RESUMO
ERI1 is a 3'-to-5' exoribonuclease involved in RNA metabolic pathways including 5.8S rRNA processing and turnover of histone mRNAs. Its biological and medical significance remain unclear. Here, we uncover a phenotypic dichotomy associated with bi-allelic ERI1 variants by reporting eight affected individuals from seven unrelated families. A severe spondyloepimetaphyseal dysplasia (SEMD) was identified in five affected individuals with missense variants but not in those with bi-allelic null variants, who showed mild intellectual disability and digital anomalies. The ERI1 missense variants cause a loss of the exoribonuclease activity, leading to defective trimming of the 5.8S rRNA 3' end and a decreased degradation of replication-dependent histone mRNAs. Affected-individual-derived induced pluripotent stem cells (iPSCs) showed impaired in vitro chondrogenesis with downregulation of genes regulating skeletal patterning. Our study establishes an entity previously unreported in OMIM and provides a model showing a more severe effect of missense alleles than null alleles within recessive genotypes, suggesting a key role of ERI1-mediated RNA metabolism in human skeletal patterning and chondrogenesis.
Assuntos
Exorribonucleases , Histonas , Humanos , Exorribonucleases/genética , Histonas/genética , Mutação de Sentido Incorreto/genética , RNA Ribossômico 5,8S , RNA , RNA Mensageiro/genéticaRESUMO
Endothelial dysfunction, a central hallmark of cardiovascular pathogenesis in diabetes mellitus, is characterized by impaired endothelial nitric oxide synthase (eNOS) and NO bioavailability. However, the underlying mechanisms remain unclear. Here in this study, we aimed to identify the role of calmodulin (CaM) in diabetic eNOS dysfunction. Human umbilical vein endothelial cells and murine endothelial progenitor cells (EPCs) treated with high glucose (HG) exhibited downregulated CaM mRNA/protein and vascular endothelial growth factor (VEGF) expression with impeded eNOS phosphorylation and cell migration/tube formation. These perturbations were reduplicated in CALM1-knockdown cells but prevented in CALM1-overexpressing cells. EPCs from type 2 diabetes animals behaved similarly to HG-treated normal EPCs, which could be rescued by CALM1-gene transduction. Consistently, diabetic animals displayed impaired eNOS phosphorylation, endothelium-dependent dilation, and CaM expression in the aorta, as well as deficient physical interaction of CaM and eNOS in the gastrocnemius. Local CALM1 gene delivery into a diabetic mouse ischemic hindlimb improved the blunted limb blood perfusion and gastrocnemius angiogenesis, and foot injuries. Diabetic patients showed insufficient foot microvascular autoregulation, eNOS phosphorylation, and NO production with downregulated CaM expression in the arterial endothelium, and abnormal CALM1 transcription in genome-wide sequencing analysis. Therefore, our findings demonstrated that downregulated CaM expression is responsible for endothelium dysfunction and angiogenesis impairment in diabetes, and provided a novel mechanism and target to protect against diabetic endothelial injury.
Assuntos
Diabetes Mellitus Tipo 2 , Humanos , Camundongos , Animais , Diabetes Mellitus Tipo 2/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Endotélio/metabolismo , Isquemia/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Neovascularização FisiológicaRESUMO
Four new xanthone glucosides, 3-hydroxy-2-methoxyxanthone-4-O-ß-D-glucopyranoside (1), 4,8-dihydroxy-2-methoxyxanthone-3-O-ß-D-glucopyranoside (2), 2-methoxyxanthone-5-O-ß-D-glucopyranoside (3), 4-hydroxy-2-methoxyxanthone-3-O-ß-D-glucopyranoside (4), a new phenolic acid, 4,4'-dihydroxy-3,3'-imino-di-benzoic acid monomethyl ester (5), and a new isoquinoline, methyl 6-hydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylate (6) were isolated from the fruit of Hypericum patulum. The structural elucidation of the isolated compounds was primarily based on HR-ESI-MS, UV, IR, 1D and 2D NMR. All compounds were evaluated for their inhibitory effect against LPS-induced NO production in RAW 264.7 cells. Compoundâ 2, 3 exhibited moderate inhibitory activity against NO production.
Assuntos
Hypericum , Hypericum/química , Frutas/química , Glucosídeos/química , Espectroscopia de Ressonância MagnéticaRESUMO
Cx43 is the major connexin in ventricular gap junctions, and plays a pivotal role in control of electrical and metabolic communication among adjacent cardiomyocytes. We previously found that Cx43 dephosphorylation at serine 282 (pS282) caused cardiomyocyte apoptosis, which is involved in cardiac ischemia/reperfusion injury. In this study we investigated whether Cx43-S282 hyper-phosphorylation could protect cardiomyocytes against apoptosis. Adenovirus carrying rat full length Cx43 gene (Cx43-wt) or a mutant gene at S282 substituted with aspartic acid (S282D) were transfected into neonatal rat ventricular myocytes (NRVMs) or injected into rat ventricular wall. Rat abdominal aorta constriction model (AAC) was used to assess Cx43-S282 phosphorylation status. We showed that Cx43 phosphorylation at S282 was increased over 2-times compared to Cx43-wt cells at 24 h after transfection, while pS262 and pS368 were unaltered. S282D-transfected cells displayed enhanced gap junctional communication, and increased basal intracellular Ca2+ concentration and spontaneous Ca2+ transients compared to Cx43-wt cells. However, spontaneous apoptosis appeared in NRVMs transfected with S282D for 34 h. Rat ventricular myocardium transfected with S282D in vivo also exhibited apoptotic responses, including increased Bax/Bcl-xL ratio, cytochrome c release as well as caspase-3 and caspase-9 activities, while factor-associated suicide (Fas)/Fas-associated death domain expression and caspase-8 activity remained unaltered. In addition, AAC-induced hypertrophic ventricles had apoptotic injury with Cx43-S282 hyper-phosphorylation compared with Sham ventricles. In conclusion, Cx43 hyper-phosphorylation at S282, as dephosphorylation, also triggers cardiomyocyte apoptosis, but through activation of mitochondrial apoptosis pathway, providing a fine-tuned Cx43-S282 phosphorylation range required for the maintenance of cardiomyocyte function and survival.
Assuntos
Apoptose , Conexina 43 , Miócitos Cardíacos , Animais , Conexina 43/genética , Conexina 43/metabolismo , Mitocôndrias , Miócitos Cardíacos/metabolismo , Fosforilação , Ratos , Serina/metabolismoRESUMO
Dysosteosclerosis (DOS) is a distinct form of sclerosing bone disease characterized by platyspondyly and progressive osteosclerosis. DOS is genetically heterogeneous. Three causal genes, SLC29A3, CSF1R, and TNFRSF11A are reported. TNFRSF11A-associated DOS has been identified in two patients; however, TNFRSF11A is also a causal gene for osteopetrosis, autosomal recessive 7 (OP-AR7). Whole-exome sequencing in a patient with sclerosing bone disease identified novel compound heterozygous variants (c.414_427 + 7del, c.1664del) in TNFRSF11A. We examined the impact of the two variants on five splicing isoforms of TNFRSF11A by RT-PCR. We found that c.1664del resulted in elongated proteins (p.S555Cfs*121, etc.), while c.414_427 + 7del generated two aberrant splicing products (p.A139Wfs*19 and p.E132Dfs*19) that lead to nonsense mediated mRNA decay (NMD). In the previous two cases of TNFRSF11A-associated DOS, their mutations produced truncated TNFRSF11A protein isoforms. The mutations in all three cases thus contrast with TNFRSF11A mutations reported in OP-AR7, which does not generated truncated or elongated TNFRSF11A proteins. Thus, we identified the third case of TNFRSF11A-associated DOS and reinforced the genotype-phenotype correlation that aberrant protein-producing TNFRSF11A mutations cause DOS.
Assuntos
Mutação , Osteosclerose/patologia , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Pré-Escolar , Feminino , Humanos , Osteosclerose/genética , Osteosclerose/metabolismo , Prognóstico , Sequenciamento do ExomaRESUMO
Dysosteosclerosis (DOS) is a rare sclerosing bone dysplasia characterized by osteosclerosis and platyspondyly. DOS is genetically heterogeneous and causally associated with mutations in three genes, SLC29A3, CSF1R, and TNFRSF11A. TNFRSF11A has been known as the causal gene for osteopetrosis, autosomal recessive 7, and is recently reported to cause DOS in three cases, which show a complex genotype-phenotype relationship. The phenotypic spectrum of TNFRSF11A-associated sclerosing bone dysplasia remains unclear and needs to be characterized further in more cases with molecular genetic diagnosis. Here, we report another TNFRSF11A-associated DOS case with a homozygous missense mutation (p.R129C). The mutation effect is different from the previous three cases, in which truncated or elongated RANK proteins were generated in isoform specific manner, thus enriching our understanding of the genotype-phenotype association in TNFRSF11A-associated sclerosing bone dysplasia. Besides DOS, our case presented with intracranial extramedullary hematopoiesis, which is an extremely rare condition and has not been identified in any other sclerosing bone dysplasias with molecular genetic diagnosis. Our findings provide the fourth case of TNFRSF11A-associated DOS and further expand its phenotypic spectrum.
Assuntos
Hematopoese/genética , Osteosclerose/genética , Receptor Ativador de Fator Nuclear kappa-B/genética , Doenças Ósseas , Criança , Pré-Escolar , Feminino , Estudos de Associação Genética , Heterogeneidade Genética , Homozigoto , Humanos , Lactente , Deficiência Intelectual , Mutação/genética , Proteínas de Transporte de Nucleosídeos/genética , Osteopetrose/genética , Osteopetrose/patologia , Osteosclerose/diagnóstico , Osteosclerose/patologia , Fenótipo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , EscleroseRESUMO
The RANKL/OPG/RANK signalling pathway is a major regulatory system for osteoclast formation and activity. Mutations in TNFSF11, TNFRSF11B and TNFRSF11A cause defects in bone metabolism and development, thereby leading to skeletal disorders with changes in bone density and/or morphology. To date, nine kinds of monogenic skeletal diseases have been found to be causally associated with TNFSF11, TNFRSF11B and TNFRSF11A mutations. These diseases can be divided into two types according to the mutation effects and the resultant pathogenesis. One is caused by the mutations inducing constitutional RANK activation or OPG deficiency, which increase osteoclastogenesis and accelerate bone turnover, resulting in juvenile Paget's disease 2, Paget disease of bone 2, familial expansile osteolysis, expansile skeletal hyperphosphatasia, panostotic expansile bone disease, and Paget disease of bone 5. The other is caused by the de-activating mutations in TNFRSF11A or TNFSF11, which decrease osteoclastogenesis and elevate bone density, resulting in osteopetrosis, autosomal recessive 2 and 7, and dysosteosclerosis. Here we reviewed the current knowledge about these genetic disorders with paying particular attention to the updating genotype-phenotype association in the TNFRSF11A-caused diseases.
Assuntos
Doenças Genéticas Inatas/metabolismo , Osteoprotegerina/metabolismo , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Transdução de Sinais , Doenças Genéticas Inatas/diagnóstico por imagem , Doenças Genéticas Inatas/terapia , Humanos , Osteoclastos/metabolismo , Osteoclastos/patologia , Receptor Ativador de Fator Nuclear kappa-B/genética , Transdução de Sinais/genéticaRESUMO
AIM: We have shown that low-dose gadolinium chloride (GdCl3) abolishes arachidonic acid (AA)-induced increase of cytoplasmic Ca(2+), which is known to play a crucial role in myocardial ischemia/reperfusion (I/R) injury. The present study sought to determine whether low-dose GdCl3 pretreatment protected rat myocardium against I/R injury in vitro and in vivo. METHODS: Cultured neonatal rat ventricular myocytes (NRVMs) were treated with GdCl3 or nifedipine, followed by exposure to anoxia/reoxygenation (A/R). Cell apoptosis was detected; the levels of related signaling molecules were assessed. SD rats were intravenously injected with GdCl3 or nifedipine. Thirty min after the administration the rats were subjected to LAD coronary artery ligation followed by reperfusion. Infarction size, the release of serum myocardial injury markers and AA were measured; cell apoptosis and related molecules were assessed. RESULTS: In A/R-treated NRVMs, pretreatment with GdCl3 (2.5, 5, 10 µmol/L) dose-dependently inhibited caspase-3 activation, death receptor-related molecules DR5/Fas/FADD/caspase-8 expression, cytochrome c release, AA release and sustained cytoplasmic Ca(2+) increases induced by exogenous AA. In I/R-treated rats, pre-administration of GdCl3 (10 mg/kg) significantly reduced the infarct size, and the serum levels of CK-MB, cardiac troponin-I, LDH and AA. Pre-administration of GdCl3 also significantly decreased the number of apoptotic cells, caspase-3 activity, death receptor-related molecules (DR5/Fas/FADD) expression and cytochrome c release in heart tissues. The positive control drug nifedipine produced comparable cardioprotective effects in vitro and in vivo. CONCLUSION: Pretreatment with low-dose GdCl3 significantly attenuates I/R-induced myocardial apoptosis in rats by suppressing activation of both death receptor and mitochondria-mediated pathways.
Assuntos
Gadolínio/uso terapêutico , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Ácido Araquidônico/farmacologia , Caspase 3/metabolismo , Ativação Enzimática , Gadolínio/administração & dosagem , Masculino , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Ratos Sprague-Dawley , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVES: The primary aim of this study was to determine whether wave intensity can discriminate cases of eccentric hypertrophy in patients with essential hypertension who have varied left ventricular configurations. METHODS: A total of 155 hypertensive patients with different ventricular configurations (27 normal configuration, 42 concentric remodeling, 62 concentric hypertrophy, and 24 eccentric hypertrophy) were recruited. We performed a noninvasive wave intensity analysis of the common carotid artery and conventional echocardiography. Blood pressure and flow velocity were measured in the right carotid artery of all patients. RESULTS: The left ventricular ejection fraction (LVEF) in the eccentric hypertrophy group was significantly lower than the values in the other groups (P < .05). The R-W1 interval/W1-W2 interval ratio (where W1 indicates the first positive peak and W2 the second positive peak) in the eccentric hypertrophy group was much higher than the values in the other groups (P < .05). However, there were no significant differences in W1, W2, and negative area among these groups. Pearson correlation analysis showed that R-W1/W1-W2, R-W1, and W1-W2were correlated with the LVEF, whereas there was no correlation between W1, W2, negative area, and the reflection coefficient with the LVEF. CONCLUSIONS: We propose that by using the R-W1/W1-W2 ratio, wave intensity analysis can identify hypertensive patients with eccentric hypertrophy without the need for echocardiography.
Assuntos
Artéria Carótida Primitiva/diagnóstico por imagem , Ecocardiografia/métodos , Hipertensão/diagnóstico por imagem , Hipertrofia Ventricular Esquerda/diagnóstico por imagem , Interpretação de Imagem Assistida por Computador/métodos , Análise de Onda de Pulso/métodos , Determinação da Pressão Arterial/métodos , Artéria Carótida Primitiva/fisiopatologia , Feminino , Humanos , Hipertensão/complicações , Hipertensão/fisiopatologia , Hipertrofia Ventricular Esquerda/complicações , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
SLC4A2 belongs to the Na+-independent solute carrier family 4 (SLC4) of anion exchangers, which regulate electroneutral exchange of Cl- for HCO3- and mediate intra- and extra-cellular pH, chloride concentration and cell volume. Slc4a2 also participates in gastric acid secretion, spermatogenesis and osteoclastogenesis. During osteoclast differentiation, Slc4a2 is exclusively expressed at the contra-lacunar membrane and is up-regulated with osteoclast maturation. Bi-allelic Slc4a2 loss-of-function mutations have been known to cause osteopetrosis in mice and cattle, but not in human. Recently, we have identified bi-allelic pathogenic variants in SLC4A2 in a patient affected by osteopetrosis with severe renal insufficiency, suggesting SLC4A2 deficiency causes a new type of autosomal recessive osteopetrosis (osteopetrosis, Ikegawa type). In this article, we review the advances in exploring the multiple functions of SLC4A2 with emphasis on its roles in osteoclast. Our review would contribute to understanding of the phenotypic spectrum and the pathomechanism of SLC4A2-associated osteopetrosis.
Assuntos
Osteoclastos , Osteopetrose , Animais , Bovinos , Humanos , Masculino , Camundongos , Antiportadores de Cloreto-Bicarbonato/genética , Antiportadores de Cloreto-Bicarbonato/metabolismo , Mutação , Osteoclastos/metabolismo , Osteogênese , Osteopetrose/patologiaRESUMO
Objectives: This study sought to derive and validate a simple model combining traditional clinical risk factors with biomarkers and imaging indicators easily obtained from routine preoperative examinations to predict functionally significant coronary artery disease (CAD) in Chinese populations. Methods: We developed five models from a derivation cohort of 320 patients retrospective collected. In the derivation cohort, we assessed each model discrimination using the area under the receiver operating characteristic curve (AUC), reclassification using the integrated discrimination improvement (IDI) and net reclassification improvement (NRI), calibration using the Hosmer-Lemeshow test, and clinical benefit using decision curve analysis (DCA) to derive the optimal model. The optimal model was internally validated by bootstrapping, and external validation was performed in another cohort including 96 patients. Results: The optimal model including 5 predictors (age, sex, hyperlipidemia, hs-cTnI and LVEF) achieved an AUC of 0.807 with positive NRI and IDI in the derivation cohort. Moreover, the Hosmer-Lemeshow test showed a good fit, and the DCA demonstrated good clinical net benefit. The C-statistic calculated by bootstrapping internal validation was 0.798, and the calibration curve showed adequate calibration (Brier score = 0.179). In the external validation cohort, the optimal model performance was acceptable (AUC = 0.704; Brier score = 0.20). Finally, a nomogram based on this model was constructed to facilitate its use in clinical practice. Conclusions: A simple model combined clinical risk factors with hs-cTnI and LVEF improving the prediction of functionally significant CAD in Chinese populations. This attractive model may be a choice for clinicians to risk stratification for CAD.
RESUMO
AIMS: Angiogenesis plays a key role in coronary collateral circulation (CCC), the compensatory formation of new blood vessels during chronic total coronary occlusion. This study aimed to determine whether plasmacytoma variant translocation 1 (PVT1), a long non-coding (lnc) RNA involved in tumor angiogenesis, plays a role in regulating angiogenesis during chronic coronary ischemia. MAIN METHODS: Patients with coronary artery disease, and ≥ 90% stenosis, were examined and divided into "Good" and "Poor" CCC groups based on Rentrop Cohen classification. RNA samples were obtained from all patients, as well as from oxygen and glucose-deprived (OGD) HUVECs. PVT1, miR-15b-5p and AKT3 levels were measured with RT-qPCR or Western blot, while HUVEC migration and angiogenesis were detected by, respectively, wound-healing and tube formation assays. Luciferase reporter assay confirmed direct PVT1-miR-15b-5p binding. KEY FINDINGS: Increased PVT1 was found in "Good CCC" patient plasma, along with being highly expressed among OGD HUVECs; PVT1 knockdown reduced HUVEC migration, tube formation, and pro-angiogenic factor expression. Conversely, OGD HUVECs had downregulated miR-15b-5p, and miR-15b-5p overexpression significantly depressed their angiogenic capabilities. These PVT1 knockdown- or miR-15b-5p overexpression-associated reductions in angiogenic effects were reversed by AKT3 overexpression. In vivo, neovascularization and functioning in both ischemic mice hind-limbs and infarcted myocardium injected with ADV-sh-PVT1 were reduced, which were ameliorated by concurrent antagomiR-15b-5p injections. SIGNIFICANCE: Circulating PVT1 may serve as a useful biomarker to distinguish between good versus poor CCC, as it is involved in orchestrating angiogenesis via the miR-15b-5p-AKT3 axis; it thus has potential as a target for treating ischemic disease.
Assuntos
MicroRNAs , RNA Longo não Codificante/genética , Indutores da Angiogênese , Animais , Antagomirs , Artérias/metabolismo , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células/genética , Glucose , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Neovascularização Patológica/genética , Oxigênio , RNA Longo não Codificante/metabolismoRESUMO
Osteopetrosis is a group of rare inherited skeletal disorders characterized by a marked increase in bone density due to deficient bone resorption. Pathogenic variants in several genes involved in osteoclast differentiation and/or function have been reported to cause osteopetrosis. Solute carrier family 4 member 2 (SLC4A2, encoding anion exchanger 2) plays an important role in osteoclast differentiation and function by exchange of Cl- with HCO3- . Biallelic Slc4a2 loss-of-function mutations in mice and cattle lead to osteopetrosis with osteoclast deficiency; however, pathogenic SLC4A2 variants in humans have not been reported. In this study, we describe a patient with autosomal recessive osteopetrosis due to biallelic pathogenic variants in SLC4A2. We identified novel compound heterozygous variants in SLC4A2 (NM_003040.4: c.556G>A [p.A186T] and c.1658T>C [p.V553A]) by exome sequencing. The measurement of intracellular Cl- showed that the variants decrease the anion exchange activity of SLC4A2. The impact of the variants on osteoclast differentiation was assessed by a gene knockout-rescue system using a mouse macrophage cell line, RAW 264.7. The Slc4a2-knockout cells show impaired osteoclastogenesis, which was rescued by the wild-type SLC4A2, but not by the mutant SLC4A2s. Immunofluorescence and pit assay revealed that the mutant SLC4A2s leads to abnormal podosome belt formation with impaired bone absorption. This is the first report on an individual affected by SLC4A2-associated osteopetrosis (osteopetrosis, Ikegawa type). With functional studies, we prove that the variants lead to SLC4A2 dysfunction, which altogether supports the importance of SLC4A2 in human osteoclast differentiation. © 2021 American Society for Bone and Mineral Research (ASBMR).
Assuntos
Reabsorção Óssea , Osteopetrose , Animais , Reabsorção Óssea/patologia , Bovinos , Linhagem Celular , Antiportadores de Cloreto-Bicarbonato/genética , Antiportadores de Cloreto-Bicarbonato/metabolismo , Humanos , Mutação/genética , Osteoclastos/metabolismo , Osteopetrose/patologiaRESUMO
Among pulmonary arterial hypertension (PAH) patients, right ventricular (RV) functioning has been considered a major determining factor for cardiac capacity and survival. However, despite the recognition of the clinical importance for preserving RV functioning, no effective treatments are currently available for RV failure. This study aims to suggest one such possible treatment, through investigating the cardio-protective capabilities of the anti-oxidant, melatonin (Mel), for treating adverse RV remodeling in PAH, along with its underlying mechanisms. Arginine vasopressin induced neonatal rat cardiomyocyte hypertrophy in vitro; in vivo, PAH was induced in rats through intraperitoneal monocrotaline (MCT) injections, and Mel was administered intraperitoneally 24 h prior to MCT. Mel reduced rat cardiomyocyte hypertrophy and mitochondrial oxidative stress in vitro by activating the Mst1-Nrf2 pathway, which were all reversed upon siRNA knockdown of Mst1. Likewise, in vivo, Mel pre-treatment significantly ameliorated MCT-induced deterioration in cardiac function, RV hypertrophy, fibrosis and dilation. These beneficial effects were also associated with Mst1-Nrf2 pathway up regulation and its associated reduction in oxidative stress, as evidenced by the decrease in RV malondialdehyde content. Notably, results from Mel treatment were similar, or even superior, to those obtained from N-acetyl cysteine (NAC), which has already been-confirmed as an anti-oxidative treatment for PAH. By contrast, co-treatment with the Mst1 inhibitor XMU-MP-1 reversed all of those Mel-associated beneficial effects. Our findings thus identified Mel as a potent cardio-protective agent against the onset of maladaptive RV remodeling, through enhancement of the anti-oxidative response via Mst1-Nrf2 pathway activation.
Assuntos
Hipertensão Pulmonar , Melatonina , Hipertensão Arterial Pulmonar , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Arginina Vasopressina , Cisteína/uso terapêutico , Modelos Animais de Doenças , Hipertensão Pulmonar Primária Familiar , Fator de Crescimento de Hepatócito/metabolismo , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/complicações , Hipertensão Pulmonar/tratamento farmacológico , Hipertrofia Ventricular Direita , Malondialdeído , Melatonina/farmacologia , Melatonina/uso terapêutico , Monocrotalina , Fator 2 Relacionado a NF-E2 , Proteínas Proto-Oncogênicas/metabolismo , Hipertensão Arterial Pulmonar/tratamento farmacológico , RNA Interferente Pequeno/uso terapêutico , Ratos , Remodelação VentricularRESUMO
Chondrodysplasias are hereditary diseases caused by mutations in the components of growth cartilage. Although the unfolded protein response (UPR) has been identified as a key disease mechanism in mouse models, no suitable in vitro system has been reported to analyze the pathology in humans. Here, we developed a three-dimensional culture protocol to differentiate hypertrophic chondrocytes from induced pluripotent stem cells (iPSCs) and examine the phenotype caused by MATN3 and COL10A1 mutations. Intracellular MATN3 or COL10 retention resulted in increased ER stress markers and ER size in most mutants, but activation of the UPR was dependent on the mutation. Transcriptome analysis confirmed a UPR with wide-ranging changes in bone homeostasis, extracellular matrix composition, and lipid metabolism in the MATN3 T120M mutant, which further showed altered cellular morphology in iPSC-derived growth-plate-like structures in vivo. We then applied our in vitro model to drug testing, whereby trimethylamine N-oxide led to a reduction of ER stress and intracellular MATN3.
Assuntos
Cartilagem/fisiologia , Condrócitos/fisiologia , Colágeno Tipo X/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Animais , Osso e Ossos/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células Cultivadas , Condrócitos/citologia , Condrogênese , Colágeno Tipo X/genética , Estresse do Retículo Endoplasmático , Matriz Extracelular/metabolismo , Edição de Genes , Perfilação da Expressão Gênica , Homeostase , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Proteínas Matrilinas/genética , Proteínas Matrilinas/metabolismo , Camundongos , Modelos Biológicos , Mutação , Osteocondrodisplasias/patologia , Fenótipo , Resposta a Proteínas não DobradasRESUMO
Bone formation represents a heritable trait regulated by many signals and complex mechanisms. Its abnormalities manifest themselves in various diseases, including sclerosing bone disorder (SBD). Exploration of genes that cause SBD has significantly improved our understanding of the mechanisms that regulate bone formation. Here, we discover a previously unknown type of SBD in four independent families caused by bi-allelic loss-of-function pathogenic variants in TMEM53, which encodes a nuclear envelope transmembrane protein. Tmem53-/- mice recapitulate the human skeletal phenotypes. Analyses of the molecular pathophysiology using the primary cells from the Tmem53-/- mice and the TMEM53 knock-out cell lines indicates that TMEM53 inhibits BMP signaling in osteoblast lineage cells by blocking cytoplasm-nucleus translocation of BMP2-activated Smad proteins. Pathogenic variants in the patients impair the TMEM53-mediated blocking effect, thus leading to overactivated BMP signaling that promotes bone formation and contributes to the SBD phenotype. Our results establish a previously unreported SBD entity (craniotubular dysplasia, Ikegawa type) and contribute to a better understanding of the regulation of BMP signaling and bone formation.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Osso e Ossos/patologia , Proteínas de Membrana/metabolismo , Esclerose/patologia , Transdução de Sinais , Proteínas Smad/metabolismo , Animais , Sequência de Bases , Diferenciação Celular , Núcleo Celular/metabolismo , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos Mutantes , Mutação/genética , Osteoblastos/patologia , Linhagem , Fosforilação , Crânio/patologia , Adulto JovemRESUMO
Dysosteosclerosis (DOS) is a distinct form of sclerosing bone disease characterized by irregular osteosclerosis and platyspondyly. DOS is genetically heterogeneous; however, only five cases with SLC29A3 mutations and a single case with a splice-site mutation of TNFRSF11A have been reported, and TNFRSF11A is also a causal gene for osteopetrosis, autosomal recessive 7 (OP-AR7). Thus, the causal genes of DOS and their genotype-phenotype associations remain unclear. In this study, we examined a Japanese patient with DOS and found a novel variant in TNFRSF11A. The homozygous variant was a G to T transversion at the first nucleotide of exon 9 (c.784G>T). Although the variant was predicted to cause a stop codon mutation (p.E262*), in silico evaluation of the exonic splicing elements followed by RT-PCR for the patient-derived cells showed that it caused aberrant splicing due to the change in the exonic splicing element and produced two types of aberrant transcripts: One caused a premature stop codon (p.E262Vfs*17) leading to nonsense mutation-mediated mRNA decay; the other produced a protein with interstitial deletion (p.E262_Q279del). The effects of the mutation on five splicing isoforms of TNFRSF11A were different from those in OP-AR7, but comparable with those in the first DOS with the TNFRSF11A mutation. Thus, we identified the second case of DOS caused by the TNFRSF11A splice-site mutation and confirmed the novel disease entity. © 2019 American Society for Bone and Mineral Research.
Assuntos
Códon de Terminação , Éxons , Osteosclerose/genética , Mutação Puntual , Receptor Ativador de Fator Nuclear kappa-B/genética , Povo Asiático , Humanos , Japão , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: To explore the effects of adenovirus vector-mediated gene transfer of ICOSIg fusion protein on experimental autoimmune myocarditis (EAM) in Lewis rats. METHODS: Expression vector containing ICOSIg (p-Adeno-ICOSIg) was constructed by fusion of human ICOS and IgGFc segment. Adenovirus vector was digested by PacI enzyme and transfected into HEK 293 cells. Adenovirus expressing ICOSIg was produced. EGFP was constructed into adenovirus vector and used as control. EAM was induced in Lewis rats by injection of porcine cardiac myosin. All immunized Lewis rats were divided into 4 groups. Group A (n = 15) and B (n = 15) received adenovirus containing ICOSIg on day 0 and day 14 respectively to study the effects of costimulatory molecules gene therapy on T cell activation and inflammation; group C (n = 10) and group D (n = 10) received adenovirus containing EGFP on day 0 and day 14 respectively as controls. Group E (n = 10) was normal controls that did not receive immunization. On day 28, all rats were killed after echocardiography examination. Histopathological examination was performed to observe myocardial inflammation. Protein levels of ICOS, ICOSL, B7-1 and B7-2 were detected by Western blot. INF-gamma, IL-2 and IL-4 mRNA were determined by realtime RT-PCR. RESULTS: On day 28, cardiac function was significantly improved and myocardial inflammation significantly attenuated in group B compared to group A, C and D (all P < 0.05). B7-1 expression at protein level was significantly lower in group B than that of group C (P < 0.05). ICOS and ICOSL expressions at protein level were significantly decreased in both group A and B compared with group C and D (P < 0.05). IFN-gamma mRNA level significantly decreased and IL-4 mRNA significantly increased in group A and B compared to group C and D (P < 0.05). CONCLUSIONS: Blockade of costimulatory pathway with gene therapy of ICOSIg alleviated autoimmune inflammatory damage and improved cardiac function in Lewis rats with EAM. Down-regulated costimulatory molecules in the myocardium and reduced inflammatory cytokine secretion might be responsible for the beneficial effects of ICOSIg in this model.
Assuntos
Antígenos de Diferenciação de Linfócitos T/genética , Doenças Autoimunes/terapia , Terapia Genética , Fragmentos Fc das Imunoglobulinas/genética , Miocardite/terapia , Adenoviridae/genética , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Vetores Genéticos , Proteína Coestimuladora de Linfócitos T Induzíveis , Masculino , Miocardite/imunologia , Miocardite/patologia , Ratos , Ratos Endogâmicos Lew , Proteínas Recombinantes de Fusão/genéticaRESUMO
BACKGROUND AND OBJECTIVES: Automatic delineation of the myocardium in echocardiography can assist radiologists to diagnosis heart problems. However, it is still challenging to distinguish myocardium from other tissue due to a low signal-to-noise ratio, low contrast, vague boundary, and speckle noise. The purpose of this study is to automatically detect myocardium region in left ventricle myocardial contrast echocardiography (LVMCE) images to help radiologists' diagnosis and further measurement on infarction size. METHODS: The LVMCE image is firstly mapped into neutrosophic similarity (NS) domain using the intensity and homogeneity features. Then, a neutrosophic active contour model (NACM) is proposed and the energy function is defined by the NS values. Finally, the ventricle is detected using the curve evolving results. The ventricle's boundary is identified as the endocardium. To speed up the evolution procedure and increase the detection accuracy, a clustering algorithm is employed to obtain the initial ventricle region. The curve evolution procedure in NACM is utilized again to obtain the epicardium, where the initial contour uses the detected endocardium and the anatomy knowledge on the thickness of the myocardium. RESULTS: Echocardiographic studies are performed on 10 male Sprague-Dawley rats using a Vivid 7 system including 5 normal cases and 5 rats with myocardial infarction. The myocardium boundaries manually outlined by an experienced radiologist are used as the reference standard for the performance evaluation. Two metrics, Hdist and AvgDist, are employed to evaluate the detection results. The NACM method was compared with those from the eliminated particle swarm optimization (EPSO) and active contour model without edges (ACMWE) methods. The mean and standard deviation of the Hdist and AvgDist on endocardium are 6.83 ± 1.12mm and 0.79 ± 0.28mm using EPSO method, 7.12 ± 0.98mm and 0.82 ± 0.32mm using ACMWE method, and 4.55 ± 0.9mm and 0.58 ± 0.18mm using NACM method, respectively. The improvement on epicardium is much more significant, and two metrics are decreased from 7.45 ± 1.24mm, and 1.47 ± 0.34mm using EPSO method, and 8.21±0.43mm, and 1.73±0.47mm using ACMWE method, to 4.94 ± 0.82mm, and 0.84 ± 0.22mm using NACM method, respectively. CONCLUSIONS: The proposed method can automatically detect myocardium accurately, and is helpful for clinical therapeutics to measure myocardial perfusion and infarct size.
Assuntos
Ecocardiografia/métodos , Endocárdio/patologia , Ventrículos do Coração/patologia , Processamento de Imagem Assistida por Computador/métodos , Miocárdio/patologia , Algoritmos , Animais , Análise por Conglomerados , Humanos , Masculino , Infarto do Miocárdio , Pericárdio/patologia , Ratos , Ratos Sprague-Dawley , Razão Sinal-RuídoRESUMO
BACKGROUND: Previous studies revealed that culprit vessels of ST-segment elevation myocardial infarction (STEMI) were often related to mild or moderate stenosis. However, recent studies suggested that severe stenosis was primarily found in culprit lesions. The objective of this study was to analyze the stenosis severity of culprit lesions in STEMI patients and to clarify the paradoxical results. METHODS: A total of 489 consecutive STEMI patients who underwent primary percutaneous coronary intervention were retrospectively studied from January 2012 to December 2014. The patients were divided into three groups based on stenosis severity using quantitative coronary analysis: Group A, 314 cases, stenosis ≥70%; Group B, 127 cases, stenosis 50-70%; and Group C, 48 cases, stenosis ≤50%. The clinical, demographic, and angiographic data of all groups were analyzed. RESULTS: Patients in Group A exhibited a significantly higher prevalence of history of angina pectoris (95.9% vs. 62.5%, P< 0.001), multivessel disease (73.2% vs. 54.2%, P = 0.007), and lower cardiac ejection fraction (53.3 ± 8.6 vs. 56.8 ± 8.4, P= 0.009) than those in Group C. Multivariable analysis revealed that history of angina pectoris (odds ratio [OR]: 13.89, 95% confidence interval [CI]: 6.21-31.11) and multivessel disease (OR: 2.32, 95% CI: 1.25-4.31) were correlated with severe stenosis of the culprit lesion in Group A. CONCLUSIONS: Most culprit lesions in STEMI patients were severe stenosis. These patients exhibited a higher prevalence of angina history and multivessel diseases.