RESUMEN

Hematopoietic stem cells (HSCs) react to various stress conditions. However, it is unclear whether and how HSCs respond to severe anemia. Here, we demonstrate that upon induction of acute anemia, HSCs rapidly proliferate and enhance their erythroid differentiation potential. In severe anemia, lipoprotein profiles largely change and the concentration of ApoE increases. In HSCs, transcription levels of lipid metabolism-related genes, such as very low-density lipoprotein receptor (Vldlr), are upregulated. Stimulation of HSCs with ApoE enhances their erythroid potential, whereas HSCs in Apoe knockout mice do not respond to anemia induction. VldlrhighHSCs show higher erythroid potential, which is enhanced after acute anemia induction. VldlrhighHSCs are epigenetically distinct because of their low chromatin accessibility, and more chromatin regions are closed upon acute anemia induction. Chromatin regions closed upon acute anemia induction are mainly binding sites of Erg. Inhibition of Erg enhanced the erythroid differentiation potential of HSCs. Our findings indicate that lipoprotein metabolism plays an important role in HSC regulation under severe anemic conditions.

Asunto(s)

Anemia , Apolipoproteínas E , Diferenciación Celular , Células Madre Hematopoyéticas , Lipoproteínas , Animales , Anemia/metabolismo , Anemia/genética , Células Madre Hematopoyéticas/metabolismo , Ratones , Apolipoproteínas E/metabolismo , Apolipoproteínas E/genética , Lipoproteínas/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de LDL/metabolismo , Receptores de LDL/genética , Masculino , Cromatina/metabolismo , Eritropoyesis/genética , Células Eritroides/metabolismo

RESUMEN

Subretinal fibrosis permanently impairs the vision of patients with neovascular age-related macular degeneration. Despite emerging evidence revealing the association between disturbed metabolism in retinal pigment epithelium (RPE) and subretinal fibrosis, the underlying mechanism remains unclear. In the present study, single-cell RNA sequencing revealed, prior to subretinal fibrosis, genes in mitochondrial fatty acid oxidation are downregulated in the RPE lacking very low-density lipoprotein receptor (VLDLR), especially the rate-limiting enzyme carnitine palmitoyltransferase 1A (CPT1A). We found that overexpression of CPT1A in the RPE of Vldlr-/- mice suppresses epithelial-to-mesenchymal transition and fibrosis. Mechanistically, TGFß2 induces fibrosis by activating a Warburg-like effect, i.e. increased glycolysis and decreased mitochondrial respiration through ERK-dependent CPT1A degradation. Moreover, VLDLR blocks the formation of the TGFß receptor I/II complex by interacting with unglycosylated TGFß receptor II. In conclusion, VLDLR suppresses fibrosis by attenuating TGFß2-induced metabolic reprogramming, and CPT1A is a potential target for treating subretinal fibrosis.

Asunto(s)

Carnitina O-Palmitoiltransferasa , Fibrosis , Degeneración Macular , Mitocondrias , Receptores de LDL , Epitelio Pigmentado de la Retina , Factor de Crecimiento Transformador beta2 , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Animales , Degeneración Macular/metabolismo , Degeneración Macular/patología , Degeneración Macular/genética , Ratones , Mitocondrias/metabolismo , Mitocondrias/patología , Carnitina O-Palmitoiltransferasa/metabolismo , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/deficiencia , Factor de Crecimiento Transformador beta2/metabolismo , Factor de Crecimiento Transformador beta2/genética , Receptores de LDL/metabolismo , Receptores de LDL/genética , Receptores de LDL/deficiencia , Humanos , Ratones Noqueados , Transición Epitelial-Mesenquimal , Metabolismo Energético , Ratones Endogámicos C57BL

RESUMEN

Hypercholesterolemia forms the background of several cardiovascular pathologies. LDL receptor-knockout (LDLR-KO) mice kept on a high-fat diet (HFD) develop high cholesterol levels and atherosclerosis (AS). Cannabinoid type 1 receptors (CB1Rs) induce vasodilation, although their role in cardiovascular pathologies is still controversial. We aimed to reveal the effects of CB1Rs on vascular function and remodeling in hypercholesterolemic AS-prone LDLR-KO mice. Experiments were performed on a newly established LDLR and CB1R double-knockout (KO) mouse model, in which KO and wild-type (WT) mice were kept on an HFD or a control diet (CD) for 5 months. The vascular functions of abdominal aorta rings were tested with wire myography. The vasorelaxation effects of acetylcholine (Ach, 1 nM-1 µM) were obtained after phenylephrine precontraction, which was repeated with inhibitors of nitric oxide synthase (NOS) and cyclooxygenase (COX), Nω-nitro-L-arginine (LNA), and indomethacin (INDO), respectively. Blood pressure was measured with the tail-cuff method. Immunostaining of endothelial NOS (eNOS) was carried out. An HFD significantly elevated the cholesterol levels in the LDLR-KO mice more than in the corresponding WT mice (mean values: 1039 ± 162 mg/dL vs. 91 ± 18 mg/dL), and they were not influenced by the presence of the CB1R gene. However, with the defect of the CB1R gene, damage to the Ach relaxation ability was moderated. The blood pressure was higher in the LDLR-KO mice compared to their WT counterparts (systolic/diastolic values: 110/84 ± 5.8/6.8 vs. 102/80 ± 3.3/2.5 mmHg), which was significantly elevated with an HFD (118/96 ± 1.9/2 vs. 100/77 ± 3.4/3.1 mmHg, p < 0.05) but attenuated in the CB1R-KO HFD mice. The expression of eNOS was depressed in the HFD WT mice compared to those on the CD, but it was augmented if CB1R was knocked out. This newly established double-knockout mouse model provides a tool for studying the involvement of CB1Rs in the development of hypercholesterolemia and atherosclerosis. Our results indicate that knocking out the CB1R gene significantly attenuates vascular damage in hypercholesterolemic mice.

Asunto(s)

Modelos Animales de Enfermedad , Hipercolesterolemia , Ratones Noqueados , Receptor Cannabinoide CB1 , Receptores de LDL , Vasodilatación , Animales , Hipercolesterolemia/metabolismo , Hipercolesterolemia/genética , Hipercolesterolemia/patología , Ratones , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB1/genética , Receptores de LDL/genética , Receptores de LDL/metabolismo , Receptores de LDL/deficiencia , Vasodilatación/efectos de los fármacos , Dieta Alta en Grasa/efectos adversos , Masculino , Óxido Nítrico Sintasa de Tipo III/metabolismo , Aterosclerosis/metabolismo , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/etiología , Remodelación Vascular/efectos de los fármacos , Ratones Endogámicos C57BL , Acetilcolina/farmacología

RESUMEN

Purpose: Retinal neovascularization is a significant feature of advanced age-related macular degeneration (AMD) and a major cause of blindness in patients with AMD. However, the underlying mechanism of this pathological neovascularization remains unknown. Iron metabolism has been implicated in various biological processes. This study was conducted to investigate the effects of iron metabolism on retinal neovascularization in neovascular AMD (nAMD). Methods: C57BL/6J and very low-density lipoprotein receptor (VLDLR) knockout (Vldlr-/-) mice, a murine model of nAMD, were used in this study. Bulk-RNA sequencing was used to identify differentially expressed genes. Western blot analysis was performed to test the expression of proteins. Iron chelator deferiprone (DFP) was administrated to the mice by oral gavage. Fundus fluorescein angiography was used to evaluate retinal vascular leakage. Immunofluorescence staining was used to detect macrophages and iron-related proteins. Results: RNA sequencing (RNA-seq) results showed altered transferrin expression in the retina and RPE of Vldlr-/- mice. Disrupted iron homeostasis was observed in the retina and RPE of Vldlr-/- mice. DFP mitigated iron overload and significantly reduced retinal neovascularization and vascular leakage. In addition, DFP suppressed the inflammation in Vldlr-/- retinas. The reduced signals of macrophages were observed at sites of neovascularization in the retina and RPE of Vldlr-/- mice after DFP treatment. Further, the IL-6/JAK2/STAT3 signaling pathway was activated in the retina and RPE of Vldlr-/- mice and reversed by DFP treatment. Conclusions: Disrupted iron metabolism may contribute to retinal neovascularization in nAMD. Restoring iron homeostasis by DFP could be a potential therapeutic approach for nAMD.

Asunto(s)

Deferiprona , Modelos Animales de Enfermedad , Homeostasis , Quelantes del Hierro , Hierro , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Retiniana , Animales , Deferiprona/farmacología , Deferiprona/uso terapéutico , Quelantes del Hierro/farmacología , Quelantes del Hierro/uso terapéutico , Ratones , Hierro/metabolismo , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/tratamiento farmacológico , Neovascularización Retiniana/etiología , Neovascularización Retiniana/patología , Angiografía con Fluoresceína , Receptores de LDL/genética , Receptores de LDL/metabolismo , Western Blotting , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/patología , Degeneración Macular Húmeda/tratamiento farmacológico , Degeneración Macular Húmeda/metabolismo , Factor de Transcripción STAT3/metabolismo , Masculino

RESUMEN

Eastern Equine Encephalitis virus (EEEV) is an alphavirus that can cause severe diseases in infected humans. The very low-density lipoprotein receptor (VLDLR) was recently identified as a receptor of EEEV. Herein, we performed cryo-electron microscopy structural and biochemistry studies on the specific interactions between EEEV and VLDLR. Our results show that VLDLR binds EEEV at three different sites A, B and C through its membrane-distal LDLR class A (LA) repeats. Site A is located in the cleft in between the E1-E2 heterodimers. Site B is located near the connecting ß ribbon of E2 and is in proximity to site A, while site C is on the domain B of E2. The binding of VLDLR LAs to EEEV is in complex modes, including the LA1-2 and LA3-5 mediated two major modes. Disruption of the LA1-2 mediated binding significantly affect the cell attachment of EEEV. However, the mutation W132G of VLDLR impairs the binding of LA3, drives the switch of the binding modes, and significantly enhances the attachment of EEEV to the cell. The W132G variant of VLDLR could be identified in human genome and SNP sequences, implying that people with similar mutations in VLDLR may be highly susceptible to EEEV infection.

Asunto(s)

Virus de la Encefalitis Equina del Este , Unión Proteica , Receptores de LDL , Humanos , Sitios de Unión , Microscopía por Crioelectrón , Virus de la Encefalitis Equina del Este/genética , Virus de la Encefalitis Equina del Este/metabolismo , Células HEK293 , Modelos Moleculares , Mutación , Receptores de LDL/metabolismo , Receptores de LDL/genética , Receptores Virales/metabolismo , Acoplamiento Viral

RESUMEN

INTRODUCTION: Hypercholesterolemia is associated with increased inflammation and impaired serotonin neurotransmission, potentially contributing to depressive symptoms. However, the role of statins, particularly pitavastatin, in modulating serotonin transporter (SERT) function within this context remains underexplored. This study aimed to investigate whether pitavastatin counteracts the neurobiological effects of hypercholesterolemia. METHODS: Low-density lipoprotein receptor knockout (LDLR-/-) mice on a C57BL/6 background were assigned to three groups: a control group fed a standard chow diet, a group fed a high-fat diet (HFD), and a third group fed a high-fat diet supplemented with pitavastatin (HFD + Pita). We evaluated the effects of HFD with or without pitavastatin on lipid profiles, inflammatory markers, and SERT availability using small-animal positron emission tomography (PET) scans with the radioligand 4-[18F]-ADAM over a 20-week period. RESULTS: Pitavastatin treatment in HFD-fed mice significantly reduced both total cholesterol and LDL cholesterol levels in HFD-fed mice compared to those on HFD alone. Elevated inflammatory markers such as IL-1α, MCP-1/CCL2, and TNF-α in HFD mice were notably decreased in the HFD + Pita group. PET scans showed reduced SERT availability in the brains of HFD mice; however, pitavastatin improved this in brain regions associated with mood regulation, suggesting enhanced serotonin neurotransmission. Additionally, the sucrose preference test showed a trend towards increased preference in the HFD + Pita group compared to the HFD group, indicating a potential reduction in depressive-like behavior. CONCLUSION: Our findings demonstrate that pitavastatin not only lowers cholesterol and reduces inflammation but also enhances SERT availability, suggesting a potential role in alleviating depressive symptoms associated with hypercholesterolemia. These results highlight the multifaceted benefits of pitavastatin, extending beyond its lipid-lowering effects to potentially improving mood regulation and neurotransmitter function.

Asunto(s)

Dieta Alta en Grasa , Hipercolesterolemia , Ratones Endogámicos C57BL , Quinolinas , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Animales , Quinolinas/farmacología , Quinolinas/uso terapéutico , Hipercolesterolemia/tratamiento farmacológico , Hipercolesterolemia/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Ratones , Dieta Alta en Grasa/efectos adversos , Masculino , Ratones Noqueados , Receptores de LDL/metabolismo , Receptores de LDL/genética , Tomografía de Emisión de Positrones , LDL-Colesterol/sangre , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/diagnóstico por imagen , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico

RESUMEN

Atherosclerosis is a cardiovascular disease caused by cholesterol-laden arterial plaques. This study evaluated the correlation between interleukin-6 (IL-6), its receptors (IL6R/CD126), and glycoprotein 130 (gp130) alongside atherosclerosis biomarkers in a cohort of 142 subjects, equally divided between lean and obese individuals. Subsequent analyses used THP-1-derived macrophages to assess the biochemical impact of inhibiting IL-6 receptors. IL-6 secretion increased with atherosclerosis in obese subjects, while IL6R/CD126 and gp130 on monocytes decreased. Pharmacological gp130 inhibition altered lipid metabolism, increasing LDLR gene expression and cholesterol synthesis via SREBF2 and mevalonate kinase, along with HMG-CoA reductase at protein levels. gp130-deficient cells produced more cholesterol and had lower ABCA1 levels, suggesting hindered cholesterol efflux. Filipin III staining confirmed cholesterol retention in gp130-inhibited cells. Ex-vivo investigation on lean PBMCs further defined the impact of gp130 inhibition on the reduction of cholesterol efflux. Our results indicates gp130 is crucial for macrophage reverse cholesterol transport and may be a target for atherosclerosis treatments.

Asunto(s)

Aterosclerosis , Colesterol , Receptor gp130 de Citocinas , Macrófagos , Receptores de Interleucina-6 , Humanos , Colesterol/metabolismo , Macrófagos/metabolismo , Receptor gp130 de Citocinas/metabolismo , Masculino , Femenino , Aterosclerosis/metabolismo , Persona de Mediana Edad , Receptores de Interleucina-6/metabolismo , Interleucina-6/metabolismo , Transducción de Señal , Células THP-1 , Obesidad/metabolismo , Adulto , Transporte Biológico , Metabolismo de los Lípidos , Receptores de LDL/metabolismo

RESUMEN

OBJECTIVE: Carotenoids are lipophilic plant molecules with antioxidant properties. Some carotenoids such as ß-carotene also serve as vitamin A precursors, playing a key role in human health. Carotenoids are transported in lipoproteins with other lipids such as cholesterol, however, the mechanisms responsible for carotenoid storage in tissues and their non-enzymatic elimination remain relatively unexplored. The goal of this study was to examine the contribution of the low-density lipoprotein receptor (LDLR) in the bodily distribution and disposal of carotenoids. METHODS: We employed mice lacking one or both carotenoid-cleaving enzymes as suitable models for carotenoid accumulation. We examined the contribution of LDLR in carotenoid distribution by crossbreeding these mice with Ldlr-/- mice or overexpressing LDLR in the liver. RESULTS: Our results show that LDLR plays a dual role in carotenoid homeostasis by simultaneously favoring carotenoid storage in the liver and adipose tissue while facilitating their fecal elimination. CONCLUSIONS: Our results highlight a novel role of the LDLR in carotenoid homeostasis, and unveil a previously unrecognized disposal pathway for these important bioactive molecules.

Asunto(s)

Carotenoides , Heces , Homeostasis , Hígado , Ratones Endogámicos C57BL , Receptores de LDL , Animales , Receptores de LDL/metabolismo , Ratones , Hígado/metabolismo , Carotenoides/metabolismo , Heces/química , Ratones Noqueados , Masculino , Tejido Adiposo/metabolismo , Femenino , Dioxigenasas , beta-Caroteno 15,15'-Monooxigenasa

RESUMEN

Hepatocytes synthesize a vast number of glycoproteins found in their membranes and secretions, many of which contain O-glycans linked to Ser/Thr residues. As the functions and distribution of O-glycans on hepatocyte-derived membrane glycoproteins and blood glycoproteins are not well understood, we generated mice with a targeted deletion of Cosmc (C1Galt1c1) in hepatocytes. Liver glycoproteins in WT mice express typical sialylated core 1 O-glycans (T antigen/CD176) (Galß1-3GalNAcα1-O-Ser/Thr), whereas the Cosmc knockout hepatocytes (HEP-Cosmc-KO) lack extended O-glycans and express the Tn antigen (CD175) (GalNAcα1-O-Ser/Thr). Tn-containing glycoproteins occur in the sera of HEP-Cosmc-KO mice but not in WT mice. The LDL-receptor (LDLR), a well-studied O-glycosylated glycoprotein in hepatocytes, behaves as a ∼145kD glycoprotein in WT liver lysates, whereas it is reduced to ∼120 kDa in lysates from HEP-Cosmc-KO mice. Interestingly, the expression of the LDLR, as well as HMG-CoA reductase, which is typically altered in response to dysregulated cholesterol metabolism, are similar between WT and HEP-Cosmc-KO mice, indicating no significant effect by Cosmc deletion on either LDLR stability or cholesterol metabolism. Consistent with this, we observed no detectable phenotype in the HEP-Cosmc-KO mice regarding development, appearance or aging compared to WT. These results provide surprising, novel information about the pathway of O-glycosylation in the liver.

Asunto(s)

Hepatocitos , Ratones Noqueados , Polisacáridos , Animales , Hepatocitos/metabolismo , Ratones , Polisacáridos/metabolismo , Glicosilación , Galactosiltransferasas/metabolismo , Galactosiltransferasas/genética , Receptores de LDL/metabolismo , Receptores de LDL/genética , Chaperonas Moleculares

RESUMEN

Eastern equine encephalitis virus (EEEV) is the most virulent alphavirus that infects humans, and many survivors develop neurological sequelae, including paralysis and intellectual disability. Alphavirus spike proteins comprise trimers of heterodimers of glycoproteins E2 and E1 that mediate binding to cellular receptors and fusion of virus and host cell membranes during entry. We recently identified very-low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) as cellular receptors for EEEV and a distantly related alphavirus, Semliki Forest virus (SFV). Here, we use single-particle cryo-electron microscopy (cryo-EM) to determine structures of the EEEV and SFV spike glycoproteins bound to the VLDLR ligand-binding domain and found that EEEV and SFV interact with the same cellular receptor through divergent binding modes. Our studies suggest that the ability of LDLR-related proteins to interact with viral spike proteins through very small footprints with flexible binding modes results in a low evolutionary barrier to the acquisition of LDLR-related proteins as cellular receptors for diverse sets of viruses.

Asunto(s)

Microscopía por Crioelectrón , Virus de la Encefalitis Equina del Este , Receptores de LDL , Receptores de LDL/metabolismo , Receptores de LDL/química , Virus de la Encefalitis Equina del Este/metabolismo , Virus de la Encefalitis Equina del Este/ultraestructura , Humanos , Animales , Virus de los Bosques Semliki/metabolismo , Unión Proteica , Receptores Virales/metabolismo , Receptores Virales/química , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/ultraestructura , Modelos Moleculares

RESUMEN

Pancreatic ductal adenocarcinoma (PDAC) poses a significant threat due to its tendency to evade early detection, frequent metastasis, and the subsequent challenges in devising effective treatments. Processes that govern epithelial-mesenchymal transition (EMT) in PDAC hold promise for advancing novel therapeutic strategies. SAMD1 (SAM domain-containing protein 1) is a CpG island-binding protein that plays a pivotal role in the repression of its target genes. Here, we revealed that SAMD1 acts as a repressor of genes associated with EMT. Upon deletion of SAMD1 in PDAC cells, we observed significantly increased migration rates. SAMD1 exerts its effects by binding to specific genomic targets, including CDH2, encoding N-cadherin, which emerged as a driver of enhanced migration upon SAMD1 knockout. Furthermore, we discovered the FBXO11-containing E3 ubiquitin ligase complex as an interactor and negative regulator of SAMD1, which inhibits SAMD1 chromatin-binding genome-wide. High FBXO11 expression in PDAC is associated with poor prognosis and increased expression of EMT-related genes, underlining an antagonistic relationship between SAMD1 and FBXO11. In summary, our findings provide insights into the regulation of EMT-related genes in PDAC, shedding light on the intricate role of SAMD1 and its interplay with FBXO11 in this cancer type.

Asunto(s)

Carcinoma Ductal Pancreático , Transición Epitelial-Mesenquimal , Proteínas F-Box , Regulación Neoplásica de la Expresión Génica , Neoplasias Pancreáticas , Receptores de LDL , Animales , Humanos , Cadherinas/metabolismo , Cadherinas/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Movimiento Celular/genética , Transición Epitelial-Mesenquimal/genética , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Pronóstico , Receptores de LDL/genética , Receptores de LDL/metabolismo

RESUMEN

Transduction of producer cells during lentiviral vector (LVV) production causes the loss of 70-90% of viable particles. This process is called retro-transduction and it is a consequence of the interaction between the LVV envelope protein, VSV-G, and the LDL receptor located on the producer cell membrane, allowing lentiviral vector transduction. Avoiding retro-transduction in LVV manufacturing is crucial to improve net production and, therefore, the efficiency of the production process. Here, we describe a method for quantifying the transduction of producer cells and three different strategies that, focused on the interaction between VSV-G and the LDLR, aim to reduce retro-transduction.

Asunto(s)

Vectores Genéticos , Lentivirus , Receptores de LDL , Transducción Genética , Vectores Genéticos/genética , Lentivirus/genética , Humanos , Receptores de LDL/metabolismo , Receptores de LDL/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Células HEK293 , Glicoproteínas de Membrana

RESUMEN

BACKGROUND: Previous epidemiological studies have repeatedly found per- and polyfluoroalkyl substances (PFAS) exposure associated with higher circulating cholesterol, one of the greatest risk factors for development of coronary artery disease. The main route of cholesterol catabolism is through its conversion to bile acids, which circulate between the liver and ileum via enterohepatic circulation. Patients with coronary artery disease have decreased bile acid excretion, indicating that PFAS-induced impacts on enterohepatic circulation may play a critical role in cardiovascular risk. OBJECTIVES: Using a mouse model with high levels of low-density and very low-density lipoprotein (LDL and VLDL, respectively) cholesterol and aortic lesion development similar to humans, the present study investigated mechanisms linking exposure to a PFAS mixture with increased cholesterol. METHODS: Male and female Ldlr-/- mice were fed an atherogenic diet (Clinton/Cybulsky low fat, 0.15% cholesterol) and exposed to a mixture of 5 PFAS representing legacy, replacement, and emerging subtypes (i.e., PFOA, PFOS, PFHxS, PFNA, GenX), each at a concentration of 2mg/L, for 7 wk. Blood was collected longitudinally for cholesterol measurements, and mass spectrometry was used to measure circulating and fecal bile acids. Transcriptomic analysis of ileal samples was performed via RNA sequencing. RESULTS: After 7 wk of PFAS exposure, average circulating PFAS levels were measured at 21.6, 20.1, 31.2, 23.5, and 1.5µg/mL in PFAS-exposed females and 12.9, 9.7, 23, 14.3, and 1.7µg/mL in PFAS-exposed males for PFOA, PFOS, PFHxS, PFNA, and GenX, respectively. Total circulating cholesterol levels were higher in PFAS-exposed mice after 7 wk (352mg/dL vs. 415mg/dL in female mice and 392mg/dL vs. 488mg/dL in male mice exposed to vehicle or PFAS, respectively). Total circulating bile acid levels were higher in PFAS-exposed mice (2,978 pg/µL vs. 8,496 pg/µL in female mice and 1,960 pg/µL vs. 4,452 pg/µL in male mice exposed to vehicle or PFAS, respectively). In addition, total fecal bile acid levels were lower in PFAS-exposed mice (1,797 ng/mg vs. 682 ng/mg in females and 1,622 ng/mg vs. 670 ng/mg in males exposed to vehicle or PFAS, respectively). In the ileum, expression levels of the apical sodium-dependent bile acid transporter (ASBT) were higher in PFAS-exposed mice. DISCUSSION: Mice exposed to a PFAS mixture displayed higher circulating cholesterol and bile acids perhaps due to impacts on enterohepatic circulation. This study implicates PFAS-mediated effects at the site of the ileum as a possible critical mediator of increased cardiovascular risk following PFAS exposure. https://doi.org/10.1289/EHP14339.

Asunto(s)

Ácidos y Sales Biliares , Fluorocarburos , Animales , Ácidos y Sales Biliares/metabolismo , Ratones , Fluorocarburos/toxicidad , Masculino , Femenino , Receptores de LDL/genética , Receptores de LDL/metabolismo , Contaminantes Ambientales/toxicidad , Lípidos/sangre , Colesterol/sangre , Colesterol/metabolismo , Ácidos Alcanesulfónicos/toxicidad

RESUMEN

Hypercholesterolemia, characterized by elevated low-density lipoprotein (LDL) cholesterol levels, is a significant risk factor for cardiovascular disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a crucial role in cholesterol metabolism by regulating LDL receptor degradation, making it a therapeutic target for mitigating hypercholesterolemia-associated risks. In this context, we aimed to engineer human H ferritin as a scaffold to present 24 copies of a PCSK9-targeting domain. The rationale behind this protein nanoparticle design was to disrupt the PCSK9-LDL receptor interaction, thereby attenuating the PCSK9-mediated impairment of LDL cholesterol clearance. The N-terminal sequence of human H ferritin was engineered to incorporate a 13-amino acid linear peptide (Pep2-8), which was previously identified as the smallest PCSK9 inhibitor. Exploiting the quaternary structure of ferritin, engineered nanoparticles were designed to display 24 copies of the targeting peptide on their surface, enabling a multivalent binding effect. Extensive biochemical characterization confirmed precise control over nanoparticle size and morphology, alongside robust PCSK9-binding affinity (KD in the high picomolar range). Subsequent efficacy assessments employing the HepG2 liver cell line demonstrated the ability of engineered ferritin's ability to disrupt PCSK9-LDL receptor interaction, thereby promoting LDL receptor recycling on cell surfaces and consequently enhancing LDL uptake. Our findings highlight the potential of ferritin-based platforms as versatile tools for targeting PCSK9 in the management of hypercholesterolemia. This study not only contributes to the advancement of ferritin-based therapeutics but also offers valuable insights into novel strategies for treating cardiovascular diseases.

Asunto(s)

LDL-Colesterol , Nanopartículas , Proproteína Convertasa 9 , Receptores de LDL , Humanos , Proproteína Convertasa 9/metabolismo , Proproteína Convertasa 9/química , Proproteína Convertasa 9/genética , Receptores de LDL/metabolismo , Receptores de LDL/química , Nanopartículas/química , LDL-Colesterol/metabolismo , Inhibidores de PCSK9/farmacología , Inhibidores de PCSK9/química , Ferritinas/química , Ferritinas/metabolismo , Unión Proteica

RESUMEN

BACKGROUND: Activation of brown adipose tissue (BAT) has gained attention due to its ability to dissipate energy and counteract cardiometabolic diseases (CMDs). METHODS: This study investigated the consequences of cold exposure on the BAT and liver proteomes of an established CMD mouse model based on LDL receptor-deficient (LdlrKO) mice fed a high-fat, high-sucrose, high-cholesterol diet for 16 weeks. We analyzed energy metabolism in vivo and performed untargeted proteomics on BAT and liver of LdlrKO mice maintained at 22 °C or 5 °C for 7 days. RESULTS: We identified several dysregulated pathways, miRNAs, and transcription factors in BAT and liver of cold-exposed Ldlrko mice that have not been previously described in this context. Networks of regulatory interactions based on shared downstream targets and analysis of ligand-receptor pairs identified fibrinogen alpha chain (FGA) and fibronectin 1 (FN1) as potential crosstalk factors between BAT and liver in response to cold exposure. Importantly, genetic variations in the genes encoding FGA and FN1 have been associated with cardiometabolic-related phenotypes and traits in humans. DISCUSSION: This study describes the key factors, pathways, and regulatory networks involved in the crosstalk between BAT and the liver in a cold-exposed CMD mouse model. These findings may provide a basis for future studies aimed at testing whether molecular mediators, as well as regulatory and signaling mechanisms involved in tissue adaption upon cold exposure, could represent a target in cardiometabolic disorders.

Asunto(s)

Tejido Adiposo Pardo , Frío , Modelos Animales de Enfermedad , Metabolismo Energético , Redes Reguladoras de Genes , Hígado , Ratones Noqueados , Proteómica , Receptores de LDL , Transducción de Señal , Animales , Tejido Adiposo Pardo/metabolismo , Hígado/metabolismo , Metabolismo Energético/genética , Receptores de LDL/genética , Receptores de LDL/metabolismo , Receptores de LDL/deficiencia , Masculino , Fibrinógeno/metabolismo , Fibrinógeno/genética , Ratones Endogámicos C57BL , MicroARNs/metabolismo , MicroARNs/genética , Fibronectinas/metabolismo , Fibronectinas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ratones , Regulación de la Expresión Génica , Mapas de Interacción de Proteínas

RESUMEN

RATIONALE: Very-low-density lipoprotein receptor (VLDLR) involves in ocular neovascularization, a major cause of severe vision loss. However, the underlying molecular mechanisms were not completely clarified. Here, we aimed to investigate roles of circular RNAs (circRNAs) in VLDLR-associated ocular neovascularization. METHODS: Vldlr knockout (Vldlr-/-, ko), Robo4 knockout (Robo4-/-, ko) and wild-type (WT) mice were used. Mouse model of oxygen induced retinopathy (OIR) and high-throughput sequence were performed to profile the differential expression of circRNA and transcripts. RNase R treatment, Sanger PCR sequencing and quantitative polymerase chain reaction (qPCR) were used to validate candidate circRNAs and their expression patterns. Choroidal sprouting assay ex vivo and laser induction choroid neovascularization were used to determine the expression and functions of QKI/CircSlc17a5 on choroidal neovascularization. RESULTS: In macrophage and ocular tissues derived from Vldlr (Vldlr-/-,Vldlr ko) or Robo4 (Robo4-/-,Robo4 ko) deficiency as well as wild-type (WT) mice, Quaking (Qki) expression was significantly down-regulated in Vldlr deficiency compared to WT and Robo4 deficiency groups. Ectopic VLDLR expression or Reelin stimulation increased expression of QKI in bEnd.3 cells. Circular RNA sequencing uncovered that VLDLR regulated the biogenesis of certain circular RNAs, including the circSlc17a5. The number of Circular RNAs increased in mice treated with OIR. QKI mediated the biogenesis of circSlc17a5, which was an important regulator of choroidal angiogenesis. CONCLUSION: CircSlc17a5 regulated by VLDLR/QKI plays important roles in the choroidal angiogenesis.

Asunto(s)

Neovascularización Coroidal , Ratones Noqueados , ARN Circular , Receptores de LDL , Animales , Neovascularización Coroidal/genética , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Receptores de LDL/genética , Receptores de LDL/metabolismo , Ratones , ARN Circular/genética , ARN Circular/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteína Reelina , Transducción de Señal , Ratones Endogámicos C57BL , Coroides/metabolismo , Coroides/irrigación sanguínea , Modelos Animales de Enfermedad , Angiogénesis

RESUMEN

Foods enriched with insects can potentially prevent several health disorders, including cardiovascular diseases, by reducing inflammation and improving antioxidant status. In this study, Tenebrio molitor and Gryllus assimilis were selected to determine the effect on the development of atherosclerosis in ApoE/LDLR-/- mice. Animals were fed AIN-93G-based diets (control) with 10% Tenebrio molitor (TM) and 10% Gryllus assimilis (GA) for 8 weeks. The nutritional value as well as antioxidant activity of selected insects were determined. The lipid profile, liver enzyme activity, and the fatty acid composition of liver and adipose tissue of model mice were evaluated. Quantitative analysis of atherosclerotic lesions in the entire aorta was performed using the en face method, and for aortic roots, the cross-section method was used. The antioxidant status of the GA cricket was significantly higher compared to the TM larvae. The results showed that the area of atherosclerosis (en face method) was not significantly different between groups. Dietary GA reduced plaque formation in the aortic root; additionally, significant differences were observed in sections at 200 and 300 µm compared to other groups. Furthermore, liver enzyme ALT activity was lower in insect-fed groups compared to the control group. The finding suggests that a diet containing edible insect GA potentially prevents atherosclerotic plaque development in the aortic root, due to its high antioxidant activity.

Asunto(s)

Apolipoproteínas E , Aterosclerosis , Receptores de LDL , Animales , Aterosclerosis/patología , Aterosclerosis/metabolismo , Ratones , Receptores de LDL/genética , Receptores de LDL/metabolismo , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Insectos Comestibles , Ratones Noqueados , Hígado/metabolismo , Hígado/patología , Antioxidantes/metabolismo , Masculino , Tenebrio , Dieta , Aorta/patología , Aorta/metabolismo , Modelos Animales de Enfermedad , Alimentación Animal , Placa Aterosclerótica/patología , Placa Aterosclerótica/metabolismo , Gryllidae

RESUMEN

There was no significant difference in the composition and content of fatty acids in eggs among different breeds initially, but following the supplementation of flaxseed oil, Dwarf Layer were observed to deposit more n-3 polyunsaturated fatty acid (PUFA) in eggs. Currently, there is limited research on the mechanisms underlying the differences in egg composition among different breeds. Therefore, in this study, 150 twenty-four-wk-old hens of each breed, including the Dwarf Layer and White Leghorn, were fed either a basal diet or a diet supplemented with 2.5% flaxseed oil. After 28 d, eggs and liver samples were collected to determine fatty acid composition, and serum, liver, intestine, and follicles were collected for subsequent biochemical, intestinal morphology, and lipid metabolism-related genes expression analysis. Duodenal contents were collected for microbial analysis. The results showed that there was no significant difference in the content and deposition efficiency of total n-3 PUFA in the liver of the 2 breeds, but the content and deposition efficiency of total n-3 PUFA in the egg of Dwarf Layer were significantly higher than those of White Leghorn after feeding flaxseed oil. Flaxseed oil and breeds did not have significant effects on cholesterol (CHO), free fatty acids (NEFA), low-density lipoprotein (LDL), and estrogen (E2) levels. After feeding with flaxseed oil, the villus height and the villus-to-crypt ratio in both breeds were increased and duodenal crypt depth was decreased. The villus-to-crypt ratio (4.78 vs. 3.60) in the duodenum of Dwarf Layer was significantly higher than that in White Leghorn after feeding with flaxseed oil. Flaxseed oil can impact the gut microbiota in the duodenum and reduce the microbiota associated with fatty acid breakdown, such as Romboutsia, Subdolibranulum, Lachnochlostridium, and Clostridium. This may mean that less ALA can be decomposed and more ALA can be absorbed into the body. Additionally, after feeding flaxseed oil, the mRNA levels of elongation enzymes 5 (ELOVL5), fatty acid desaturase 1 (FADS1), and fatty acid transporter 1 (FATP1) in the liver of Dwarf Layer were significantly higher than those in White Leghorn, while the mRNA levels of peroxisome proliferator-activated receptor alpha (PPAR), carnitine palmitoyl transferase 1 (CPT1), Acyl CoA oxidase 1 (ACOX1), and Acyl-CoA synthetase (ACSL) were significantly lower than those in White Leghorn. The mRNA level of FABP1 in the duodenum of Dwarf Layer was significantly higher than that of White Leghorn, while the mRNA level of FATP1 was significantly lower than that of White Leghorn. The protein levels of ELOVL5 in the liver of Dwarf Layer and very low-density lipoprotein receptor (VLDLR) in the follicles were significantly higher than those of White Leghorn. In summary, after feeding flaxseed oil, the higher ratio of villus height to crypt depth in Dwarf Layer allows more α-linolenic acid (ALA) to be absorbed into the body. The higher mRNA expression of FADS1, ELOVL5, and FATP1, as well as the higher protein expression of ELOVL5 in the liver of Dwarf Layer enhance the conversion of ALA into DHA. The higher protein expression of VLDLR in follicles of Dwarf Layer allows more n-3 PUFA to deposit in the follicles. These combined factors contribute to the Dwarf Layer's ability to deposit higher levels of n-3 PUFA in eggs, as well as improving the deposition efficiency of n-3 PUFA.

Asunto(s)

Alimentación Animal , Pollos , Ácidos Grasos Omega-3 , Aceite de Linaza , Animales , Femenino , Alimentación Animal/análisis , Proteínas Aviares/metabolismo , Proteínas Aviares/genética , Pollos/fisiología , Pollos/metabolismo , Pollos/genética , Dieta/veterinaria , Suplementos Dietéticos/análisis , Ácidos Grasos Omega-3/metabolismo , Ácidos Grasos Omega-3/análisis , Ácidos Grasos Omega-3/administración & dosificación , Aceite de Linaza/administración & dosificación , Aceite de Linaza/metabolismo , Hígado/metabolismo , Óvulo/química , Receptores de LDL/metabolismo , Receptores de LDL/genética

RESUMEN

BACKGROUND: In early atherosclerosis, circulating LDLs (low-density lipoproteins) traverse individual endothelial cells by an active process termed transcytosis. The CANTOS trial (Canakinumab Antiinflammatory Thrombosis Outcome Study) treated advanced atherosclerosis using a blocking antibody for IL-1ß (interleukin-1ß); this significantly reduced cardiovascular events. However, whether IL-1ß regulates early disease, particularly LDL transcytosis, remains unknown. METHODS: We used total internal reflection fluorescence microscopy to quantify transcytosis by human coronary artery endothelial cells exposed to IL-1ß. To investigate transcytosis in vivo, we injected wild-type and knockout mice with IL-1ß and LDL to visualize acute LDL deposition in the aortic arch. RESULTS: Exposure to picomolar concentrations of IL-1ß induced transcytosis of LDL but not of albumin by human coronary artery endothelial cells. Surprisingly, expression of the 2 known receptors for LDL transcytosis, ALK-1 (activin receptor-like kinase-1) and SR-BI (scavenger receptor BI), was unchanged or decreased. Instead, IL-1ß increased the expression of the LDLR (LDL receptor); this was unexpected because LDLR is not required for LDL transcytosis. Overexpression of LDLR had no effect on basal LDL transcytosis. However, knockdown of LDLR abrogated the effect of IL-1ß on transcytosis rates while the depletion of Cav-1 (caveolin-1) did not. Since LDLR was necessary but overexpression had no effect, we reasoned that another player must be involved. Using public RNA sequencing data to curate a list of Rab (Ras-associated binding) GTPases affected by IL-1ß, we identified Rab27a. Overexpression of Rab27a alone had no effect on basal transcytosis, but its knockdown prevented induction by IL-1ß. This was phenocopied by depletion of the Rab27a effector JFC1 (synaptotagmin-like protein 1). In vivo, IL-1ß increased LDL transcytosis in the aortic arch of wild-type but not Ldlr-/- or Rab27a-deficient mice. The JFC1 inhibitor nexinhib20 also blocked IL-1ß-induced LDL accumulation in the aorta. CONCLUSIONS: IL-1ß induces LDL transcytosis by a distinct pathway requiring LDLR and Rab27a; this route differs from basal transcytosis. We speculate that induction of transcytosis by IL-1ß may contribute to the acceleration of early disease.

Asunto(s)

Vasos Coronarios , Células Endoteliales , Interleucina-1beta , Lipoproteínas LDL , Ratones Noqueados , Receptores de LDL , Transducción de Señal , Transcitosis , Proteínas de Unión al GTP rab , Interleucina-1beta/metabolismo , Animales , Humanos , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Lipoproteínas LDL/metabolismo , Vasos Coronarios/metabolismo , Vasos Coronarios/efectos de los fármacos , Células Cultivadas , Ratones Endogámicos C57BL , Caveolina 1/metabolismo , Caveolina 1/genética , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Modelos Animales de Enfermedad , Aorta Torácica/metabolismo , Aorta Torácica/efectos de los fármacos , Aterosclerosis/metabolismo , Aterosclerosis/genética , Aterosclerosis/patología , Masculino , Ratones

RESUMEN

Western equine encephalitis virus (WEEV) is an arthropod-borne virus (arbovirus) that frequently caused major outbreaks of encephalitis in humans and horses in the early twentieth century, but the frequency of outbreaks has since decreased markedly, and strains of this alphavirus isolated in the past two decades are less virulent in mammals than strains isolated in the 1930s and 1940s1-3. The basis for this phenotypic change in WEEV strains and coincident decrease in epizootic activity (known as viral submergence3) is unclear, as is the possibility of re-emergence of highly virulent strains. Here we identify protocadherin 10 (PCDH10) as a cellular receptor for WEEV. We show that multiple highly virulent ancestral WEEV strains isolated in the 1930s and 1940s, in addition to binding human PCDH10, could also bind very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), which are recognized by another encephalitic alphavirus as receptors4. However, whereas most of the WEEV strains that we examined bind to PCDH10, a contemporary strain has lost the ability to recognize mammalian PCDH10 while retaining the ability to bind avian receptors, suggesting WEEV adaptation to a main reservoir host during enzootic circulation. PCDH10 supports WEEV E2-E1 glycoprotein-mediated infection of primary mouse cortical neurons, and administration of a soluble form of PCDH10 protects mice from lethal WEEV challenge. Our results have implications for the development of medical countermeasures and for risk assessment for re-emerging WEEV strains.

Asunto(s)

Virus de la Encefalitis Equina del Oeste , Especificidad del Huésped , Protocadherinas , Receptores Virales , Animales , Femenino , Humanos , Masculino , Ratones , Aves/metabolismo , Aves/virología , Enfermedades Transmisibles Emergentes/epidemiología , Enfermedades Transmisibles Emergentes/virología , Virus de la Encefalitis Equina del Oeste/clasificación , Virus de la Encefalitis Equina del Oeste/metabolismo , Virus de la Encefalitis Equina del Oeste/patogenicidad , Encefalomielitis Equina/epidemiología , Encefalomielitis Equina/virología , Proteínas Relacionadas con Receptor de LDL/metabolismo , Neuronas/metabolismo , Neuronas/virología , Fenotipo , Protocadherinas/metabolismo , Receptores de LDL/metabolismo , Receptores de LDL/genética , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Zoonosis Virales/epidemiología , Zoonosis Virales/virología