RESUMEN
BACKGROUND: Increasing evidence highlights the importance of novel players in Alzheimer's disease (AD) pathophysiology, including alterations of lipid metabolism and neuroinflammation. Indeed, a potential involvement of Proprotein convertase subtilisin/kexin type 9 (PCSK9) in AD has been recently postulated. Here, we first investigated the effects of PCSK9 on neuroinflammation in vitro. Then, we examined the impact of a genetic ablation of PCSK9 on cognitive performance in a severe mouse model of AD. Finally, in the same animals we evaluated the effect of PCSK9 loss on Aß pathology, neuroinflammation, and brain lipids. METHODS: For in vitro studies, U373 human astrocytoma cells were treated with Aß fibrils and human recombinant PCSK9. mRNA expression of the proinflammatory cytokines and inflammasome-related genes were evaluated by q-PCR, while MCP-1 secretion was measured by ELISA. For in vivo studies, the cognitive performance of a newly generated mouse line - obtained by crossing 5XFADHet with PCSK9KO mice - was tested by the Morris water maze test. After sacrifice, immunohistochemical analyses were performed to evaluate Aß plaque deposition, distribution and composition, BACE1 immunoreactivity, as well as microglia and astrocyte reactivity. Cholesterol and hydroxysterols levels in mouse brains were quantified by fluorometric and LC-MS/MS analyses, respectively. Statistical comparisons were performed according to one- or two-way ANOVA, two-way repeated measure ANOVA or Chi-square test. RESULTS: In vitro, PCSK9 significantly increased IL6, IL1B and TNFΑ mRNA levels in Aß fibrils-treated U373 cells, without influencing inflammasome gene expression, except for an increase in NLRC4 mRNA levels. In vivo, PCSK9 ablation in 5XFAD mice significantly improved the performance at the Morris water maze test; these changes were accompanied by a reduced corticohippocampal Aß burden without affecting plaque spatial/regional distribution and composition or global BACE1 expression. Furthermore, PCSK9 loss in 5XFAD mice induced decreased microgliosis and astrocyte reactivity in several brain regions. Conversely, knocking out PCSK9 had minimal impact on brain cholesterol and hydroxysterol levels. CONCLUSIONS: In vitro studies showed a pro-inflammatory effect of PCSK9. Consistently, in vivo data indicated a protective role of PCSK9 ablation against cognitive impairments, associated with improved Aß pathology and attenuated neuroinflammation in a severe mouse model of AD. PCSK9 may thus be considered a novel pharmacological target for the treatment of AD.
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Enfermedad de Alzheimer , Disfunción Cognitiva , Ratones , Humanos , Animales , Ratones Transgénicos , Proproteína Convertasa 9/uso terapéutico , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Secretasas de la Proteína Precursora del Amiloide/uso terapéutico , Enfermedades Neuroinflamatorias , Cromatografía Liquida , Inflamasomas , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Ácido Aspártico Endopeptidasas/uso terapéutico , Espectrometría de Masas en Tándem , Enfermedad de Alzheimer/metabolismo , ARN Mensajero , Colesterol , Péptidos beta-Amiloides/metabolismo , Modelos Animales de EnfermedadRESUMEN
STUDY QUESTION: Does LH addition to FSH in vitro recover the human primary granulosa lutein cell (hGLC) sub/poor-response? SUMMARY ANSWER: A picomolar concentration of LH may recover the FSH-induced cAMP and progesterone production of hGLC from sub/poor-responder women. WHAT IS KNOWN ALREADY: Clinical studies suggested that FSH and LH co-treatment may be beneficial for the ovarian response of sub/poor-responders undergoing ovarian stimulation during ART. STUDY DESIGN, SIZE, DURATION: hGLC samples from 286 anonymous women undergoing oocyte retrieval for ART were collected from October 2017 to February 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: hGLCs from women undergoing ovarian stimulation during ART were blindly purified, cultured, genotyped and treated in vitro by increasing concentrations of FSH (nM) ±0.5 nM LH. cAMP and progesterone levels produced after 3 and 24 h, respectively, were measured. In vitro data were stratified a posteriori, according to the donors' ovarian response, into normo-, sub- and poor-responder groups and statistically compared. The effects of LH addition to FSH were compared with those obtained by FSH alone in all the groups as well. MAIN RESULTS AND THE ROLE OF CHANCE: hGLCs from normo-responders were shown to have higher sensitivity to FSH treatment than sub-/poor-responders in vitro. Equimolar FSH concentrations induced higher cAMP (about 2.5- to 4.2-fold), and progesterone plateau levels (1.2- to 2.1-fold), in cells from normo-responder women than those from sub-/poor-responders (ANOVA; P < 0.05). The addition of LH to the cell treatment significantly increased overall FSH efficacy, indicated by cAMP and progesterone levels, within all groups (P > 0.05). Interestingly, these in vitro endpoints, collected from the normo-responder group treated with FSH alone, were similar to those obtained in the sub-/poor-responder group under FSH + LH treatment. No different allele frequencies and FSH receptor (FSHR) gene expression levels between groups were found, excluding genetics of gonadotropin and their receptors as a factor linked to the normo-, sub- and poor-response. In conclusion, FSH elicits phenotype-specific ovarian lutein cell response. Most importantly, LH addition may fill the gap between cAMP and steroid production patterns between normo- and sub/poor-responders. LIMITATIONS, REASONS FOR CAUTION: Although the number of experimental replicates is overall high for an in vitro study, clinical trials are required to demonstrate if the endpoints evaluated herein reflect parameters of successful ART. hGLC retrieved after ovarian stimulation may not fully reproduce the response to hormones of granulosa cells from the antral follicular stage. WIDER IMPLICATIONS OF THE FINDINGS: This in vitro assay may describe the individual response to personalize ART stimulation protocol, according to the normo-, sub- and poor-responder status. Moreover, this in vitro study supports the need to conduct optimally designed, randomized clinical trials exploring the personalized use of LH in assisted reproduction. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Merck KGaA. M.L. and C.C. are employees of Merck KGaA or of the affiliate Merck Serono SpA. Other authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.
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Hormona Folículo Estimulante , Células Lúteas , Humanos , Femenino , Hormona Folículo Estimulante/uso terapéutico , Células Lúteas/metabolismo , Progesterona , Gonadotropinas , Reproducción , Inducción de la Ovulación/métodos , Fertilización In Vitro/métodosRESUMEN
OBJECTIVE: HDL (high-density lipoprotein) particles are known to possess several antiatherogenic properties that include the removal of excess cholesterol from peripheral tissues, the maintenance of endothelial integrity, antioxidant, and anti-inflammatory activities. ApoA-I overexpression in apoE-deficient (EKO) mice has been shown to increase HDL levels and to strongly reduce atherosclerosis development. The aim of the study was to investigate gene expression patterns associated with atherosclerosis development in the aorta of EKO mice and how HDL plasma levels relate to gene expression patterns at different stages of atherosclerosis development and with different dietary treatments. Approach and Results: Eight-week-old EKO mice, EKO mice overexpressing human apoA-I, and wild-type mice as controls were fed either normal laboratory or Western diet for 6 or 22 weeks. Cholesterol distribution among lipoproteins was evaluated, and atherosclerosis of the aorta was quantified. High-throughput sequencing technologies were used to analyze the transcriptome of the aorta of the 3 genotypes in each experimental condition. In addition to the well-known activation of inflammation and immune response, the impairment of sphingolipid metabolism, phagosome-lysosome system, and osteoclast differentiation emerged as relevant players in atherosclerosis development. The reduced atherosclerotic burden in the aorta of EKO mice expressing high levels of apoA-I was accompanied by a reduced activation of immune system markers, as well as reduced perturbation of lysosomal activity and a better regulation of the sphingolipid synthesis pathway. CONCLUSIONS: ApoA-I modulates atherosclerosis development in the aorta of EKO mice affecting the expression of pathways additional to those associated with inflammation and immune response.
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Aorta/metabolismo , Enfermedades de la Aorta/genética , Apolipoproteína A-I/metabolismo , Aterosclerosis/genética , Inflamación/genética , Lisosomas/genética , Esfingolípidos/metabolismo , Transcriptoma , Animales , Aorta/patología , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Apolipoproteína A-I/sangre , Apolipoproteína A-I/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Células Cultivadas , Colesterol/sangre , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Inflamación/metabolismo , Inflamación/patología , Lisosomas/metabolismo , Lisosomas/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Placa Aterosclerótica , Mapas de Interacción de Proteínas , Transducción de Señal , Esfingolípidos/sangre , Factores de Tiempo , Triglicéridos/sangreRESUMEN
Objective: ApoM enriches S1P (sphingosine-1-phosphate) within HDL (high-density lipoproteins) and facilitates the activation of the S1P1 (S1P receptor type 1) by S1P, thereby preserving endothelial barrier function. Many protective functions exerted by HDL in extravascular tissues raise the question of how S1P regulates transendothelial HDL transport. Approach and Results: HDL were isolated from plasma of wild-type mice, Apom knockout mice, human apoM transgenic mice or humans and radioiodinated to trace its binding, association, and transport by bovine or human aortic endothelial cells. We also compared the transport of fluorescently-labeled HDL or Evans Blue, which labels albumin, from the tail vein into the peritoneal cavity of apoE-haploinsufficient mice with (apoE-haploinsufficient mice with endothelium-specific knockin of S1P1) or without (control mice, ie, apoE-haploinsufficient mice without endothelium-specific knockin of S1P1) endothelium-specific knockin of S1P1. The binding, association, and transport of HDL from Apom knockout mice and human apoM-depleted HDL by bovine aortic endothelial cells was significantly lower than that of HDL from wild-type mice and human apoM-containing HDL, respectively. The binding, uptake, and transport of 125I-HDL by human aortic endothelial cells was increased by an S1P1 agonist but decreased by an S1P1 inhibitor. Silencing of SR-BI (scavenger receptor BI) abrogated the stimulation of 125I-HDL transport by the S1P1 agonist. Compared with control mice, that is, apoE-haploinsufficient mice without endothelium-specific knockin of S1P1, apoE-haploinsufficient mice with endothelium-specific knockin of S1P1 showed decreased transport of Evans Blue but increased transport of HDL from blood into the peritoneal cavity and SR-BI expression in the aortal endothelium. Conclusions: ApoM and S1P1 promote transendothelial HDL transport. Their opposite effect on transendothelial transport of albumin and HDL indicates that HDL passes endothelial barriers by specific mechanisms rather than passive filtration.
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Apolipoproteínas M/metabolismo , Aterosclerosis/metabolismo , Células Endoteliales/metabolismo , Lipoproteínas HDL/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Animales , Aterosclerosis/genética , Aterosclerosis/patología , Transporte Biológico , Bovinos , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/patología , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Permeabilidad , Placa Aterosclerótica , Receptores Depuradores de Clase B/genética , Receptores Depuradores de Clase B/metabolismo , Receptores de Esfingosina-1-Fosfato/genéticaRESUMEN
Endothelial dysfunction is an early event in the pathogenesis of atherosclerosis and represents the first step in the pathogenesis of cardiovascular diseases. The evaluation of endothelial health is fundamental in clinical practice and several direct and indirect markers have been suggested so far to identify any alterations in endothelial homeostasis. Alongside the known endothelial role on vascular health, several pieces of evidence have demonstrated that proper endothelial functioning plays a key role in human fertility and reproduction. Therefore, this state-of-the-art review updates the endothelial health markers discriminating between those available for clinical practice or for research purposes and their application in human fertility. Moreover, new molecules potentially helpful to clarify the link between endothelial and reproductive health are evaluated herein.
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Biomarcadores/metabolismo , Enfermedades Cardiovasculares/metabolismo , Endotelio Vascular/metabolismo , Fertilidad/fisiología , Animales , Homeostasis , Humanos , Reproducción/fisiologíaRESUMEN
Multiple factors may affect the metabolic fate of carbohydrates. Today, well-standardised and accepted methods may allow for the definitions of the changes in the glucose and insulin curves following the ingestion of either carbohydrate-based and other foods. More debate is still raised on the clinical meaning of these classifications when used at a population level, while emphasis is raised on the approach to carbohydrate metabolism on an individual basis. Within these ranges of applications, other compounds, such as plant polyphenols, may favourably add synergic effects through the modulation of carbohydrate digestion and glucose metabolic steps, resulting in lowering postprandial glucose and insulin levels. Finally, a growing knowledge suggests that the balance of dietary fructose and individual physical activity represent the key point to address the compound towards either positive, energy sparing effects, or a degenerative metabolic burden. The carbohydrate quality within a whole dietary and lifestyle pattern may therefore challenge the individual balance towards health or disease.
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Carbohidratos de la Dieta/administración & dosificación , Índice Glucémico , Glucemia , Dieta , Fructosa , Humanos , Metabolismo de los Lípidos , Comidas , Ciencias de la Nutrición , Periodo Posprandial , InvestigaciónRESUMEN
Clusterin (CLU) is a chaperone-like protein with multiple functions. sCLU is frequently upregulated in prostate tumor cells after chemo- or radiotherapy and after surgical or pharmacological castration. Moreover, CLU has been documented to modulate the cellular homolog of murine thymoma virus akt8 oncogene (AKT) activity. Here, we investigated how CLU overexpression influences phosphatidylinositol 3'-kinase (PI3K)/AKT signaling in human normal and cancer epithelial prostate cells. Human prostate cells stably transfected with CLU were broadly profiled by reverse phase protein array (RPPA), with particular emphasis on the PI3K/AKT pathway. The effect of CLU overexpression on normal and cancer cell motility was also tested. Our results clearly indicate that CLU overexpression enhances phosphorylation of AKT restricted to isoform 2. Mechanistically, this can be explained by the finding that the phosphatase PH domain leucine-rich repeat-containing protein phosphatase 1 (PHLPP1), known to dephosphorylate AKT2 at S474, is markedly downregulated by CLU, whereas miR-190, a negative regulator of PHLPP1, is upregulated. Moreover, we found that phosphatase and tensin homolog (PTEN) was heavily phosphorylated at the inhibitory site S380, contributing to the hyperactivation of AKT signaling. By keeping AKT2 phosphorylation high, CLU dramatically enhances the migratory behavior of prostate epithelial cell lines with different migratory and invasive phenotypes, namely prostate normal epithelial 1A (PNT1A) and prostatic carcinoma 3 (PC3) cells. Altogether, our results unravel for the first time a circuit by which CLU can switch a low migration phenotype toward a high migration phenotype, through miR-190-dependent downmodulation of PHLPP1 expression and, in turn, stabilization of AKT2 phosphorylation.
Asunto(s)
Clusterina/metabolismo , Proteínas Nucleares/metabolismo , Fosfohidrolasa PTEN/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células 3T3 , Animales , Línea Celular Tumoral , Movimiento Celular/fisiología , Clusterina/genética , Células Epiteliales/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , Ratones , MicroARNs/genética , Células PC-3 , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Próstata/patología , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal/genéticaRESUMEN
RESEARCH QUESTION: What is the cumulative effect of two follicle-stimulating hormone receptor (FSHR) mutations in spontaneous ovarian hyperstimulation syndrome (sOHSS) pathogenesis? Are these mutations in the mono- or biallelic state? DESIGN: Two FSHR mutations were found in a pregnant patient affected by sOHSS with no predisposing conditions. While the p.Asn106His mutation is novel, the p.Ser128Tyr mutation has been associated with sOHSS previously. The patient's FSHR gene was analysed by Sanger sequencing, and FSHR cDNAs carrying a single or both point mutations were created by mutagenesis in vitro. cAMP activation by recombinant FSH, luteinizing hormone (LH), human chorionic gonadotropin (HCG) and thyroid-stimulating hormone (TSH) was evaluated in transfected HEK293 cells by bioluminescence resonance energy transfer. RESULTS: All mutations decreased the 50% effective concentration of FSH calculated for cAMP (P < 0.05, nâ¯=â¯6), resulting in two- to 10-fold lower ligand potency. TSH failed to induce an FSHR-mediated increase in intracellular cAMP, while LH was approximately four-fold more potent than HCG in p.Ser128Tyr FSHR-expressing HEK293 cells despite lower cAMP plateau levels (P < 0.05, nâ¯=â¯5). The p.Ser128Tyr FSHR mutation was found to be responsible for an LH-/HCG-induced increase in cAMP when it was in the biallelic heterozygous state with p.Asn106His, but no increase in cAMP was induced in the monoallelic state. CONCLUSION: In-vitro data support that, in pregnant patients with sOHSS, the two FSHR mutations have an opposing effect on the pathogenesis of sOHSS and are in the biallelic heterozygous form, allowing HCG to induce a p.Ser128Tyr FSHR-mediated increase in cAMP.
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Síndrome de Hiperestimulación Ovárica/genética , Receptores de HFE/genética , Adulto , AMP Cíclico/metabolismo , Femenino , Hormona Folículo Estimulante/metabolismo , Células HEK293 , Humanos , Síndrome de Hiperestimulación Ovárica/metabolismo , Receptores de HFE/metabolismoRESUMEN
Several studies have demonstrated that polyphenol-enriched diets may have beneficial effects against the development of degenerative diseases, including atherosclerosis and disorders affecting the central nervous system. This activity has been associated not only with antioxidant and anti-inflammatory properties, but also with additional mechanisms, such as the modulation of lipid metabolism and gut microbiota function. However, long-term studies on humans provided controversial results, making the prediction of polyphenol impact on health uncertain. The aim of this review is to provide an overview and critical analysis of the literature related to the effects of the principal dietary polyphenols on cardiovascular and neurodegenerative disorders. We critically considered and meta-analyzed randomized controlled clinical trials involving subjects taking polyphenol-based supplements. Although some polyphenols might improve specific markers of cardiovascular risk and cognitive status, many inconsistent data are present in literature. Therefore, definitive recommendations for the use of these compounds in the prevention of cardiovascular disease and cognitive decline are currently not applicable. Once pivotal aspects for the definition of polyphenol bioactivity, such as the characterization of pharmacokinetics and safety, are addressed, it will be possible to have a clear picture of the realistic potential of polyphenols for disease prevention.
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Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Neurodegenerativas/tratamiento farmacológico , Polifenoles/uso terapéutico , Presión Sanguínea/efectos de los fármacos , Enfermedades Cardiovasculares/fisiopatología , Cognición/efectos de los fármacos , Humanos , Enfermedades Neurodegenerativas/fisiopatología , Sustancias ProtectorasRESUMEN
Commercial gonadotropin-releasing hormone (GnRH) antagonists differ by 1-2 amino acids and are used to inhibit gonadotropin production during assisted reproduction technologies (ART). In this study, potencies of three GnRH antagonists, Cetrorelix, Ganirelix and Teverelix, in inhibiting GnRH-mediated intracellular signaling, were compared in vitro. GnRH receptor (GnRHR)-transfected HEK293 and neuroblastoma-derived SH-SY5Y cell lines, as well as mouse pituitary LßT2 cells endogenously expressing the murine GnRHR, were treated with GnRH in the presence or absence of the antagonist. We evaluated intracellular calcium (Ca2+) and cAMP increases, cAMP-responsive element binding-protein (CREB) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation, ß-catenin activation and mouse luteinizing-hormone ß-encoding gene (Lhb) transcription by bioluminescence resonance energy transfer (BRET), Western blotting, immunostaining and real-time PCR as appropriate. The kinetics of GnRH-induced Ca2+ rapid increase revealed dose-response accumulation with potency (EC50) of 23 nM in transfected HEK293 cells, transfected SH-SY5Y and LßT2 cells. Cetrorelix inhibited the 3 × EC50 GnRH-activated calcium signaling at concentrations of 1 nM-1 µM, demonstrating higher potency than Ganirelix and Teverelix, whose inhibitory doses fell within the 100 nM-1 µM range in both transfected HEK293 and SH-SY5Y cells in vitro. In transfected SH-SY5Y, Cetrorelix was also significantly more potent than other antagonists in reducing GnRH-mediated cAMP accumulation. All antagonists inhibited pERK1/2 and pCREB activation at similar doses, in LßT2 and transfected HEK293 cells treated with 100 nM GnRH. Although immunostainings suggested that Teverelix could be less effective than Cetrorelix and Ganirelix in inhibiting 1 µM GnRH-induced ß-catenin activation, Lhb gene expression increase occurring upon LßT2 cell treatment by 1 µM GnRH was similarly inhibited by all antagonists. To conclude, this study has demonstrated Cetrorelix-, Ganirelix- and Teverelix-specific biased effects at the intracellular level, not affecting the efficacy of antagonists in inhibiting Lhb gene transcription.
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Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Hormona Liberadora de Gonadotropina/antagonistas & inhibidores , Antagonistas de Hormonas/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Receptores LHRH/metabolismo , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Hormona Liberadora de Gonadotropina/metabolismo , Células HEK293 , Humanos , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismoRESUMEN
BACKGROUND: Human luteinizing hormone (LH) and chorionic gonadotropin (hCG) are glycoprotein hormones regulating development and reproductive functions by acting on the same receptor (LHCGR). We compared the LH and hCG activity in gonadal cells from male mouse in vitro, i.e. primary Leydig cells, which is a common tool used for gonadotropin bioassay. Murine Leydig cells are naturally expressing the murine LH receptor (mLhr), which binds human LH/hCG. METHODS: Cultured Leydig cells were treated by increasing doses of recombinant LH and hCG, and cell signaling, gene expression and steroid synthesis were evaluated. RESULTS: We found that hCG is about 10-fold more potent than LH in cAMP recruitment, and slightly but significantly more potent on cAMP-dependent Erk1/2 phosphorylation. However, no significant differences occur between LH and hCG treatments, measured as activation of downstream signals, such as Creb phosphorylation, Stard1 gene expression and testosterone synthesis. CONCLUSIONS: These data demonstrate that the responses to human LH/hCG are only quantitatively and not qualitatively different in murine cells, at least in terms of cAMP and Erk1/2 activation, and equal in activating downstream steroidogenic events. This is at odds with what we previously described in human primary granulosa cells, where LHCGR mediates a different pattern of signaling cascades, depending on the natural ligand. This finding is relevant for gonadotropin quantification used in the official pharmacopoeia, which are based on murine, in vivo bioassay and rely on the evaluation of long-term, testosterone-dependent effects mediated by rodent receptor.
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Gonadotropina Coriónica/farmacología , Células Intersticiales del Testículo/metabolismo , Hormona Luteinizante/farmacología , Transducción de Señal/fisiología , Testosterona/biosíntesis , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Células Intersticiales del Testículo/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacosRESUMEN
OBJECTIVE: Sphingosine 1-phosphate (S1P) partly accounts for antiatherogenic properties of high-density lipoproteins. We previously demonstrated that FTY720, a synthetic S1P analog targeting all S1P receptors but S1P receptor type 2, inhibits murine atherosclerosis. Here, we addressed the identity of S1P receptor mediating atheroprotective effects of S1P. APPROACH AND RESULTS: Low-density lipoprotein receptor-deficient mice on cholesterol-rich diet were given selective S1P receptor type 1 agonist KRP-203 (3.0 mg/kg per day; 6 and 16 weeks). KRP-203 substantially reduced atherosclerotic lesion formation without affecting plasma lipid concentrations. However, KRP-203 induced lymphopenia, reduced total (CD4(+), CD8(+)) and activated (CD69(+)/CD8(+), CD69(+)/CD4(+)) T cells in peripheral lymphoid organs, and interfered with lymphocyte function, as evidenced by decreased T-cell proliferation and interleukin-2 and interferon-γ production in activated splenocytes. Cyto- and chemokine (tumor necrosis factor-α, regulated and normal T cell expressed and secreted) levels in plasma and aortas were reduced by KRP-203 administration. Moreover, macrophages from KRP-203-treated mice showed reduced expression of activation marker MCH-II and poly(I:C)-elicited production of tumor necrosis factor-α, monocyte chemoattractant protein-1, and interleukin-6. In vitro studies demonstrated that KRP-203 reduced tumor necrosis factor-α, interleukin-6, and interferon-γ-induced protein-10 production; IκB and signal transducer and activator of transcription-1 phosphorylation; and nuclear factor κB and signal transducer and activator of transcription-1 activation in poly(I:C)-, lipopolysaccharide-, or interferon-γ-stimulated bone marrow macrophages, respectively. CONCLUSIONS: Present results demonstrate that activation of S1P signaling pathways inhibit atherosclerosis by modulating lymphocyte and macrophage function and suggest that S1P receptor type 1 at least partially mediates antiatherogenic effects of S1P.
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Aorta/efectos de los fármacos , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Fármacos Cardiovasculares/farmacología , Inflamación/prevención & control , Receptores de LDL/deficiencia , Receptores de Lisoesfingolípidos/agonistas , Compuestos de Sulfhidrilo/farmacología , Animales , Aorta/inmunología , Aorta/metabolismo , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Biomarcadores/sangre , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Mediadores de Inflamación/metabolismo , Lípidos/sangre , Activación de Linfocitos/efectos de los fármacos , Linfopenia/inmunología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Noqueados , Receptores de LDL/genética , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal/efectos de los fármacos , Células U937RESUMEN
AIMS: The entry of lipoproteins from blood into the arterial wall is a rate-limiting step in atherosclerosis. It is controversial whether this happens by filtration or regulated transendothelial transport.Because sphingosine-1-phosphate (S1P) preserves the endothelial barrier, we investigated in vivo and in vitro, whether S1P and its cognate S1P-receptor 3 (S1P3) regulate the transendothelial transport of lipoproteins. METHODS AND RESULTS: Compared to apoE-haploinsufficient mice (CTRL), apoE-haploinsufficient mice with additional endothelium-specific knock-in of S1P3 (S1P3-iECKI) showed decreased transport of LDL and Evan's Blue but increased transport of HDL from blood into the peritoneal cave. After 30 weeks of high-fat diet feeding, S1P3-iECKI mice had lower levels of non-HDL-cholesterol and less atherosclerosis than CTRL mice. In vitro stimulation with an S1P3 agonist increased the transport of 125I-HDL but decreased the transport of 125I-LDL through human aortic endothelial cells (HAECs). Conversely, inhibition or knock-down of S1P3 decreased the transport of 125I-HDL but increased the transport of 125I-LDL. Silencing of SCARB1 encoding scavenger receptor B1 (SR-BI) abrogated the stimulation of 125I-HDL transport by the S1P3 agonist. The transendothelial transport of 125I-LDL was decreased by silencing of SCARB1 or ACVLR1 encoding activin-like kinase 1 but not by interference with LDLR. None of the three knock-downs prevented the stimulatory effect of S1P3 inhibition on transendothelial 125I-LDL transport. CONCLUSION: S1P3 regulates the transendothelial transport of HDL and LDL oppositely by SR-BI-dependent and SR-BI-independent mechanisms, respectively. This divergence supports a contention that lipoproteins pass the endothelial barrier by specifically regulated mechanisms rather than passive filtration.
Asunto(s)
Aterosclerosis , Células Endoteliales , Lipoproteínas HDL , Lipoproteínas LDL , Transporte de Proteínas , Receptores de Esfingosina-1-Fosfato , Animales , Humanos , Ratones , Aterosclerosis/metabolismo , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/prevención & control , Transporte Biológico , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Lisofosfolípidos , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Receptores Depuradores de Clase B/metabolismo , Receptores Depuradores de Clase B/genética , Receptores de Esfingosina-1-Fosfato/metabolismo , Receptores de Esfingosina-1-Fosfato/genética , Transporte de Proteínas/genéticaRESUMEN
Among the strategies to overcome the underperformance of statins in cardiovascular diseases (CVDs), the development of drugs targeting the Proprotein Convertase Subtilisin-like Kexin type 9 (PCSK9) is considered one of the most promising. However, only anti-PCSK9 biological drugs have been approved to date, and orally available small-molecules for the treatment of hypercholesterolemic conditions are still missing on the market. In the present work, we describe the application of a phenotypic approach to the identification and optimization of 4-amino-2-pyridone derivatives as a new chemotype with anti-PCSK9 activity. Starting from an in-house collection of compounds, functional assays on HepG2 cells followed by a chemistry-driven hit optimization campaign, led to the potent anti-PCSK9 candidate 5c. This compound, at 5 µM, totally blocked PCSK9 secretion from HepG2 cells, significantly increased LDL receptor (LDLR) expression, and acted cooperatively with simvastatin by reducing its induction of PCSK9 expression. Finally, compound 5c also proved to be well tolerated in C57BL/6J mice at the tested concentration (40 mg/kg) with no sign of toxicity or behavior modifications.
Asunto(s)
Inhibidores de PCSK9 , Proproteína Convertasa 9 , Animales , Humanos , Ratones , Células Hep G2 , Ratones Endogámicos C57BL , Proproteína Convertasa 9/metabolismo , Receptores de LDL/metabolismo , Piridonas/química , Piridonas/metabolismoRESUMEN
Scavenger receptor SR-BI significantly contributes to HDL cholesterol metabolism and atherogenesis in mice. However, the role of SR-BI may not be as pronounced in humans due to cholesteryl ester transfer protein (CETP) activity. To address the impact of CETP expression on the adverse effects associated with SR-BI deficiency, we cross-bred our SR-BI conditional knock-out mouse model with CETP transgenic mice. CETP almost completely restored the abnormal HDL-C distribution in SR-BI-deficient mice. However, it did not normalize the elevated plasma free to total cholesterol ratio characteristic of hepatic SR-BI deficiency. Red blood cell and platelet count abnormalities observed in mice liver deficient for SR-BI were partially restored by CETP, but the elevated erythrocyte cholesterol to phospholipid ratio remained unchanged. Complete deletion of SR-BI was associated with diminished adrenal cholesterol stores, whereas hepatic SR-BI deficiency resulted in a significant increase in adrenal gland cholesterol content. In both mouse models, CETP had no impact on adrenal cholesterol metabolism. In diet-induced atherosclerosis studies, hepatic SR-BI deficiency accelerated aortic lipid lesion formation in both CETP-expressing (4-fold) and non-CETP-expressing (8-fold) mice when compared with controls. Impaired macrophage to feces reverse cholesterol transport in mice deficient for SR-BI in liver, which was not corrected by CETP, most likely contributed by such an increase in atherosclerosis susceptibility. Finally, comparison of the atherosclerosis burden in SR-BI liver-deficient and fully deficient mice demonstrated that SR-BI exerted an atheroprotective activity in extra-hepatic tissues whether CETP was present or not. These findings support the contention that the SR-BI pathway contributes in unique ways to cholesterol metabolism and atherosclerosis susceptibility even in the presence of CETP.
Asunto(s)
Aterosclerosis/metabolismo , Antígenos CD36/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol/biosíntesis , Colesterol/metabolismo , Regulación de la Expresión Génica , Animales , HDL-Colesterol/metabolismo , Eritrocitos/citología , Femenino , Humanos , Hígado/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
OBJECTIVE: To assess the role of apolipoprotein (apo) E in macrophage reverse cholesterol transport (RCT) in vivo. METHODS AND RESULTS: ApoE exerts an antiatherosclerotic activity by regulating lipoprotein metabolism and promoting cell cholesterol efflux. We discriminated between macrophage and systemic apoE contribution using an assay of macrophage RCT in mice. The complete absence of apoE lead to an overall impairment of the process and, similarly, the absence of apoE exclusively in macrophages resulted in the reduction of cholesterol mobilization from macrophages to plasma, liver, and feces. Conversely, expression of apoE in macrophages is sufficient to promote normal RCT even in apoE-deficient mice. The mechanisms accounting for these results were investigated by evaluating the first step of RCT (ie, cholesterol efflux from cells). Macrophages isolated from apoE-deficient mice showed a reduced ability to release cholesterol into the culture medium, whereas the apoB-depleted plasma from apoE-deficient and healthy mice possessed a similar capacity to promote cellular lipid release from cultured macrophages. CONCLUSIONS: Our data demonstrate, for the first time to our knowledge, that apoE significantly contributes to macrophage RCT in vivo and that this role is fully attributable to apoE expressed in macrophages.
Asunto(s)
Apolipoproteínas E/metabolismo , Colesterol/metabolismo , Macrófagos Peritoneales/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Apolipoproteínas E/sangre , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Transporte Biológico , Células Cultivadas , Colesterol/sangre , HDL-Colesterol/metabolismo , Heces/química , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
The ability to accept cholesterol from cells and to promote reverse cholesterol transport (RCT) represents the best characterized antiatherogenic function of HDL. Studies carried out in animal models have unraveled the multiple mechanisms by which these lipoproteins drive cholesterol efflux from macrophages and cholesterol uptake to the liver. Moreover, the influence of HDL composition and the role of lipid transporters have been clarified by using suitable transgenic models or through experimental design employing pharmacological or nutritional interventions. Cholesterol efflux capacity (CEC), an in vitro assay developed to offer a measure of the first step of RCT, has been shown to associate with cardiovascular risk in several human cohorts, supporting the atheroprotective role of RCT in humans as well. However, negative data in other cohorts have raised concerns on the validity of this biomarker. In this review we will present the most relevant data documenting the role of HDL in RCT, as assessed in classical or innovative methodological approaches.
Asunto(s)
HDL-Colesterol/metabolismo , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Hígado/metabolismo , Animales , Transporte Biológico/genética , HDL-Colesterol/genética , Humanos , Lipoproteínas HDL/genéticaRESUMEN
Chronic kidney disease (CKD) is a long-term condition characterized by a gradual loss of kidney functions, usually accompanied by other comorbidities including cardiovascular diseases (hypertension, heart failure and stroke) and diabetes mellitus. Therefore, multiple pharmacological prescriptions are very common in these patients. Epidemiological and clinical observations have shown that polypharmacy may increase the probability of adverse drug reactions (ADRs), possibly through a higher risk of drug-drug interactions (DDIs). Renal impairment may further worsen this scenario by affecting the physiological and biochemical pathways underlying pharmacokinetics and ultimately modifying the pharmacodynamic responses. It has been estimated that the prevalence of DDIs in CKD patients ranged between 56.9% and 89.1%, accounting for a significant increase in healthcare costs, length and frequency of hospitalization, with a detrimental impact on health and quality of life of these patients. Despite these recognized high-risk conditions, scientific literature released on this topic is still limited. Basing on the most commonly prescribed therapies in patients with CKD, the present short review summarizes the current state of knowledge of the putative DDIs occurring in CKD patients undergoing polytherapy. The most relevant underlying mechanisms and their clinical significance are also debated.
RESUMEN
Reverse cholesterol transport (RCT) is a physiological mechanism protecting cells from an excessive accumulation of cholesterol. When this process begins in vascular macrophages, it acquires antiatherogenic properties, as has been widely demonstrated in animal models. Dietary lipids, despite representing a fundamental source of energy and exerting multiple biological functions, may induce detrimental effects on cardiovascular health. In the present review we summarize the current knowledge on the mechanisms of action of the most relevant classes of dietary lipids, such as fatty acids, sterols and liposoluble vitamins, with effects on different steps of RCT. We also provide a critical analysis of data obtained from experimental models which can serve as a valuable tool to clarify the effects of dietary lipids on cardiovascular disease.
Asunto(s)
Transporte Biológico , Colesterol , Grasas de la Dieta , Animales , Enfermedades Cardiovasculares , HDL-Colesterol , Ácidos Grasos , Humanos , Macrófagos/metabolismo , Modelos Animales , RoedoresRESUMEN
From February 2019 the World faces the Covid19 pandemic. The data in our possession are still insufficient to effectively combat this pathology. The gold standard for diagnosis remains molecular testing, while clinical and instrumental and serological diagnostics are highly nonspecific leading to a slowdown in the battle against covid19.[3] Can Artificial Intelligence (AI) and Machine Learning (ML) help us? The use of large databases to cross-reference data to stratify the diagnostic scores, to quickly differentiate a critical Covid-19 patient from a non-critical one is the challenge of the future. All to achieve better management of resources in the field and a more effective therapeutic approach.[2].