disease burden. Significant clinical heterogeneity is present in individuals homozygous for the NPC1I1061T variant and in siblings. Analysis of the SOAT1 polymorphism, rs1044925 (A>C), showed a significant association of the C-allele with earlier age of neurological onset. The C-allele may be associated with a higher Annualized Severity Index Score as well as increased frequency of liver disease and seizures. A polymorphism associated with decreased expression of SOAT1 appears to be a genetic modifier of the NPC1 phenotype. This finding is consistent with prior data showing decreased phenotypic severity in Npc1-/-:Soat1-/- mice and supports efforts to investigate the potential of SOAT1 inhibitors as a potential therapy for NPC1.

ALOX15B controls macrophage cholesterol homeostasis via lipid peroxidation, ERK1/2 and SREBP2

Macrophage cholesterol homeostasis is crucial for health and disease and has been linked to the lipid-peroxidizing enzyme arachidonate 15-lipoxygenase type B (ALOX15B), albeit molecular mechanisms remain obscure. We performed global transcriptome and immunofluorescence analysis in ALOX15B-silenced primary human macrophages and observed a reduction of nuclear sterol regulatory element-binding protein (SREBP) 2, the master transcription factor of cellular cholesterol biosynthesis. Consequently, SREBP2-target gene expression was reduced as were the sterol biosynthetic intermediates desmosterol and lathosterol as well as 25- and 27-hydroxycholesterol. Mechanistically, suppression of ALOX15B reduced lipid peroxidation in primary human macrophages and thereby attenuated activation of mitogen-activated protein kinase ERK1/2, which lowered SREBP2 abundance and activity. Low nuclear SREBP2 rendered both, ALOX15B-silenced and ERK1/2-inhibited macrophages refractory to SREBP2 activation upon blocking the NPC intracellular cholesterol transporter 1. These studies suggest a regulatory mechanism controlling macrophage cholesterol homeostasis based on ALOX15B-mediated lipid peroxidation and concomitant ERK1/2 activation.

Long-term efficacy of intrathecal cyclodextrin in patients with Niemann-Pick disease type C

Background and objectivesNiemann–Pick type C (NPC) is a rare lysosomal storage disease characterized by hepatosplenomegaly and progressive neurological deterioration due to abnormal intracellular cholesterol transport. Cyclic oligosaccharide 2-hydroxypropyl-β-cyclodextrin (HPBCD) is an effective treatment for NPC; however, few reports have shown its long-term efficacy and safety. To demonstrate long-term efficacy and safety of intrathecal HPBCD (IT-HPBCD) treatment for NPC, we herein reports five patients with NPC treated using IT-HPBCD for 4–11 years. Cases and resultsPatients’ ages at the onset ranged from 1.5 to 20 years. Notably, all patients showed rapid disease progression despite treatment with miglustat before IT-HPBCD treatment. Similarly, some patients showed transient improvement; however, all patients’ conditions stabilized after long-term IT-HPBCD therapy. Mild-to-moderate hearing loss was observed in three patients. Furthermore, long-term treatment with IT-HPBCD may suppress neurological deterioration in patients with NPC; however, patients still experience some disease progression. ConclusionsLong-term treatment with IT-HPBCD may suppress neurological deterioration in patients with NPC; however, the treatment outcome is dependent on the neurological status at the time of diagnosis, and disease progression is not completely inhibited. Awareness of the disease and newborn screening is needed for earlier disease detection. In addition, further optimization of the treatment protocol and additional treatments are needed to improve patient outcomes.

Sperm traits and seminal plasma proteome of locally adapted hairy rams subjected to intermittent scrotal insulation

The present study evaluated the effects of heat stress on reproductive parameters of hairy rams. Six animals were subjected to scrotal insulation during four consecutive nights (6 PM – 6 AM). Day (D) 0 was the first day of insulation. Scrotal circumference increased from 30.5 ± 0.3 cm (at pre-insulation) to 31.8 ± 0.4 cm on D4, decreased 3.9 cm on D28, returning to 30.6 ± 0.6 cm on D57. Sperm concentration decreased from 3.7 ± 0.12 ×109 sperm/mL before insulation to 2.6 ± 0.1 ×109 on D23, returning to normal on D57. Sperm motility averaged 75 ± 2.9% before insulation, was undetectable on D23, and became normal on D77. Sperm with normal morphology reached 5.9 ± 2.6% on D35 but recovered (86.8 ± 2.1%) on D91. Sperm DNA integrity decreased from 86.5 ± 4.7% before insulation to 11.1 ± 3.7% on D63, returning to pre-insulation values on D120. Sperm BSP immunostaining was reduced after scrotal insulation. Variations in seminal protein abundances coincided with changes in sperm parameters. Seminal plasma superoxide dismutase, carboxypeptidase Q-precursor and NPC intracellular cholesterol transporter 2 decreased on D18, returning to normal after D28. Albumin, inhibitor of carbonic anhydrase precursor, EGF-like repeat and discoid I-like domain-containing protein 3 and polymeric immunoglobulin receptor increased after insulation. In summary, intermittent scrotal insulation drastically altered ram sperm attributes and seminal proteins, especially those associated with oxidative stress. Knowledge of animal´s response to thermal stress is vital in the scenario of climate changes.

Repositioning of ezetimibe for the treatment of idiopathic pulmonary fibrosis.

We previously identified ezetimibe, an inhibitor of Niemann-Pick C1-like intracellular cholesterol transporter 1 and European Medicines Agency-approved lipid-lowering agent, as a potent autophagy activator. However, its efficacy against pulmonary fibrosis has not yet been evaluated. This study aimed to determine whether ezetimibe has therapeutic potential against idiopathic pulmonary fibrosis. Primary lung fibroblasts isolated from both humans and mice were employed for mechanistic in vitro experiments. mRNA sequencing of human lung fibroblasts and gene set enrichment analysis were performed to explore the therapeutic mechanism of ezetimibe. A bleomycin-induced pulmonary fibrosis mouse model was used to examine in vivo efficacy of the drug. Tandem fluorescent-tagged microtubule-associated protein 1 light chain 3 transgenic mice were used to measure autophagic flux. Finally, the medical records of patients with idiopathic pulmonary fibrosis from three different hospitals were reviewed retrospectively, and analyses on survival and lung function were conducted to determine the benefits of ezetimibe. Ezetimibe inhibited myofibroblast differentiation by restoring the mechanistic target of rapamycin complex 1-autophagy axis with fine control of intracellular cholesterol distribution. Serum response factor, a potential autophagic substrate, was identified as a primary downstream effector in this process. Similarly, ezetimibe ameliorated bleomycin-induced pulmonary fibrosis in mice by inhibiting mechanistic target of rapamycin complex 1 activity and increasing autophagic flux, as observed in mouse lung samples. Patients with idiopathic pulmonary fibrosis who regularly used ezetimibe showed decreased rates of all-cause mortality and lung function decline. Our study presents ezetimibe as a potential novel therapeutic for idiopathic pulmonary fibrosis.

Fut2 Deficiency Promotes Intestinal Stem Cell Aging by Damaging Mitochondrial Functions via Down-Regulating α1,2-Fucosylation of Asah2 and Npc1.

Fut2-mediated α1,2-fucosylation is important for gut homeostasis, including the intestinal stem cell (ISC). The stemness of ISC declines with age, and aging-associated ISC dysfunction is closely related to many age-related intestinal diseases. We previously found intestinal epithelial dysfunction in some aged Fut2 knockout mice. However, how Fut2-mediated α1,2-fucosylation affects ISC aging is still unknown. On this basis, the herein study aims to investigate the role of Fut2-mediated α1,2-fucosylation in ISC aging. Aging models in ISC-specific Fut2 knockout mice were established. ISCs were isolated for proteomics and N-glycoproteomics analysis. ISC functions and mitochondrial functions were examined in mice and organoids. Ulex europaeus agglutinin I chromatography and site-directed mutagenesis were used to validate the key target fucosylated proteins of Fut2. As a result, Fut2 knockout impaired ISC stemness and promoted aging marker expression in aged mice. Proteomics analysis indicated mitochondrial dysfunction in Fut2 knockout ISC. More injured mitochondria, elevated levels of reactive oxygen species, and decreased levels of adenosine 5'-triphosphate (ATP) in Fut2 knockout ISC were found. Moreover, respiratory chain complex impairment and mitophagy dysfunction in Fut2 knockout ISC were further noted. Finally, Fut2 was demonstrated to regulate mitochondrial functions mainly by regulating the α1,2-fucosylation of N-acyl sphingosine amidohydrolase 2 (Asah2) and Niemann-Pick type C intracellular cholesterol transporter 1 (Npc1). In conclusion, this study demonstrated the substantial role of Fut2 in regulating ISC functions during aging by affecting mitochondrial function. These findings provide novel insights into the molecular mechanisms of ISC aging and therapeutic strategies for age-related intestinal diseases.

Glucose-mediated mitochondrial reprogramming by cholesterol export at TM4SF5-enriched mitochondria-lysosome contact sites.

Transmembrane 4 L six family member 5 (TM4SF5) translocates subcellularly and functions metabolically, although it is unclear how intracellular TM4SF5 translocation is linked to metabolic contexts. It is thus of interests to understand how the traffic dynamics of TM4SF5 to subcellular endosomal membranes are correlated to regulatory roles of metabolisms. Here, we explored the metabolic significance of TM4SF5 localization at mitochondria-lysosome contact sites (MLCSs), using in vitro cells and in vivo animal systems, via approaches by immunofluorescence, proximity labelling based proteomics analysis, organelle reconstitution etc. RESULTS: Upon extracellular glucose repletion following depletion, TM4SF5 became enriched at MLCSs via an interaction between mitochondrial FK506-binding protein 8 (FKBP8) and lysosomal TM4SF5. Proximity labeling showed molecular clustering of phospho-dynamic-related protein I (DRP1) and certain mitophagy receptors at TM4SF5-enriched MLCSs, leading to mitochondrial fission and autophagy. TM4SF5 bound NPC intracellular cholesterol transporter 1 (NPC1) and free cholesterol, and mediated export of lysosomal cholesterol to mitochondria, leading to impaired oxidative phosphorylation but intact tricarboxylic acid (TCA) cycle and β-oxidation. In mouse models, hepatocyte Tm4sf5 promoted mitophagy and cholesterol transport to mitochondria, both with positive relations to liver malignancy. Our findings suggested that TM4SF5-enriched MLCSs regulate glucose catabolism by facilitating cholesterol export for mitochondrial reprogramming, presumably while hepatocellular carcinogenesis, recapitulating aspects for hepatocellular carcinoma metabolism with mitochondrial reprogramming to support biomolecule synthesis in addition to glycolytic energetics.

Identification of Genetic Loci That Modify the Scarb1 and Apoe Atherothrombotic Phenotype

Introduction: Atherothrombosis, or the formation of a thrombus on a ruptured atherosclerotic plaque, is a major health problem in western societies and can lead to myocardial infarction and ischemic stroke. Atherosclerotic plaques form on the inner walls of blood vessels and consist of macrophages, lipids, and prothrombotic molecules such as tissue factor that initiate the blood clotting process. Despite the complexity of atherothrombotic disease, with major (and evolving) environmental contributors, genetic variants play a role in modifying the disease process. In mice, deficiency of the Scavenger Receptor Class B type 1 (Scarb1) and Apolipoprotein E (ApoE) genes play important roles in the proper maintenance of lipid levels in the body. The Scarb1 protein is an HDL receptor known for its role in the process of transporting cholesterol from peripheral tissues toward the liver for excretion. The ApoE protein combines with lipids to form lipoproteins that package cholesterol and carry it throughout the circulation. In order to study mice deficient in these proteins, our lab produced Scarb1 and ApoE double knockout (dKO) mice on the C57BL/6J (B6) and 129S1/SvImJ (129S1) background and discovered major phenotypic differences in size and survival. Mice with a mixed 129S1/B6 background succumb to lethal atherothrombosis at approximately 8 weeks of age (as seen in Figure 1), whereas dKOs generated on the B6 background die at approximately 5 days old. We hypothesized that specific 129S1 genetic variants modify the severity of atherothrombosis in dKO mice. Methods: We produced F1-F3 generation dKO mice on the mixed 129S1/B6 background for experiments. Weights were taken for the 38 dKO mice and their littermates from 3 weeks old until the death of the knockout mice at around 8 weeks of age. Purified DNA from the dKO mice was subjected to whole genome genotyping for 143,000 markers using the GigaMUGA genotyping platform (Neogen). Weight data were used as a phenotype to perform quantitative trait loci analysis (QTL) to identify potential loci responsible for the phenotypic weight differences. Results: dKO mice from F1-F3 generations were born at the expected Mendelian frequencies but exhibited 3 distinct phenotypes based on their weights relative to their littermates. dKO mouse weights fell into 3 distinct categories: (1) indistinguishable from littermates, (2) 14%-39% less than littermates, (3) greater than 39% less than littermates. QTL analysis identified 6 loci above the significance threshold of 4.04 on Chromosomes (Chr) 1 (LOD 6.82), 2 (LOD 6.44) , 3 (LOD 4.65), 6 (LOD 4.30), 12 (LOD 4.22) and 14 (LOD 5.36; Figure 2). In addition, interaction analyses identified significant interactions of Chr 12 with Chrs 1, 2, 3, and 14. The Chr 12 candidate region is between 83 and 93 Mb consisting of 89 genes. Niemann Pick type C2 ( Npc2), an intracellular cholesterol transporter, is within the candidate region. Thus, 129S1 variants in this region affecting Npc2 transcription are strong candidates for modifying the dKO phenotypes. Discussion: We have analyzed 38 dKO mice for genetic variants affecting their health, as measured by weights relative to their non dKO littermates. We identified 6 significant genetic loci, with Npc2 as a candidate gene for the Chromosome 12 locus. Studies to reinforce these findings include the analysis of an additional cohort of dKO mice to narrow the candidate QTL intervals, analysis of Npc2 expression in the B6 and 129 strain, histology of the liver to visualize lipid accumulation, blood smears to examine the blood cell size difference in dKOs, and sequencing with GigaMUGA. The identification of atherothrombosis modifier genes could result in novel treatment strategies for atherothrombosis.

Identifying genes involved in the secretory physiological response to feeding in Pacific White Shrimp (Litopenaeus vannamei) using transcriptomics

The physiological response to feeding is important for production aspects that include feed utilization and growth, and the responses require the action of numerous secretory factors. However, as an important aquaculture animal, the secretory response of Pacific White Shrimp (Litopenaeus vannamei) after feeding has not been comprehensively characterized. In this study, transcriptome analysis showed that 3172 differentially expressed genes were involved in the post-feeding response, including 289 new genes not annotated in the L. vannamei reference genome. Subsequently, 715 differentially expressed secretory reference genes and 18 new differentially expressed secretory genes were obtained through the identification of signal peptides in secreted proteins. Functional classification revealed that differentially expressed secretory genes were enriched in pathways pertaining to lipid metabolism (20 genes), carbohydrate metabolism (21 genes), glycan biosynthesis and metabolism (27 genes), digestive system (40 genes), and transport and metabolism (43 genes). The 14 pathways most enriched by differentially expressed secretory genes involved 83 genes, 71 of which encoded enzymes involved in food digestion and metabolism. Specific enzymes such as lipase 3-like and NPC intracellular cholesterol transporter 1-like in lipid metabolism, alpha-amylase-like and glucosylceramidase-like in carbohydrate metabolism, and cysteine proteinase 4-like and trypsin-1-like in the digestive system were found to be differentially expressed. Furthermore, we discovered a new gene, MSTRG.2504, that participates in the digestive system and carbohydrate metabolism. The study provides valuable insights into the secretory response (especially metabolism-related enzymes) to feeding in L. vannamei, uncovering the significant roles of both known and new genes. Furthermore, this study will improve our understanding of the feeding physiology of L. vannamei and provide a reference basis for further feeding endocrine research in the future.

Ocular manifestation of an adult Niemann-Pick disease type B

Niemann-Pick disease is a rare, autosomal recessive inherited lysosomal storage disorder. The pathophysiological background for this condition is the deficiency or reduced function of the enzyme sphingomyelinase, as well as a deficiency in the intracellular cholesterol transporter protein. Due to the breakdown defect, sphingomyelin and cholesterol accumulate in the lysosomes of cells. The disease is divided into 5 subtypes (A, A/B, B, C, D). The authors present the case of a 24-year-old young man diagnosed with Niemann-Pick disease type B as a child, focusing on the ophthalmic manifestation of the disease. During the examination of the patient, fundus photographs and fundus autofluorescence imaging were taken, and optical coherence tomography (OCT), optical coherence tomography angiography (OCTA), and visual field (perimetry) examinations were performed. The characteristic macular halo and the cherry-red spot in the fovea were clearly visible during ophthalmoscopy and on the fundus photographs. The OCT images showed focal thickening with high reflectivity in the ganglion cell layer corresponding to the macular halo, and the area of the foveola was spared. With visual field examination, an intact field of vision was found on both eyes. Similar to the presented patient, symptoms in patients with the B subtype are milder, and besides the visceral symptoms, there are no neurological symptoms, and the specific ophthalmic abnormalities do not cause visual impairment. Currently, Niemann-Pick disease is considered a rare disease, and the diagnosis of the patients is often delayed or even missed due to non-specific or mild symptoms. Through consultation between medical specialties, ophthalmological examination can also contribute to the correct diagnosis in cases with mild general symptoms. Timely diagnosis can potentially lead to mitigation of symptoms thanks to the ever-expanding therapeutic options, stabilization of the disease progression, and increase of the patients' life expectancy. Orv Hetil. 2023; 164(46): 1838-1844.

Role of S100A4 in the crosstalk between smooth muscle and inflammatory cells in atherosclerosis

Abstract Background During atherosclerotic plaque formation, smooth muscle cells (SMCs) accumulate in the intima and undergo a transition from a contractile to a synthetic phenotype. We previously showed that S100A4 is a marker of the synthetic SMCs in vitro and intimal SMCs in pig, human and mouse. In vitro, S100A4-treated SMCs acquire a pro-inflammatory phenotype. The systemic neutralization of extracellular S100A4 in ApoE-/- mice induces atherosclerotic plaque stabilization. Purpose To characterize the role of S100A4-expressing SMCs in atherosclerosis. Methods and results We established a coculture model between S100A4-stimulated human aortic SMCs and human blood-derived monocytes. Flow cytometry analysis of monocytes exposed to S100A4-treated SMCs for 3 days showed an upregulation of markers typical of inflammatory M1 (HLA-DR) and anti-inflammatory M2 (CD206) macrophages (fig. 1). Macrophages also upregulated mRNA expression of NPC Intracellular cholesterol transporter 1 and 2 by qPCR, suggesting modifications in their cholesterol metabolism. In vivo, in an SMC-lineage-tracing ApoE-/-mouse model, we identified S100A4-expressing SMCs at the onset of atherosclerosis in aortic media of 11-week-old mice fed a high cholesterol diet (HCD) for 3 weeks. Medial SMCs were characterized by means of single-cell RNA sequencing. Interestingly, 10 out of 11 identified clusters expressed S100A4, demonstrating broad and early involvement of S100A4 in shaping medial SMC identity. We assumed that blood-derived monocytes might be a driving force of early S100A4 activation in SMCs. To mitigate the impact of monocytes, 8-week-old ApoE-/- mice were fed an HCD for 9 weeks and injected with either clodronate-encapsulated or empty liposomes during the first 3 weeks. No differences in S100A4 expression in the media were observed by qPCR. Unexpectedly, ApoE-/- mice treated with clodronate demonstrated larger atherosclerotic lesions compared to control animals as measured by OilRedO staining (fig. 2). Studies are in progress to define the plaque composition. Conclusion Our findings shed light on the role of S100A4 in the crosstalk between SMCs and inflammatory cells in atherosclerosis and the fine-tuning of the phenotype of medial SMCs prior to their accumulation in the intima. We suggest that temporary monocyte depletion at the onset of the disease alters the mechanisms underlying plaque formation, worsening the outcome of the disease, while not affecting S100A4 expression in medial SMCs.

The Sterol Transporter Npc2c Controls Intestinal Stem Cell Mitosis and Host-Microbiome Interactions in Drosophila.

Cholesterol is necessary for all cells to function. The intracellular cholesterol transporters Npc1 and Npc2 control sterol trafficking and their malfunction leads to Neimann-Pick Type C disease, a rare disorder affecting the nervous system and the intestine. Unlike humans that encode single Npc1 and Npc2 transporters, flies encompass two Npc1 (Npc1a-1b) and eight Npc2 (Npc2a-2h) members, and most of the Npc2 family genes remain unexplored. Here, we focus on the intestinal function of Npc2c in the adult. We find that Npc2c is necessary for intestinal stem cell (ISC) mitosis, maintenance of the ISC lineage, survival upon pathogenic infection, as well as tumor growth. Impaired mitosis of Npc2c-silenced midguts is accompanied by reduced expression of Cyclin genes, and genes encoding ISC regulators, such as Delta, unpaired1 and Socs36E. ISC-specific Npc2c silencing induces Attacin-A expression, a phenotype reminiscent of Gram-negative bacteria overabundance. Metagenomic analysis of Npc2c-depleted midguts indicates intestinal dysbiosis, whereby decreased commensal complexity is accompanied by increased gamma-proteobacteria. ISC-specific Npc2c silencing also results in increased cholesterol aggregation. Interestingly, administration of the non-steroidal ecdysone receptor agonist, RH5849, rescues mitosis of Npc2c-silenced midguts and increases expression of the ecdysone response gene Broad, underscoring the role of Npc2c and sterols in ecdysone signaling. Assessment of additional Npc2 family members indicates potential redundant roles with Npc2c in ISC control and response to ecdysone signaling. Our results highlight a previously unidentified essential role of Npc2c in ISC mitosis, as well as an important role in ecdysone signaling and microbiome composition in the Drosophila midgut.

Bile acids-mediated intracellular cholesterol transport promotes intestinal cholesterol absorption and NPC1L1 recycling

Niemann-Pick C1-like 1 (NPC1L1) is essential for intestinal cholesterol absorption. Together with the cholesterol-rich and Flotillin-positive membrane microdomain, NPC1L1 is internalized via clathrin-mediated endocytosis and transported to endocytic recycling compartment (ERC). When ERC cholesterol level decreases, NPC1L1 interacts with LIMA1 and moves back to plasma membrane. However, how cholesterol leaves ERC is unknown. Here, we find that, in male mice, intracellular bile acids facilitate cholesterol transport to other organelles, such as endoplasmic reticulum, in a non-micellar fashion. When cholesterol level in ERC is decreased by bile acids, the NPC1L1 carboxyl terminus that previously interacts with the cholesterol-rich membranes via the A1272LAL residues dissociates from membrane, exposing the Q1277KR motif for LIMA1 recruitment. Then NPC1L1 moves back to plasma membrane. This study demonstrates an intracellular cholesterol transport function of bile acids and explains how the substantial amount of cholesterol in NPC1L1-positive compartments is unloaded in enterocytes during cholesterol absorption.

Early plasma proteomic biomarkers and prediction model of acute respiratory distress syndrome after cardiopulmonary bypass: a prospective nested cohort study.

Early recognition of the risk of acute respiratory distress syndrome (ARDS) after cardiopulmonary bypass (CPB) may improve clinical outcomes. The main objective of this study was to identify proteomic biomarkers and develop an early prediction model for CPB-ARDS. We conducted three prospective nested cohort studies of all consecutive patients undergoing cardiac surgery with CPB at XXX Hospital. Plasma proteomic profiling was performed in ARDS patients and matched controls (Cohort 1, April, 2021 to July, 2021) at multiple timepoints: before CPB (T1), at end of CPB (T2), and 24 h after CPB (T3). Then, for Cohort 2 (August, 2021 to July, 2022), biomarker expression was measured and verified in the plasma. Furthermore, lung ischemia/reperfusion injury (LIRI) models and sham-operation were established in 50 rats to explore the tissue-level expression of biomarkers identified in the aforementioned clinical cohort. Subsequently, a machine learning-based prediction model incorporating protein and clinical predictors from Cohort 2 for CPB-ARDS was developed and internally validated. Model performance was externally validated on Cohort 3 (January, 2023 to March, 2023). A total of 709 proteins were identified, with 9, 29, and 35 altered proteins between ARDS cases and controls at T1, T2, and T3, respectively, in Cohort 1. Following quantitative verification of several predictive proteins in Cohort 2, higher levels of thioredoxin domain containing 5 (TXNDC5), cathepsin L (CTSL), and NPC intracellular cholesterol transporter 2 (NPC2) at T2 were observed in CPB-ARDS patients. A dynamic online predictive nomogram was developed based on three proteins (TXNDC5, CTSL, and NPC2) and two clinical risk factors (CPB time, massive blood transfusion), with excellent performance (precision: 83.33%, sensitivity: 93.33%, specificity: 61.16%, F1 score: 85.05%). Mean area under the receiver operating characteristics curve (AUC) of the model after 10-fold cross-validation was 0.839 (95% confidence interval (CI): 0.824 to 0.855). Model discrimination and calibration were maintained during external validation dataset testing, with AUC of 0.820 (95% CI: 0.685 to 0.955) and Brier Score of 0.177 (95% CI: 0.147 to 0.206). Moreover, the considerably overexpressed TXNDC5 and CTSL proteins identified in the plasma of patients with CPB-ARDS, exhibited a significant upregulation in the lung tissue of LIRI rats. This study identified several novel predictive biomarkers, developed and validated a practical prediction tool using biomarker and clinical factor combinations for individual prediction of CPB-ARDS risk. Assessing the plasma TXNDC5, CTSL, and NPC2 levels might identify patients who warrant closer follow-up and intensified therapy for ARDS prevention following major surgery.