RESUMO
Homeostatic adaptation to systemic iron overload involves transcriptional induction of bone morphogenetic protein 6 (BMP6) in liver sinusoidal endothelial cells (LSECs). BMP6 is then secreted to activate signaling of the iron hormone hepcidin (HAMP) in neighboring hepatocytes. To explore the mechanism of iron sensing by LSECs, we generated TfrcTek-Cre mice with endothelial cell-specific ablation of transferrin receptor 1 (Tfr1). We also used control Tfrcfl/fl mice to characterize the LSEC-specific molecular responses to iron using single-cell transcriptomics. TfrcTek-Cre animals tended to have modestly increased liver iron content (LIC) compared with Tfrcfl/fl controls but expressed physiological Bmp6 and Hamp messenger RNA (mRNA). Despite a transient inability to upregulate Bmp6, they eventually respond to iron challenges with Bmp6 and Hamp induction, yet occasionally to levels slightly lower relative to LIC. High dietary iron intake triggered the accumulation of serum nontransferrin bound iron (NTBI), which significantly correlated with liver Bmp6 and Hamp mRNA levels and elicited more profound alterations in the LSEC transcriptome than holo-transferrin injection. This culminated in the robust induction of Bmp6 and other nuclear factor erythroid 2-related factor 2 (Nrf2) target genes, as well as Myc target genes involved in ribosomal biogenesis and protein synthesis. LSECs and midzonal hepatocytes were the most responsive liver cells to iron challenges and exhibited the highest expression of Bmp6 and Hamp mRNAs, respectively. Our data suggest that during systemic iron overload, LSECs internalize NTBI, which promotes oxidative stress and thereby transcriptionally induces Bmp6 via Nrf2. Tfr1 appears to contribute to iron sensing by LSECs, mostly under low iron conditions.
Assuntos
Sobrecarga de Ferro , Ferro , Camundongos , Animais , Ferro/metabolismo , Transferrina/metabolismo , Células Endoteliais/metabolismo , Proteína Morfogenética Óssea 6/genética , Proteína Morfogenética Óssea 6/metabolismo , Fator 2 Relacionado a NF-E2 , Hepatócitos/metabolismo , Fígado/metabolismo , Hepcidinas/genética , Hepcidinas/metabolismo , Sobrecarga de Ferro/genética , Sobrecarga de Ferro/metabolismo , RNA Mensageiro/metabolismoRESUMO
The GPCR HCAR1 is known to be the sole receptor for lactate, which modulates its metabolic effects. Despite its significant role in many processes, mice deficient in HCAR1 exhibit no visible phenotype and are healthy and fertile. We performed transcriptomic analysis on HCAR1 deficient cells, in combination with lactate, to explore pathophysiologically altered processes. Processes such as immune regulation, various cancers, and neurodegenerative diseases were significantly enriched for HCAR1 transcriptomic signature. However, the most affected process of all was autism spectrum disorder. We performed behavioral tests on HCAR1 KO mice and observed that these mice manifest autistic-like behavior. Our data opens new avenues for research on HCAR1 and lactate effect at a pathological level. Video Abstract.
Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Camundongos , Animais , Ácido Láctico/metabolismo , Transdução de Sinais , Receptores Acoplados a Proteínas G/metabolismoRESUMO
The roles of nitric oxide (NO) and endothelial NO synthase (eNOS) in the regulation of angiogenesis are well documented. However, the involvement of eNOS in the sprouting of endothelial tip-cells at the vascular front during sprouting angiogenesis remains poorly defined. In this study, we show that downregulation of eNOS markedly inhibits VEGF-stimulated migration of endothelial cells but increases their polarization, as evidenced by the reorientation of the Golgi in migrating monolayers and by the fewer filopodia on tip cells at ends of sprouts in endothelial cell spheroids. The effect of eNOS inhibition on EC polarization was prevented in Par3-depleted cells. Importantly, downregulation of eNOS increased the expression of polarity genes, such as PARD3B, PARD6A, PARD6B, PKCΖ, TJP3, and CRB1 in endothelial cells. In retinas of eNOS knockout mice, vascular development is retarded with decreased vessel density and vascular branching. Furthermore, tip cells at the extremities of the vascular front have a marked reduction in the number of filopodia per cell and are more oriented. In a model of oxygen-induced retinopathy (OIR), eNOS deficient mice are protected during the initial vaso-obliterative phase, have reduced pathological neovascularization, and retinal endothelial tip cells have fewer filopodia. Single-cell RNA sequencing of endothelial cells from OIR retinas revealed enrichment of genes related to cell polarity in the endothelial tip-cell subtype of eNOS deficient mice. These results indicate that inhibition of eNOS alters the polarity program of endothelial cells, which increases cell polarization, regulates sprouting angiogenesis and normalizes pathological neovascularization during retinopathy.
Assuntos
Neovascularização Patológica , Neovascularização Fisiológica , Óxido Nítrico Sintase Tipo III/fisiologia , Retina/metabolismo , Neovascularização Retiniana , Vasos Retinianos , Animais , Bovinos , Linhagem Celular , Movimento Celular , Polaridade Celular , Células Endoteliais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Retina/citologia , Retina/patologia , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Vasos Retinianos/citologia , Vasos Retinianos/patologiaRESUMO
Diabetic macular edema is a major complication of diabetes resulting in loss of central vision. Although heightened vessel leakiness has been linked to glial and neuronal-derived factors, relatively little is known on the mechanisms by which mature endothelial cells exit from a quiescent state and compromise barrier function. Here we report that endothelial NOTCH1 signaling in mature diabetic retinas contributes to increased vascular permeability. By providing both human and mouse data, we show that NOTCH1 ligands JAGGED1 and DELTA LIKE-4 are up-regulated secondary to hyperglycemia and activate both canonical and rapid noncanonical NOTCH1 pathways that ultimately disrupt endothelial adherens junctions in diabetic retinas by causing dissociation of vascular endothelial-cadherin from ß-catenin. We further demonstrate that neutralization of NOTCH1 ligands prevents diabetes-induced retinal edema. Collectively, these results identify a fundamental process in diabetes-mediated vascular permeability and provide translational rational for targeting the NOTCH pathway (primarily JAGGED1) in conditions characterized by compromised vascular barrier function.
Assuntos
Permeabilidade Capilar , Retinopatia Diabética/patologia , Receptor Notch1/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Proteínas de Ligação ao Cálcio/biossíntese , Ativação Enzimática , Hiperglicemia/metabolismo , Proteína Jagged-1/biossíntese , Camundongos , Óxido Nítrico/biossíntese , Vasos Retinianos/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Quinases da Família src/metabolismoRESUMO
AIMS/HYPOTHESIS: Proliferative diabetic retinopathy (PDR) with retinal neovascularisation (NV) is a leading cause of vision loss. This study identified a set of metabolites that were altered in the vitreous humour of PDR patients compared with non-diabetic control participants. We corroborated changes in vitreous metabolites identified in prior studies and identified novel dysregulated metabolites that may lead to treatment strategies for PDR. METHODS: We analysed metabolites in vitreous samples from 43 PDR patients and 21 non-diabetic epiretinal membrane control patients from Japan (age 27-80 years) via ultra-high-performance liquid chromatography-mass spectrometry. We then investigated the association of a novel metabolite (creatine) with retinal NV in mouse oxygen-induced retinopathy (OIR). Creatine or vehicle was administered from postnatal day (P)12 to P16 (during induced NV) via oral gavage. P17 retinas were quantified for NV and vaso-obliteration. RESULTS: We identified 158 metabolites in vitreous samples that were altered in PDR patients vs control participants. We corroborated increases in pyruvate, lactate, proline and allantoin in PDR, which were identified in prior studies. We also found changes in metabolites not previously identified, including creatine. In human vitreous humour, creatine levels were decreased in PDR patients compared with epiretinal membrane control participants (false-discovery rate <0.001). We validated that lower creatine levels were associated with vascular proliferation in mouse retina in the OIR model (p = 0.027) using retinal metabolomics. Oral creatine supplementation reduced NV compared with vehicle (P12 to P16) in OIR (p = 0.0024). CONCLUSIONS/INTERPRETATION: These results suggest that metabolites from vitreous humour may reflect changes in metabolism that can be used to find pathways influencing retinopathy. Creatine supplementation could be useful to suppress NV in PDR. Graphical abstract.
Assuntos
Retinopatia Diabética/metabolismo , Metabolômica , Corpo Vítreo/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Aminoácidos/análise , Animais , Cromatografia Líquida de Alta Pressão , Creatina/administração & dosagem , Creatina/análise , Retinopatia Diabética/fisiopatologia , Feminino , Humanos , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Neovascularização Retiniana/metabolismo , Corpo Vítreo/químicaRESUMO
BACKGROUND: Inflammation and particularly interleukin-1ß (IL-1ß), a pro-inflammatory cytokine highly secreted by activated immune cells during early AMD pathological events, contribute significantly to retinal neurodegeneration. Here, we identify specific cell types that generate IL-1ß and harbor the IL-1 receptor (IL-1R) and pharmacologically validate IL-1ß's contribution to neuro-retinal degeneration using the IL-1R allosteric modulator composed of the amino acid sequence rytvela (as well as the orthosteric antagonist, Kineret) in a model of blue light-induced retinal degeneration. METHODS: Mice were exposed to blue light for 6 h and sacrificed 3 days later. Mice were intraperitoneally injected with rytvela, Kineret, or vehicle twice daily for 3 days. The inflammatory markers F4/80, NLRP3, caspase-1, and IL-1ß were assessed in the retinas. Single-cell RNA sequencing was used to determine the cell-specific expression patterns of retinal Il1b and Il1r1. Macrophage-induced photoreceptor death was assessed ex vivo using retinal explants co-cultured with LPS-activated bone marrow-derived macrophages. Photoreceptor cell death was evaluated by the TUNEL assay. Retinal function was assessed by flash electroretinography. RESULTS: Blue light markedly increased the mononuclear phagocyte recruitment and levels of inflammatory markers associated with photoreceptor death. Co-localization of NLRP3, caspase-1, and IL-1ß with F4/80+ mononuclear phagocytes was clearly detected in the subretinal space, suggesting that these inflammatory cells are the main source of IL-1ß. Single-cell RNA sequencing confirmed the immune-specific expression of Il1b and notably perivascular macrophages in light-challenged mice, while Il1r1 expression was found primarily in astrocytes, bipolar, and vascular cells. Retinal explants co-cultured with LPS/ATP-activated bone marrow-derived macrophages displayed a high number of TUNEL-positive photoreceptors, which was abrogated by rytvela treatment. IL-1R antagonism significantly mitigated the inflammatory response triggered in vivo by blue light exposure, and rytvela was superior to Kineret in preserving photoreceptor density and retinal function. CONCLUSION: These findings substantiate the importance of IL-1ß in neuro-retinal degeneration and revealed specific sources of Il1b from perivascular MPs, with its receptor Ilr1 being separately expressed on surrounding neuro-vascular and astroglial cells. They also validate the efficacy of rytvela-induced IL-1R modulation in suppressing detrimental inflammatory responses and preserving photoreceptor density and function in these conditions, reinforcing the rationale for clinical translation.
Assuntos
Interleucina-1beta/imunologia , Peptídeos/farmacologia , Células Fotorreceptoras/patologia , Receptores de Interleucina-1/antagonistas & inibidores , Degeneração Retiniana/patologia , Animais , Modelos Animais de Doenças , Inflamação/imunologia , Inflamação/patologia , Interleucina-1beta/metabolismo , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Camundongos , Células Fotorreceptoras/efeitos dos fármacos , Degeneração Retiniana/imunologiaRESUMO
Recent genomic studies have shed light on the impact of in vitro culture (IVC) on embryonic homeostasis and the differential gene expression profiles associated with lower developmental competence. Consistently, the embryonic stress responses to IVC conditions correlate with transcriptomic changes in pathways related to energetic metabolism, extracellular matrix remodelling and inflammatory signalling. These changes appear to result from a developmental adaptation that enhances a Warburg-like effect known to occur naturally during blastulation. First discovered in cancer cells, the Warburg effect (increased glycolysis under aerobic conditions) is thought to result from mitochondrial dysfunction. In the case of IVC embryos, culture conditions may interfere with mitochondrial maturation and oxidative phosphorylation, forcing cells to rely on glycolysis in order to maintain energetic homeostasis. While beneficial in the short term, such adaptations may lead to epigenetic changes with potential long-term effects on implantation, foetal growth and post-natal health. We conclude that lessening the detrimental effects of IVC on mitochondrial activity would lead to significantly improved embryo quality.
Assuntos
Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/genética , Genômica/métodos , Estresse Fisiológico/genética , Animais , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , MitocôndriasRESUMO
PURPOSE OF REVIEW: There has been increasing interest in developing assisted reproductive technologies to overcome failure at fertilization and early embryonic arrest. Some of the affected patients harbor too few copies of mitochondrial DNA (mtDNA) in their eggs at the time of fertilization, which results in developmental failure. RECENT FINDINGS: In the last 3-5 years, there has been a drive to overcome mtDNA deficiency through mitochondrial supplementation protocols using autologous populations of mitochondrial DNA so as not to perturb the offspring's genetic identity or mediate a series of side-effects because of the mixing of two distinct populations of mitochondrial DNA. It is evident that there is strict regulation of mitochondrial DNA copy number from the primordial germ cell through to the time when tissues are specified during organogenesis. Supplementation of oocytes can give rise to better quality embryos, enhanced blastocyst rates, and ongoing pregnancies. It utilizes a key mitochondrial DNA replication event that takes place shortly after fertilization to stabilize the embryonic genome. SUMMARY: These findings provide a rationale for undertaking mitochondrial supplementation and propose a mechanism to explain why the process can enhance embryo development. They also take the approach a step closer to clinical practice.
Assuntos
Blastocisto/fisiologia , DNA Mitocondrial/genética , Oócitos/fisiologia , Técnicas de Reprodução Assistida , Replicação do DNA , Desenvolvimento Embrionário , Feminino , HumanosRESUMO
Although the oocyte is the largest cell in the body and an unavoidable phase in life, its physiology is still poorly understood, and other cell types provide little insight into its unique nature. Even basic cellular functions in the oocyte such as energy metabolism are not yet fully understood. It is known that the mitochondria of the female gamete exhibit an immature form characterized by limited energy production from glucose and oxidative phosphorylation. We show that the bovine oocyte uses alternative means to maintain ATP production during maturation, namely, the adenosine salvage pathway. Meiosis resumption is triggered by destruction of cyclic AMP by phosphodiesterases producing adenosine monophosphate that is converted into ATP by adenylate kinases and creatine kinases. Inhibition of these enzymes decreased ATP production, and addition of their substrates restored ATP production in denuded oocytes. Addition of phosphocreatine to the oocyte maturation medium influenced the phenotype of the resulting blastocysts. We propose a model in which adenylate kinases and creatine kinases act as drivers of ATP production from added AMP during oocyte maturation.
Assuntos
Trifosfato de Adenosina/metabolismo , Adenosina/metabolismo , Adenilato Quinase/metabolismo , Creatina Quinase/metabolismo , Mitocôndrias/metabolismo , Oócitos/metabolismo , Oogênese , Matadouros , Adenilato Quinase/antagonistas & inibidores , Adenilato Quinase/genética , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Blastocisto/ultraestrutura , Bovinos , Creatina Quinase/antagonistas & inibidores , Creatina Quinase/genética , Ectogênese/efeitos dos fármacos , Técnicas de Cultura Embrionária , Inibidores Enzimáticos/farmacologia , Feminino , Fertilização in vitro , Técnicas de Maturação in Vitro de Oócitos , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Análise de Sequência com Séries de Oligonucleotídeos , Oócitos/efeitos dos fármacos , Oócitos/ultraestrutura , Oogênese/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacosRESUMO
Zonula occludens-1 (ZO-1) is involved in the regulation of cell-cell junctions between endothelial cells (ECs). Here we identify the ZO-1 protein interactome and uncover ZO-1 interactions with RNA-binding proteins that are part of stress granules (SGs). Downregulation of ZO-1 increased SG formation in response to stress and protected ECs from cellular insults. The ZO-1 interactome uncovered an association between ZO-1 and Y-box binding protein 1 (YB-1), a constituent of SGs. Arsenite treatment of ECs decreased the interaction between ZO-1 and YB-1, and drove SG assembly. YB-1 expression is essential for SG formation and for the cytoprotective effects induced by ZO-1 downregulation. In the developing retinal vascular plexus of newborn mice, ECs at the front of growing vessels express less ZO-1 but display more YB-1-positive granules than ECs located in the vascular plexus. Endothelial-specific deletion of ZO-1 in mice at post-natal day 7 markedly increased the presence of YB-1-positive granules in ECs of retinal blood vessels, altered tip EC morphology and vascular patterning, resulting in aberrant endothelial proliferation, and arrest in the expansion of the retinal vasculature. Our findings suggest that, through its interaction with YB-1, ZO-1 controls SG formation and the response of ECs to stress during angiogenesis.
Assuntos
Células Endoteliais , Proteína 1 de Ligação a Y-Box , Proteína da Zônula de Oclusão-1 , Animais , Proteína 1 de Ligação a Y-Box/metabolismo , Proteína 1 de Ligação a Y-Box/genética , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genética , Camundongos , Humanos , Células Endoteliais/metabolismo , Grânulos de Estresse/metabolismo , Neovascularização Fisiológica , Vasos Retinianos/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Angiogênese , Fatores de TranscriçãoRESUMO
Pathological neovascularization in retinopathy of prematurity (ROP) can cause visual impairment in preterm infants. Current ROP treatments which are not preventative and only address late neovascular ROP, are costly and can lead to severe complications. We showed that topical 0.1% dexamethasone eye drops administered prior to peak neovessel formation prevented neovascularization in five extremely preterm infants at high risk for ROP and suppressed neovascularization by 30% in mouse oxygen-induced retinopathy (OIR) modeling ROP. In contrast, in OIR, topical dexamethasone treatment before any neovessel formation had limited efficacy in preventing later neovascularization, while treatment after peak neovessel formation had a non-statistically significant trend to exacerbating disease. Optimally timed topical dexamethasone suppression of neovascularization in OIR was associated with increased retinal mitochondrial gene expression and decreased inflammatory marker expression, predominantly found in immune cells. Blocking mitochondrial ATP synthetase reversed the inhibitory effect of dexamethasone on neovascularization in OIR. This study provides new insights into topical steroid effects in retinal neovascularization and into mitochondrial function in phase II ROP, and suggests a simple clinical approach to prevent severe ROP.
RESUMO
Compromised vascular endothelial barrier function is a salient feature of diabetic complications such as sight-threatening diabetic macular edema (DME). Current standards of care for DME manage aspects of the disease, but require frequent intravitreal administration and are poorly effective in large subsets of patients. Here we provide evidence that an elevated burden of senescent cells in the retina triggers cardinal features of DME pathology and conduct an initial test of senolytic therapy in patients with DME. In cell culture models, sustained hyperglycemia provoked cellular senescence in subsets of vascular endothelial cells displaying perturbed transendothelial junctions associated with poor barrier function and leading to micro-inflammation. Pharmacological elimination of senescent cells in a mouse model of DME reduces diabetes-induced retinal vascular leakage and preserves retinal function. We then conducted a phase 1 single ascending dose safety study of UBX1325 (foselutoclax), a senolytic small-molecule inhibitor of BCL-xL, in patients with advanced DME for whom anti-vascular endothelial growth factor therapy was no longer considered beneficial. The primary objective of assessment of safety and tolerability of UBX1325 was achieved. Collectively, our data suggest that therapeutic targeting of senescent cells in the diabetic retina with a BCL-xL inhibitor may provide a long-lasting, disease-modifying intervention for DME. This hypothesis will need to be verified in larger clinical trials. ClinicalTrials.gov identifier: NCT04537884 .
Assuntos
Diabetes Mellitus , Retinopatia Diabética , Edema Macular , Animais , Camundongos , Humanos , Edema Macular/tratamento farmacológico , Edema Macular/etiologia , Retinopatia Diabética/tratamento farmacológico , Inibidores da Angiogênese/uso terapêutico , Células Endoteliais , Senoterapia , Senescência CelularRESUMO
In order to understand how in vitro culture affects embryonic quality, we analyzed survival and global gene expression in bovine blastocysts after exposure to increased oxidative stress conditions. Two pro-oxidant agents, one that acts extracellularly by promoting reactive oxygen species (ROS) production (0.01 mM 2,2'-azobis (2-amidinopropane) dihydrochloride [AAPH]) or another that acts intracellularly by inhibiting glutathione synthesis (0.4 mM buthionine sulfoximine [BSO]) were added separately to in vitro culture media from Day 3 (8-16-cell stage) onward. Transcriptomic analysis was then performed on resulting Day-7 blastocysts. In the literature, these two pro-oxidant conditions were shown to induce delayed degeneration in a proportion of Day-8 blastocysts. In our experiment, no morphological difference was visible, but AAPH tended to decrease the blastocyst rate while BSO significantly reduced it, indicating a differential impact on the surviving population. At the transcriptomic level, blastocysts that survived either pro-oxidant exposure showed oxidative stress and an inflammatory response (ARRB2), although AAPH induced higher disturbances in cellular homeostasis (SERPINE1). Functional genomics of the BSO profile, however, identified differential expression of genes related to glycine metabolism and energy metabolism (TPI1). These differential features might be indicative of pre-degenerative blastocysts (IGFBP7) in the AAPH population whereas BSO exposure would select the most viable individuals (TKDP1). Together, these results illustrate how oxidative disruption of pre-attachment development is associated with systematic up-regulation of several metabolic markers. Moreover, it indicates that a better capacity to survive anti-oxidant depletion may allow for the survival of blastocysts with a quieter metabolism after compaction.
Assuntos
Blastocisto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genoma , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Transcriptoma/fisiologia , Amidinas/farmacologia , Animais , Antioxidantes/metabolismo , Blastocisto/citologia , Bovinos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fatores de Tempo , Transcriptoma/efeitos dos fármacosRESUMO
To understand the compromised survival of embryos derived from assisted reproductive techniques, transcriptome survey of early embryonic development has shown the impact of in vitro culture environment on gene expression in bovine or other living species. However, how the differentially expressed genes translate into developmentally compromised embryos is unresolved. We therefore aimed to characterize transcriptomic markers expressed by bovine blastocysts cultured in conditions that are known to impair embryo development. As increasing glucose concentrations has been shown to be stressful for early cleavage stages of mammalian embryos and to decrease subsequent blastocyst survival, in vitro-matured/fertilized bovine zygotes were cultured in control (0.2 mM) or high-glucose (5 mM) conditions until the 8- to 16-cell stage, and then transferred to control media until they reached the blastocyst stage. The concentration of 5 mM glucose was chosen as a stress treatment because there was a significant effect on blastocyst rate without the treatment's being lethal as with 10 mM. Microarray analysis revealed gene expression differences unrelated to embryo sex or hatching. Overrepresented processes among differentially expressed genes in treated blastocysts were extracellular matrix signalling, calcium signaling, and energy metabolism. On a pathophysiological level, higher glucose treatment impacts pathways associated with diabetes and tumorigenesis through genes controlling the Warburg effect, i.e., emphasis on use of anaerobic glycolysis rather than oxidative phosphorylation. These results allowed us to conclude that disruption of in vitro preattachment development is concomitant with gene expression modifications involved in metabolic control.
Assuntos
Blastocisto/metabolismo , Fase de Clivagem do Zigoto/metabolismo , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Perfilação da Expressão Gênica , Hiperglicemia/metabolismo , Animais , Blastocisto/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Bovinos , Células Cultivadas , Fase de Clivagem do Zigoto/efeitos dos fármacos , Relação Dose-Resposta a Droga , Embrião de Mamíferos/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Glucose/farmacologia , Técnicas In Vitro , Modelos AnimaisRESUMO
BACKGROUND: Individuals born preterm present left ventricle changes and increased risk of cardiac diseases and heart failure. The pathophysiology of heart disease after preterm birth is incompletely understood. Mitochondria dysfunction is a hallmark of cardiomyopathy resulting in heart failure. We hypothesized that neonatal hyperoxia in rats, a recognized model simulating preterm birth conditions and resulting in oxygen-induced cardiomyopathy, induce left ventricle mitochondrial changes in juvenile rats. We also hypothesized that humanin, a mitochondrial-derived peptide, would be reduced in young adults born preterm. METHODS: Sprague-Dawley pups were exposed to room air (controls) or 80% O2 at postnatal days 3 to 10 (oxygen-induced cardiomyopathy). We studied left ventricle mitochondrial changes in 4 weeks old males. In a cohort of young adults born preterm (n=55) and age-matched term (n=54), we compared circulating levels of humanin. RESULTS: Compared with controls, oxygen-exposed rats showed smaller left ventricle mitochondria with disrupted integrity on electron microscopy, decreased oxidative phosphorylation, increased glycolysis markers, and reduced mitochondrial biogenesis and abundance. In oxygen-exposed rats, we observed lipid deposits, increased superoxide production (isolated cardiomyocytes), and reduced Nrf2 gene expression. In the cohort, left ventricle ejection fraction and peak global longitudinal strain were similar between groups however humanin levels were lower in preterm and associated with left ventricle ejection fraction and peak global longitudinal strain. CONCLUSIONS: In conclusion, neonatal hyperoxia impaired left ventricle mitochondrial structure and function in juvenile animals. Serum humanin level was reduced in preterm adults. This study suggests that preterm birth-related conditions entail left ventricle mitochondrial alterations that may underlie cardiac changes perpetuated into adulthood. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03261609.
Assuntos
Cardiomiopatias/etiologia , Hiperóxia/complicações , Mitocôndrias/metabolismo , Nascimento Prematuro , Disfunção Ventricular Esquerda/etiologia , Adolescente , Adulto , Animais , Cardiomiopatias/metabolismo , Cardiomiopatias/fisiopatologia , Feminino , Humanos , Hiperóxia/metabolismo , Hiperóxia/fisiopatologia , Peptídeos e Proteínas de Sinalização Intracelular/sangue , Masculino , Miócitos Cardíacos/metabolismo , Fosforilação Oxidativa , Ratos , Ratos Sprague-Dawley , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/fisiopatologia , Adulto JovemRESUMO
Pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs) have shown great potential as an alternative to primary human hepatocytes (PHHs) for in vitro modeling. Several differentiation protocols have been described to direct PSCs toward the hepatic fate. Here, by leveraging recent knowledge of the signaling pathways involved in liver development, we describe a robust, scalable protocol that allowed us to consistently generate high-quality bipotent human hepatoblasts and HLCs from both embryonic stem cells and induced PSC (iPSCs). Although not yet fully mature, such HLCs were more similar to adult PHHs than were cells obtained with previously described protocols, showing good potential as a physiologically representative alternative to PHHs for in vitro modeling. PSC-derived hepatoblasts effectively generated with this protocol could differentiate into mature hepatocytes and cholangiocytes within syngeneic liver organoids, thus opening the way for representative human 3D in vitro modeling of liver development and pathophysiology.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Adulto , Diferenciação Celular , Células-Tronco Embrionárias , Hepatócitos , Humanos , Transdução de SinaisRESUMO
Dyslipidemia and autophagy have been implicated in the pathogenesis of blinding neovascular age-related macular degeneration (NV-AMD). VLDL receptor (VLDLR), expressed in photoreceptors with a high metabolic rate, facilitates the uptake of triglyceride-derived fatty acids. Since fatty acid uptake is reduced in Vldlr-/- tissues, more remain in circulation, and the retina is fuel deficient, driving the formation in mice of neovascular lesions reminiscent of retinal angiomatous proliferation (RAP), a subtype of NV-AMD. Nutrient scarcity and energy failure are classically mitigated by increasing autophagy. We found that excess circulating lipids restrained retinal autophagy, which contributed to pathological angiogenesis in the Vldlr-/- RAP model. Triglyceride-derived fatty acid sensed by free fatty acid receptor 1 (FFAR1) restricted autophagy and oxidative metabolism in photoreceptors. FFAR1 suppressed transcription factor EB (TFEB), a master regulator of autophagy and lipid metabolism. Reduced TFEB, in turn, decreased sirtuin-3 expression and mitochondrial respiration. Metabolomic signatures of mouse RAP-like retinas were consistent with a role in promoting angiogenesis. This signature was also found in human NV-AMD vitreous. Restoring photoreceptor autophagy in Vldlr-/- retinas, either pharmacologically or by deleting Ffar1, enhanced metabolic efficiency and suppressed pathological angiogenesis. Dysregulated autophagy by circulating lipids might therefore contribute to the energy failure of photoreceptors driving neovascular eye diseases, and FFAR1 may be a target for intervention.
Assuntos
Degeneração Macular , Neovascularização Retiniana , Animais , Autofagia , Proliferação de Células , Ácidos Graxos , Degeneração Macular/patologia , Camundongos , Neovascularização Patológica , Receptores Acoplados a Proteínas G , Neovascularização Retiniana/patologia , TriglicerídeosRESUMO
While most assisted reproductive technologies (ART) are considered routine for the reproduction of species of economical importance, such as the bovine, the impact of these manipulations on the developing embryo remains largely unknown. In an effort to obtain a comprehensive survey of the bovine embryo transcriptome and how it is modified by ART, resources were combined to design an embryo-specific microarray. Close to one million high-quality reads were produced from subtracted bovine embryo libraries using Roche 454 Titanium deep sequencing technology, which enabled the creation of an augmented bovine genome catalog. This catalog was enriched with bovine embryo transcripts, and included newly discovered indel type and 3'UTR variants. Using this augmented bovine genome catalog, the EmbryoGENE Bovine Microarray was designed and is composed of a total of 42,242 probes, including 21,139 known reference genes; 9,322 probes for novel transcribed regions (NTRs); 3,677 alternatively spliced exons; 3,353 3'-tiling probes; and 3,723 controls. A suite of bioinformatics tools was also developed to facilitate microrarray data analysis and database creation; it includes a quality control module, a Laboratory Information Management System (LIMS) and microarray analysis software. Results obtained during this study have already led to the identification of differentially expressed blastocyst targets, NTRs, splice variants of the indel type, and 3'UTR variants. We were able to confirm microarray results by real-time PCR, indicating that the EmbryoGENE bovine microarray has the power to detect physiologically relevant changes in gene expression.
Assuntos
Bovinos/embriologia , Bovinos/genética , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Transcriptoma/fisiologia , Animais , Biologia Computacional , Sistemas de Gerenciamento de Base de Dados , Bases de Dados Genéticas , Embrião de Mamíferos , Feminino , Perfilação da Expressão Gênica/normas , Células da Granulosa/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Técnicas de Reprodução Assistida , Interface Usuário-ComputadorRESUMO
The kallikrein-kinin system (KKS) contributes to retinal inflammation and neovascularization, notably in diabetic retinopathy (DR) and neovascular age-related macular degeneration (AMD). Bradykinin type 1 (B1R) and type 2 (B2R) receptors are G-protein-coupled receptors that sense and mediate the effects of kinins. While B2R is constitutively expressed and regulates a plethora of physiological processes, B1R is almost undetectable under physiological conditions and contributes to pathological inflammation. Several KKS components (kininogens, tissue and plasma kallikreins, and kinin receptors) are overexpressed in human and animal models of retinal diseases, and their inhibition, particularly B1R, reduces inflammation and pathological neovascularization. In this review, we provide an overview of the KKS with emphasis on kinin receptors in the healthy retina and their detrimental roles in DR and AMD. We highlight the crosstalk between the KKS and the renin-angiotensin system (RAS), which is known to be detrimental in ocular pathologies. Targeting the KKS, particularly the B1R, is a promising therapy in retinal diseases, and B1R may represent an effector of the detrimental effects of RAS (Ang II-AT1R).
Assuntos
Cininas/metabolismo , Degeneração Macular/patologia , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/metabolismo , Retina/metabolismo , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Humanos , Sistema Calicreína-Cinina , Degeneração Macular/metabolismo , Neovascularização Patológica , Sistema Renina-Angiotensina , Retina/patologiaRESUMO
The group of retinal degenerations, retinitis pigmentosa (RP), comprises more than 150 genetic abnormalities affecting photoreceptors. Finding degenerative pathways common to all genetic abnormalities may allow general treatment such as neuroprotection. Neuroprotection may include enhancing the function of cells that directly support photoreceptors, retinal pigment epithelial cells, and Müller glia. Treatment with fibroblast growth factor 21 (FGF21), a neuroprotectant, from postnatal week 4-10, during rod and cone loss in P23H mice (an RP model) with retinal degeneration, preserved photoreceptor function and normalized Müller glial cell morphology. Single-cell transcriptomics of retinal cells showed that FGF21 receptor Fgfr1 was specifically expressed in Müller glia/astrocytes. Of all retinal cells, FGF21 predominantly affected genes in Müller glia/astrocytes with increased expression of axon development and synapse formation pathway genes. Therefore, enhancing retinal glial axon and synapse formation with neurons may preserve retinal function in RP and may suggest a general therapeutic approach for retinal degenerative diseases.