RESUMO
The underlying mechanisms of atherosclerosis, the second leading cause of death among Werner syndrome (WS) patients, are not fully understood. Here, we establish an in vitro co-culture system using macrophages (iMφs), vascular endothelial cells (iVECs), and vascular smooth muscle cells (iVSMCs) derived from induced pluripotent stem cells. In co-culture, WS-iMφs induces endothelial dysfunction in WS-iVECs and characteristics of the synthetic phenotype in WS-iVSMCs. Transcriptomics and open chromatin analysis reveal accelerated activation of type I interferon signaling and reduced chromatin accessibility of several transcriptional binding sites required for cellular homeostasis in WS-iMφs. Furthermore, the H3K9me3 levels show an inverse correlation with retrotransposable elements, and retrotransposable element-derived double-stranded RNA activates the DExH-box helicase 58 (DHX58)-dependent cytoplasmic RNA sensing pathway in WS-iMφs. Conversely, silencing type I interferon signaling in WS-iMφs rescues cell proliferation and suppresses cellular senescence and inflammation. These findings suggest that Mφ-specific inhibition of type I interferon signaling could be targeted to treat atherosclerosis in WS patients.
Assuntos
Aterosclerose , Inflamação , Interferon Tipo I , Macrófagos , Retroelementos , Síndrome de Werner , Interferon Tipo I/metabolismo , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Humanos , Aterosclerose/metabolismo , Aterosclerose/imunologia , Aterosclerose/genética , Aterosclerose/patologia , Macrófagos/metabolismo , Macrófagos/imunologia , Retroelementos/genética , Inflamação/metabolismo , Inflamação/patologia , Inflamação/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Transdução de Sinais , Técnicas de Cocultura , Miócitos de Músculo Liso/metabolismo , Células Endoteliais/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Senescência Celular , Proliferação de CélulasRESUMO
Tissue regeneration following an injury requires dynamic cell-state transitions that allow for establishing the cell identities required for the restoration of tissue homeostasis and function. Here, we present a biochemical intervention that induces an intermediate cell state mirroring a transition identified during normal differentiation of myoblasts and other multipotent and pluripotent cells to mature cells. When applied in somatic differentiated cells, the intervention, composed of one-carbon metabolites, reduces some dedifferentiation markers without losing the lineage identity, thus inducing limited reprogramming into a more flexible cell state. Moreover, the intervention enabled accelerated repair after muscle injury in young and aged mice. Overall, our study uncovers a conserved biochemical transitional phase that enhances cellular plasticity in vivo and hints at potential and scalable biochemical interventions of use in regenerative medicine and rejuvenation interventions that may be more tractable than genetic ones.
Assuntos
Músculos , Mioblastos , Camundongos , Animais , Diferenciação Celular , Mioblastos/metabolismoRESUMO
Werner syndrome (WS) is a hereditary premature aging disorder characterized by visceral fat accumulation and subcutaneous lipoatrophy, resulting in severe insulin resistance. However, its underlying mechanism remains unclear. In this study, we show that senescence-associated inflammation and suppressed adipogenesis play a role in subcutaneous adipose tissue reduction and dysfunction in WS. Clinical data from four Japanese patients with WS revealed significant associations between the decrease of areas of subcutaneous fat and increased insulin resistance measured by the glucose clamp. Adipose-derived stem cells from the stromal vascular fraction derived from WS subcutaneous adipose tissues (WSVF) showed early replicative senescence and a significant increase in the expression of senescence-associated secretory phenotype (SASP) markers. Additionally, adipogenesis and insulin signaling were suppressed in WSVF, and the expression of adipogenesis suppressor genes and SASP-related genes was increased. Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), alleviated premature cellular senescence, rescued the decrease in insulin signaling, and extended the lifespan of WS model of C. elegans. To the best of our knowledge, this study is the first to reveal the critical role of cellular senescence in subcutaneous lipoatrophy and severe insulin resistance in WS, highlighting the therapeutic potential of rapamycin for this disease.
Assuntos
Resistência à Insulina , Insulinas , Lipodistrofia , Síndrome de Werner , Animais , Humanos , Síndrome de Werner/genética , Adipogenia/genética , Caenorhabditis elegans , Senescência Celular/genética , Gordura Subcutânea/metabolismo , Inflamação , Sirolimo , MamíferosRESUMO
STUDY OBJECTIVE: The effects of the sodium-dependent glucose transporter-2 inhibitor ipragliflozin were compared with metformin in a previous study, which revealed that ipragliflozin reduced visceral fat content by 12%; however, the underlying mechanism was unclear. Therefore, this sub-analysis aimed to compare metabolomic changes associated with ipragliflozin and metformin that may contribute to their biological effects. DESIGN: A sub-analysis of a randomized controlled study. SETTING: Chiba University Hospital and ten hospitals in Japan. PATIENTS: Fifteen patients with type 2 diabetes in the ipragliflozin group and 15 patients with type 2 diabetes in the metformin group with matching characteristics, such as age, sex, baseline A1C, baseline visceral fat area, smoking status, and concomitant medication. INTERVENTIONS: Ipragliflozin 50 mg or metformin 1000 mg daily. MEASUREMENTS: The clinical data were reanalyzed, and metabolomic analysis of serum samples collected before and 24 weeks after drug administration was performed using capillary electrophoresis time-of-flight mass spectrometry. MAIN RESULTS: The reduction in the mean visceral fat area after 24 weeks of treatment was significantly larger (p = 0.002) in the ipragliflozin group (-19.8%) than in the metformin group (-2.5%), as were the subcutaneous fat area and body weight. The A1C and blood glucose levels decreased in both groups. Glutamic pyruvic oxaloacetic transaminase, γ-glutamyl transferase, uric acid, and triglyceride levels decreased in the ipragliflozin group. Low-density lipoprotein cholesterol levels decreased in the metformin group. After ipragliflozin administration, N2-phenylacetylglutamine, inosine, guanosine, and 1-methyladenosine levels increased, whereas galactosamine, glucosamine, 11-aminoundecanoic acid, morpholine, and choline levels decreased. After metformin administration, metformin, hypotaurine, methionine, methyl-2-oxovaleric acid, 3-nitrotyrosine, and cyclohexylamine levels increased, whereas citrulline, octanoic acid, indole-3-acetaldehyde, and hexanoic acid levels decreased. CONCLUSIONS: Metabolites that may affect visceral fat reduction were detected in the ipragliflozin group. Studies are required to further elucidate the underlying mechanisms.
Assuntos
Diabetes Mellitus Tipo 2 , Metformina , Inibidores do Transportador 2 de Sódio-Glicose , Humanos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Metformina/uso terapêutico , Hipoglicemiantes/efeitos adversos , Japão , Hemoglobinas Glicadas , Gordura Intra-Abdominal/metabolismo , Glicemia , Quimioterapia Combinada , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêuticoRESUMO
In the retina, microglia are resident immune cells that are essential for development and function. Retinal microglia play a central role in mediating pathological degeneration in diseases such as glaucoma, retinitis pigmentosa, age-related neurodegeneration, ischemic retinopathy, and diabetic retinopathy. Current models of mature human retinal organoids (ROs) derived from iPS cell (hiPSC) do not contain resident microglia integrated into retinal layers. Increasing cellular diversity in ROs by including resident microglia would more accurately represent the native retina and better model diseases in which microglia play a key role. In this study, we develop a new 3D in vitro tissue model of microglia-containing retinal organoids by co-culturing ROs and hiPSC-derived macrophage precursor cells (MPCs). We optimized the parameters for successful integration of MPCs into retinal organoids. We show that while in the ROs, MPCs migrate to the equivalent of the outer plexiform layer where retinal microglia cells reside in healthy retinal tissue. While there, they develop a mature morphology characterized by small cell bodies and long branching processes which is only observed in vivo. During this maturation process these MPCs cycle through an activated phase followed by a stable mature microglial phase as seen by the down regulation of pro-inflammatory cytokines and upregulation of anti-inflammatory cytokines. Finally, we characterized mature ROs with integrated MPCs using RNAseq showing an enrichment of cell-type specific microglia markers. We propose that this co-culture system may be useful for understanding the pathogenesis of retinal diseases involving retinal microglia and for drug discovery directly in human tissue.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doenças Retinianas , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Microglia/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Retina , Doenças Retinianas/patologia , Organoides/patologia , Macrófagos/patologia , Citocinas/metabolismoRESUMO
Transgenerational epigenetic inheritance in mammals remains a debated subject. Here, we demonstrate that DNA methylation of promoter-associated CpG islands (CGIs) can be transmitted from parents to their offspring in mice. We generated DNA methylation-edited mouse embryonic stem cells (ESCs), in which CGIs of two metabolism-related genes, the Ankyrin repeat domain 26 and the low-density lipoprotein receptor, were specifically methylated and silenced. DNA methylation-edited mice generated by microinjection of the methylated ESCs exhibited abnormal metabolic phenotypes. Acquired methylation of the targeted CGI and the phenotypic traits were maintained and transmitted across multiple generations. The heritable CGI methylation was subjected to reprogramming in parental PGCs and subsequently reestablished in the next generation at post-implantation stages. These observations provide a concrete step toward demonstrating transgenerational epigenetic inheritance in mammals, which may have implications in our understanding of evolutionary biology as well as the etiology, diagnosis, and prevention of non-genetically inherited human diseases.
Assuntos
Metilação de DNA , Epigênese Genética , Camundongos , Humanos , Animais , Ilhas de CpG , Padrões de Herança , Mamíferos/genéticaRESUMO
Retinal neovascularization (NV) is the major cause of severe visual impairment in patients with ischemic eye diseases. While it is known that retinal microglia contribute to both physiological and pathological angiogenesis, the molecular mechanisms by which these glia regulate pathological NV have not been fully elucidated. In this study, we utilized a retinal microglia-specific Transforming Growth Factor-ß (Tgfß) receptor knock out mouse model and human iPSC-derived microglia to examine the role of Tgfß signaling in activated microglia during retinal NV. Using a tamoxifen-inducible, microglia-specific Tgfß receptor type 2 (Tgfßr2) knockout mouse [Tgfßr2 KO (ΔMG)] we show that Tgfß signaling in microglia actively represses leukostasis in retinal vessels. Furthermore, we show that Tgfß signaling represses expression of the pro-angiogenic factor, Insulin-like growth factor 1 (Igf1), independent of Vegf regulation. Using the mouse model of oxygen-induced retinopathy (OIR) we show that Tgfß signaling in activated microglia plays a role in hypoxia-induced NV where a loss in Tgfß signaling microglia exacerbates and prolongs retinal NV in OIR. Using human iPSC-derived microglia cells in an in vitro assay, we validate the role of Transforming Growth Factor-ß1 (Tgfß1) in regulating Igf1 expression in hypoxic conditions. Finally, we show that Tgfß signaling in microglia is essential for microglial homeostasis and that the disruption of Tgfß signaling in microglia exacerbates retinal NV in OIR by promoting leukostasis and Igf1 expression.
Assuntos
Leucostasia , Doenças Retinianas , Neovascularização Retiniana , Animais , Modelos Animais de Doenças , Hipóxia/complicações , Hipóxia/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Leucostasia/complicações , Leucostasia/metabolismo , Leucostasia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/metabolismo , Neovascularização Patológica/metabolismo , Oxigênio/metabolismo , Doenças Retinianas/metabolismo , Neovascularização Retiniana/etiologia , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Host lipid metabolism and viral responses are intimately connected. However, the process by which the acquired immune systems adapts lipid metabolism to meet demands, and whether or not the metabolic rewiring confers a selective advantage to host immunity, remains unclear. Here we show that viral infection attenuates the expression of genes related to lipid metabolism in murine CD4+ T cells, which in turn increases the expression of antiviral genes. Inhibition of the fatty acid synthesis pathway substantially increases the basal expression of antiviral genes via the spontaneous production of type I interferon (IFN). Using a combination of CRISPR/Cas9-mediated genome editing technology and a global lipidomics analysis, we found that the decrease in monounsaturated fatty acid caused by genetic deletion of Scd2 in mice was crucial for the induction of an antiviral response through activation of the cGAS-STING pathway. These findings demonstrate the important relationship between fatty acid biosynthesis and type I IFN responses that enhances the antiviral response.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Ácidos Graxos Monoinsaturados/metabolismo , Interferon Tipo I/farmacologia , Proteínas de Membrana/fisiologia , Nucleotidiltransferases/fisiologia , Estearoil-CoA Dessaturase/fisiologia , Viroses/imunologia , Animais , Interações Hospedeiro-Patógeno , Metabolismo dos Lipídeos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transdução de Sinais , Viroses/metabolismoRESUMO
Adult progeria Werner syndrome (WS), a rare autosomal recessive disorder, is characterized by accelerated aging symptoms after puberty. The causative gene, WRN, is a member of the RecQ DNA helicase family and is predominantly involved in DNA replication, repair, and telomere maintenance. Here, we report the generation of iPS cells from a patient with WS and correction of the WRN gene by the CRISPR/Cas9-mediated method. These iPSC lines would be a valuable resource for deciphering the pathogenesis of WS.
Assuntos
Células-Tronco Pluripotentes Induzidas , Síndrome de Werner , Adulto , Sistemas CRISPR-Cas/genética , Exodesoxirribonucleases/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Síndrome de Werner/genética , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismoRESUMO
Werner syndrome (WS), also known as adult progeria, is characterized by accelerated aging symptoms from a young age. Patients with WS experience painful intractable skin ulcers with calcifications in their extremities, subcutaneous lipoatrophy, and sarcopenia. However, there is no significant abnormality in the trunk skin, where the subcutaneous fat relatively accumulates. The cause of such differences between the limbs and trunk is unknown. To investigate the underlying mechanism behind these phenomena, we established and analyzed dermal fibroblasts from the foot and trunk of two WS patients. As a result, WS foot-derived fibroblasts showed decreased proliferative potential compared to that from the trunk, which correlated with the telomere shortening. Transcriptome analysis showed increased expression of genes involved in osteogenesis in the foot fibroblasts, while adipogenic and chondrogenic genes were downregulated in comparison with the trunk. Consistent with these findings, the adipogenic and chondrogenic differentiation capacity was significantly decreased in the foot fibroblasts in vitro. On the other hand, the osteogenic potential was mutually maintained and comparable in the foot and trunk fibroblasts. These distinct phenotypes in the foot and trunk fibroblasts are consistent with the clinical symptoms of WS and may partially explain the underlying mechanism of this disease phenotype.
Assuntos
Abdome/fisiologia , Envelhecimento/genética , Fibroblastos/patologia , Pé/fisiopatologia , Corpo Humano , Fenótipo , Síndrome de Werner/genética , Senescência Celular , Perfilação da Expressão Gênica , Humanos , Osteogênese , Helicase da Síndrome de Werner/genéticaRESUMO
Mutations in transforming growth factor-beta-induced (TGFBI) gene cause clinically distinct types of corneal dystrophies. To delineate the mechanisms driving these dystrophies, we focused on the R124C mutation in TGFBI that causes lattice corneal dystrophy type1 (LCD1) and generated novel transgenic mice harbouring a single amino acid substitution of arginine 124 with cysteine in TGFBI via ssODN-mediated base-pair substitution using CRISPR/Cas9 technology. Eighty percent of homozygous and 9.1% of heterozygous TGFBI-R124C mice developed a corneal opacity at 40 weeks of age. Hematoxylin and eosin and Masson trichrome staining showed eosinophilic deposits in subepithelial corneal stroma that stained negative for Congo-red. Although amyloid deposition was not observed in TGFBI-R124C mice, irregular amorphous deposits were clearly observed via transmission electron microscopy near the basement membrane. Interestingly, we found that the corneal deposition of TGFBI protein (TGFBIp) was significantly increased in homozygous TGFBI-R124C mice, suggesting a pathogenic role for the mutant protein accumulation. Furthermore, as observed in the LCD1 patients, corneal epithelial wound healing was significantly delayed in TGFBI-R124C mice. In conclusion, our novel mouse model of TGFBI-R124C corneal dystrophy reproduces features of the human disease. This mouse model will help delineate the pathogenic mechanisms of human corneal dystrophy.
Assuntos
Distrofias Hereditárias da Córnea/genética , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/genética , Fator de Crescimento Transformador beta/genética , Substituição de Aminoácidos , Animais , Arginina/genética , Sistemas CRISPR-Cas , Distrofias Hereditárias da Córnea/patologia , Substância Própria/patologia , Substância Própria/ultraestrutura , Cisteína/genética , Feminino , Heterozigoto , Homozigoto , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Mutação , Reparo de DNA por RecombinaçãoRESUMO
ST2hi memory-type Th2 cells are identified as a pathogenic subpopulation in eosinophilic airway inflammation. These ST2hi pathogenic Th2 cells produce large amount of IL-5 upon T cell receptor stimulation, but not in response to IL-33 treatment. By contrast, IL-33 alone induces cytokine production in ST2+ group 2 innate lymphoid cells (ILC2). Here we show that a MAPK phosphatase Dusp10 is a key negative regulator of IL-33-induced cytokine production in Th2 cells. In this regard, Dusp10 is expressed by ST2hi pathogenic Th2 cells but not by ILC2, and Dusp10 expression inhibits IL-33-induced cytokine production. Mechanistically, this inhibition is mediated by DUSP10-mediated dephosphorylation and inactivation of p38 MAPK, resulting in reduced GATA3 activity. The deletion of Dusp10 renders ST2hi Th2 cells capable of producing IL-5 by IL-33 stimulation. Our data thus suggest that DUSP10 restricts IL-33-induced cytokine production in ST2hi pathogenic Th2 cells by controlling p38-GATA3 activity.
Assuntos
Citocinas/metabolismo , Fosfatases de Especificidade Dupla/metabolismo , Interleucina-33/farmacologia , Células Th2/efeitos dos fármacos , Células Th2/metabolismo , Animais , Animais Geneticamente Modificados , Imunoprecipitação da Cromatina , Fosfatases de Especificidade Dupla/genética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Fator de Transcrição GATA3/metabolismo , Humanos , Immunoblotting , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The gut is an extremely complicated ecosystem where micro-organisms, nutrients and host cells interact vigorously. Although the function of the intestine and its barrier system weakens with age, some probiotics can potentially prevent age-related intestinal dysfunction. Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131, which are the constituents of LB81 yogurt, are representative probiotics. However, it is unclear whether their long-term intake has a beneficial influence on systemic function. Here, we examined the gut microbiome, fecal metabolites and gene expression profiles of various organs in mice. Although age-related alterations were apparent in them, long-term LB81 yogurt intake led to an increased Bacteroidetes to Firmicutes ratio and elevated abundance of the bacterial family S24-7 (Bacteroidetes), which is known to be associated with butyrate and propanoate production. According to our fecal metabolite analysis to detect enrichment, long-term LB81 yogurt intake altered the intestinal metabolic pathways associated with propanoate and butanoate in the mice. Gene ontology analysis also revealed that long-term LB81 yogurt intake influenced many physiological functions related to the defense response. The profiles of various genes associated with antimicrobial peptides-, tight junctions-, adherens junctions- and mucus-associated intestinal barrier functions were also drastically altered in the LB81 yogurt-fed mice. Thus, long-term intake of LB81 yogurt has the potential to maintain systemic homeostasis, such as the gut barrier function, by controlling the intestinal microbiome and its metabolites.
Assuntos
Fermentação , Lactobacillus delbrueckii/metabolismo , Probióticos/metabolismo , Streptococcus thermophilus/metabolismo , Iogurte/microbiologia , Animais , Intestinos/imunologia , Intestinos/microbiologia , Lactobacillus delbrueckii/genética , Lactobacillus delbrueckii/imunologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Streptococcus thermophilus/genética , Streptococcus thermophilus/imunologiaRESUMO
Immunotherapies have led to the successful development of novel therapies for cancer. However, there is increasing concern regarding the adverse effects caused by non-tumor-specific immune responses. Here, we report an effective strategy to generate high-avidity tumor-antigen-specific CTLs, using Cas9/single-guide RNA (sgRNA) ribonucleoprotein (RNP) delivery. As a proof-of-principle demonstration, we selected the gp100 melanoma-associated tumor antigen, and cloned the gp100-specific high-avidity TCR from gp100-immunized mice. To enable rapid structural dissection of the TCR, we developed a 3D protein structure modeling system for the TCR/antigen-major histocompatibility complex (pMHC) interaction. Combining these technologies, we efficiently generated gp100-specific PD-1(-) CD8+ T cells, and demonstrated that the genetically engineered CD8+ T cells have high avidity against melanoma cells both in vitro and in vivo. Our methodology offers computational prediction of the TCR response, and enables efficient generation of tumor antigen-specific CD8+ T cells that can neutralize tumor-induced immune suppression leading to a potentially powerful cancer therapeutic.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Sistemas CRISPR-Cas , Edição de Genes , Neoplasias/genética , Neoplasias/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Animais , Antígenos de Neoplasias/química , Linhagem Celular Tumoral , Feminino , Técnicas de Inativação de Genes , Genes Reporter , Melanoma Experimental , Camundongos , Modelos Moleculares , Complexos Multiproteicos , Neoplasias/metabolismo , Peptídeos/química , Peptídeos/imunologia , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Antígeno gp100 de Melanoma/química , Antígeno gp100 de Melanoma/imunologia , Antígeno gp100 de Melanoma/metabolismoRESUMO
Radiation-induced intestinal fibrosis (RIF) is a serious complication after abdominal radiotherapy for pelvic tumor or peritoneal metastasis. Herein, we show that RIF is mediated by eosinophil interactions with α-smooth muscle actin-positive (α-SMA+) stromal cells. Abdominal irradiation caused RIF especially in the submucosa (SM) of the small intestine, which was associated with the excessive accumulation of eosinophils in both human and mouse. Eosinophil-deficient mice showed markedly ameliorated RIF, suggesting the importance of eosinophils. After abdominal irradiation, chronic crypt cell death caused elevation of extracellular adenosine triphosphate, which in turn activated expression of C-C motif chemokine 11 (CCL11) by pericryptal α-SMA+ cells in the SM to attract eosinophils in mice. Inhibition of C-C chemokine receptor 3 (CCR3) by genetic deficiency or neutralizing antibody (Ab) treatment suppressed eosinophil accumulation in the SM after irradiation in mice, suggesting a critical role of the CCL11/CCR3 axis in the eosinophil recruitment. Activated α-SMA+ cells also expressed granulocyte-macrophage colony-stimulating factor (GM-CSF) to activate eosinophils. Transforming growth factor-ß1 from GM-CSF-stimulated eosinophils promoted collagen expression by α-SMA+ cells. In translational studies, treatment with a newly developed interleukin-5 receptor α-targeting Ab, analogous to the human agent benralizumab, depleted intestinal eosinophils and suppressed RIF in mice. Collectively, we identified eosinophils as a crucial factor in the pathogenesis of RIF and showed potential therapeutic strategies for RIF by targeting eosinophils.
Assuntos
Eosinófilos/metabolismo , Fibrose/etiologia , Fibrose/metabolismo , Intestino Delgado/efeitos da radiação , Lesões Experimentais por Radiação/metabolismo , Animais , Modelos Animais de Doenças , Fibrose/prevenção & controle , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Lesões Experimentais por Radiação/prevenção & controleRESUMO
Granular corneal dystrophy (GCD) is an autosomal dominant hereditary disease in which multiple discrete and irregularly shaped granular opacities are deposited in the corneal stroma. GCD is caused by a point mutation in the transforming growth factor-ß-induced (TGFBI) gene, located on chromosome 5q31. Here, we report the first successful application of CRISPR-Cas9-mediated genome editing for the correction of a TGFBI mutation in GCD patient-derived primary corneal keratocytes via homology-directed repair (HDR). To correct genetic defects in GCD patient cells, we designed a disease-specific guide RNA (gRNA) targeting the R124H mutation of TGFBI, which causes GCD type 2 (GCD2). An R124H mutation in primary human corneal keratocytes derived from a GCD2 patient was corrected by delivering a CRISPR plasmid expressing Cas9/gRNA and a single-stranded oligodeoxynucleotide HDR donor template in vitro. The gene correction efficiency was 20.6% in heterozygous cells and 41.3% in homozygous cells. No off-target effects were detected. These results reveal a new therapeutic strategy for GCD2; this method may also be applicable to other heredity corneal diseases.
Assuntos
Sistemas CRISPR-Cas/genética , Fator de Crescimento Transformador beta/genética , Sequência de Bases , Células Cultivadas , Distrofias Hereditárias da Córnea/metabolismo , Distrofias Hereditárias da Córnea/patologia , Ceratócitos da Córnea/citologia , Ceratócitos da Córnea/metabolismo , Edição de Genes , Heterozigoto , Homozigoto , Humanos , Mutagênese Sítio-Dirigida , Análise de Sequência de DNARESUMO
A randomized clinical trial showed the beneficial effects of the selective peroxisome proliferator-activated receptor (PPAR)-α agonist, fenofibrate, in reducing the progression of diabetic retinopathy independent of serum lipid levels. All subtypes of PPAR (PPAR-α, PPAR-γ, and PPAR-ß/δ) have been reported to play a key role in microvascular inflammation and angiogenesis. Therefore, the agonistic function of fenofibrate against the PPAR-α has been suggested to contribute to its medicinal effect. Furthermore, bezafibrate is a fibrate drug commonly used as a lipid-lowering agent to treat hyperlipidemia and acts as a pan-agonist of all PPARs subtypes. However, the effects of bezafibrate in diabetic retinopathy remain unclear. Therefore, the purpose of this study was to investigate the effects of bezafibrate on retinal microvascular inflammation. Bezafibrate was not cytotoxic against human retinal microvascular endothelial cells (HRMECs) and human retinal pigment epithelial cells (ARPE-19 cells) treated with <100 and 200µM bezafibrate, respectively. In HRMECs, the expression levels of tumor necrosis factor (TNF)-α-induced monocyte chemoattractant protein (MCP)-1, intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1 were significantly suppressed by bezafibrate in a dose-dependent manner. TNF-α-induced nuclear translocation of nuclear factor (NF)-κB p65 and cell migration were also significantly inhibited in bezafibrate-treated HRMECs. Furthermore, bezafibrate treatment significantly suppressed interleukin (IL)-1ß-induced vascular endothelial growth factor (VEGF) production in ARPE-19 cells. These results suggest that bezafibrate has beneficial effects on retinal microvascular inflammation. Our study demonstrates the therapeutic potential of bezafibrate for managing diabetic retinopathy.
Assuntos
Anti-Inflamatórios/farmacologia , Bezafibrato/farmacologia , Retinopatia Diabética/tratamento farmacológico , Células Endoteliais/efeitos dos fármacos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Receptores Ativados por Proliferador de Peroxissomo/agonistas , Retina/patologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL2/metabolismo , Retinopatia Diabética/imunologia , Células Endoteliais/imunologia , Humanos , NF-kappa B/metabolismo , Epitélio Pigmentado da Retina/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by post-transcriptional inhibition of mRNA translation. Dysregulation of miRNAs, including circulating miRNAs, has been reported to play an important role in the development of various diseases, including fibrotic diseases. Aberrant expression of miRNAs in the vitreous humor of vitreoretinal diseased eyes has been reported. However, the expression pattern of miRNAs present in the vitreous humor of proliferative vitreoretinal disease (PVD) patients, including proliferative diabetic retinopathy (PDR), and proliferative vitreoretinopathy (PVR), remains unknown. To investigate the factors important for the development of PVD, we characterized the miRNAs present in the vitreous humor of PVD patients and analyzed the expression profiles of 377 miRNAs using quantitative polymerase chain reaction-based miRNA arrays. The expression of a specific subset of miRNAs, previously reported to be associated with the development of angiogenesis and fibrosis, was significantly altered in the vitreous of PVD patients. Among these miRNAs, we identified miR-21 as a candidate fibrotic miRNA with an important role in the pathogenesis of PVD. Increased miR-21 levels in the vitreous were associated with retinal fibrosis, including PVR and PDR. Because epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPECs) plays a critical role in retinal fibrosis, the expression of miR-21 in human RPECs was determined. Its expression in RPECs was induced by transforming growth factor-ß, a key growth factor involved in fibrogenesis, and was enhanced by high glucose culture conditions, suggesting that miR-21 expression positively correlates with disease progression. Gain- and loss-of-function studies revealed that miR-21 promoted cell proliferation and migration of ARPE-19 cells without affecting EMT-related gene expression. Together, our studies have identified miR-21 as a potential disease-modifying miRNA in the vitreous humor that is involved in the development of retinal fibrosis and may be a novel marker of PVD.
Assuntos
Proliferação de Células , Retinopatia Diabética/metabolismo , MicroRNAs/genética , Epitélio Pigmentado da Retina/metabolismo , Regulação para Cima , Corpo Vítreo/metabolismo , Idoso , Estudos de Casos e Controles , Linhagem Celular , Movimento Celular , Retinopatia Diabética/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Epitélio Pigmentado da Retina/patologia , Fator de Crescimento Transformador beta/metabolismo , Corpo Vítreo/patologiaRESUMO
In the current aging society, cognitive dysfunction is one of the most serious issues that should be urgently resolved. It also affects a wide range of age groups harboring neurological and psychiatric disorders, such as Alzheimer's disease and schizophrenia. Although the molecular mechanism of memory impairment still remains to be determined, neuronal loss and dysfunction has been revealed to mainly attribute to its pathology. The discovery of neural stem cells in the adult brain that are proliferating and able to generate functional neurons has given rise to the idea that neuronal loss could be rescued by manipulating endogenous neural progenitor and stem cells. To this end, we must characterize them in detail and their developmental programming must be better understood. A growing body of evidence has indicated that insulin-like peptides are involved in learning and memory and maintenance of neural progenitor and stem cells, and clinical trials of insulin as a memory enhancer have begun. In contrast to the expectation of insulin and IGF1, the roles of IGF2 in cognitive ability have been poorly understood. However, recent evidence demonstrated in rodents suggests that IGF2 may play a pivotal role in adult neurogenesis and cognitive function. Here, we would like to review the rapidly growing world of IGF2 in cognitive neuroscience and introduce the evidence that its deficit is indeed involved in the impairment of the hippocampal neurogenesis and cognitive dysfunction in the model mouse of 22q11.2 deletion syndrome, which deletes Dgcr8, a critical gene for microRNA processing.