RESUMO
Calcific aortic valve disease (CAVD) is characterized by the fibrosis and mineralization of the aortic valve, which leads to aortic stenosis and heart failure. At the cellular level, this is due to the osteoblastic-like differentiation of valve interstitial cells (VICs), resulting in the calcification of the tissue. Unfortunately, human VICs are not readily available to study CAVD pathogenesis and the implicated mechanisms in vitro; however, adipose-derived stromal/stem cells (ASCs), carrying the patient's specific genomic features, have emerged as a promising cell source to model cardiovascular diseases due to their multipotent nature, availability, and patient-specific characteristics. In this study, we describe a comprehensive transcriptomic analysis of tissue-engineered, scaffold-free, ASC-embedded mineralized tissue sheets using bulk RNA sequencing. Bioinformatic and gene set enrichment analyses revealed the up-regulation of genes associated with the organization of the extracellular matrix (ECM), suggesting that the ECM could play a vital role in the enhanced mineralization observed in these tissue-engineered ASC-embedded sheets. Upon comparison with publicly available gene expression datasets from CAVD patients, striking similarities emerged regarding cardiovascular diseases and ECM functions, suggesting a potential link between ECM gene expression and CAVDs pathogenesis. A matrisome-related sub-analysis revealed the ECM microenvironment promotes the transcriptional activation of the master gene runt-related transcription factor 2 (RUNX2), which is essential in CAVD development. Tissue-engineered ASC-embedded sheets with enhanced mineralization could be a valuable tool for research and a promising avenue for the identification of more effective aortic valve replacement therapies.
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Valvopatia Aórtica , Estenose da Valva Aórtica , Valva Aórtica/patologia , Calcinose , Humanos , Estenose da Valva Aórtica/metabolismo , Calcinose/metabolismo , Valvopatia Aórtica/metabolismo , Células-Tronco/metabolismo , Células CultivadasRESUMO
Neovascularization is a critical process in tumor progression and malignant transformation associated with neurofibromatosis type 1 (NF1). Indeed, fibroblasts are known to play a key role in the tumoral microenvironment modification by producing an abundant collagenous matrix, but their contribution in paracrine communication pathways is poorly understood. Here, we hypothesized that NF1 heterozygosis in human dermal fibroblasts could promote angiogenesis through exosomes secretion. The purposes of this study are to identify the NF1 fibroblast-derived exosome protein contents and to determine their proangiogenic activity. Angiogenic proteome measurement confirmed the overexpression of VEGF and other proteins involved in vascularization. Tube formation of microvascular endothelial cells was also enhanced in presence of exosomes derived from NF1 skin fibroblasts. NF1 tissue-engineered skin (NF1-TES) generation showed a significantly denser microvessels networks compared to healthy controls. The reduction of exosomes production with an inhibitor treatment demonstrated a drastic decrease in blood vessel formation within the dermis. Our results suggest that NF1 haploinsufficiency alters the dermal fibroblast function and creates a pro-angiogenic signal via exosomes, which increases the capillary formation. This study highlights the potential of targeting exosome secretion and angiogenesis for therapeutic interventions in NF1.
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Exossomos , Neurofibromatose 1 , Humanos , Células Endoteliais/metabolismo , Neurofibromatose 1/metabolismo , Neovascularização Patológica , Fibroblastos , Pele , Exossomos/metabolismo , Microambiente TumoralRESUMO
BACKGROUND: Variants in the ring finger protein 213 (RNF213) gene are known to be associated with increased predisposition to cerebrovascular diseases development. Genomic studies have identified RNF213 as a major risk factor of Moyamoya disease in East Asian descendants. However, little is known about the RNF213 (ring finger protein 213) biological functions or its associated pathogenic mechanisms underlying Moyamoya disease. METHODS: To investigate RNF213 loss-of-function effect in endothelial cell, stable RNF213-deficient human cerebral endothelial cells were generated using the CRISPR-Cas9 genome editing technology. RESULTS: In vitro assays, using RNF213 knockout brain endothelial cells, showed clear morphological changes and increased blood-brain barrier permeability. Downregulation and delocalization of essential interendothelial junction proteins involved in the blood-brain barrier maintenance, such as PECAM-1 (platelet endothelial cell adhesion molecule-1), was also observed. Brain endothelial RNF213-deficient cells also showed an abnormal potential to transmigration of leukocytes and secreted high amounts of proinflammatory cytokines. CONCLUSIONS: Taken together, these results indicate that RNF213 could be a key regulator of cerebral endothelium integrity, whose disruption could be an early pathological mechanism leading to Moyamoya disease. This study also further reinforces the importance of blood-brain barrier integrity in the development of Moyamoya disease and other RNF213-associated diseases.
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Adenosina Trifosfatases , Doença de Moyamoya , Ubiquitina-Proteína Ligases , Adenosina Trifosfatases/genética , Células Endoteliais/metabolismo , Endotélio , Predisposição Genética para Doença , Humanos , Doença de Moyamoya/patologia , Fatores de Transcrição , Ubiquitina-Proteína Ligases/genéticaRESUMO
Olfactory and gustatory dysfunctions (OD, GD) are prevalent symptoms following COVID-19 and persist in 6%-44% of individuals post-infection. As only few reports have described their prognosis after 6 months, our main objective was to assess the prevalence of OD and GD 11-month post-COVID-19. We also aimed to determine intraclass correlation coefficients (ICC) of chemosensory self-ratings for the follow-up of chemosensory sensitivity. We designed an observational study and distributed an online questionnaire assessing chemosensory function to healthcare workers with a RT-PCR-confirmed SARS-CoV-2 infection 5- and 11-month post-COVID-19. Specifically, we assessed olfaction, gustation, and trigeminal sensitivity (10-point visual analog scale) and function (4-point Likert scale). We further measured clinically relevant OD using the Chemosensory Perception Test, a psychophysical test designed to provide a reliable remote olfactory evaluation. We included a total of 366 participants (mean [SD] age of 44.8 (11.7) years old). They completed the last online questionnaire 10.6 months (0.7) after the onset of COVID-19 symptoms. Of all participants, 307 (83.9%) and 301 (82.2%) individuals retrospectively reported lower olfactory or gustatory sensitivity during the acute phase of COVID-19. At the time of evaluation, 184 (50.3%) and 163 (44.5%) indicated reduced chemosensory sensitivity, 32.2% reported impairment of olfactory function while 24.9% exhibited clinically relevant OD. Olfactory sensitivity had a high test-retest reliability (ICC: 0.818; 95% CI: 0.760-0.860). This study suggests that chemosensory dysfunctions persist in a third of COVID-19 patients 11 months after COVID-19. OD appears to be a common symptom of post-COVID-19 important to consider when treating patients.
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COVID-19 , Transtornos do Olfato , Adulto , COVID-19/epidemiologia , Seguimentos , Pessoal de Saúde , Humanos , Pessoa de Meia-Idade , Transtornos do Olfato/diagnóstico , Transtornos do Olfato/epidemiologia , Transtornos do Olfato/etiologia , Reprodutibilidade dos Testes , Estudos Retrospectivos , SARS-CoV-2 , Distúrbios do Paladar/diagnóstico , Distúrbios do Paladar/epidemiologia , Distúrbios do Paladar/etiologiaRESUMO
Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease affecting upper and lower motor neurons (MNs). To investigate whether Schwann cells could be involved in the disease pathogenesis, we developed a tissue-engineered three-dimensional (3D) in vitro model that combined MNs cocultured with astrocytes and microglia seeded on top of a collagen sponge populated with epineurium fibroblasts to enable 3D axonal migration. C2C12 myoblasts were seeded underneath the sponge in the presence or absence of Schwann cells. To reproduce an ALS cellular microenvironment, MNs, astrocytes, and microglia were extracted from SOD1G93A mice recapitulating many aspects of the human disease. This 3D ALS in vitro model was compared with a 3D control made of cells isolated from SOD1WT mice. We showed that normal Schwann cells strongly enhanced MN axonal migration in the 3D control model but had no effect in the ALS model. However, ALS-derived Schwann cells isolated from SOD1G93A mice failed to significantly improve axonal migration in both models. These results suggest that a cell therapy using healthy Schwann cells may not be effective in promoting axonal regeneration in ALS. In addition, this 3D ALS model could be used to study the impact of other cell types on ALS by various combinations of normal and diseased cells.
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Esclerose Lateral Amiotrófica , Doenças Neurodegenerativas , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Modelos Animais de Doenças , Camundongos , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Células de Schwann/metabolismo , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismoRESUMO
Corneal wound healing involves communication between the different cell types that constitute the three cellular layers of the cornea (epithelium, stroma and endothelium), a process ensured in part by a category of extracellular vesicles called exosomes. In the present study, we isolated exosomes released by primary cultured human corneal epithelial cells (hCECs), corneal fibroblasts (hCFs) and corneal endothelial cells (hCEnCs) and determined whether they have wound healing characteristics of their own and to which point they modify the genetic and proteomic pattern of these cell types. Exosomes released by all three cell types significantly accelerated wound closure of scratch-wounded hCECs in vitro compared to controls (without exosomes). Profiling of activated kinases revealed that exosomes from human corneal cells caused the activation of signal transduction mediators that belong to the HSP27, STAT, ß-catenin, GSK-3ß and p38 pathways. Most of all, data from gene profiling analyses indicated that exosomes, irrespective of their cellular origin, alter a restricted subset of genes that are completely different between each targeted cell type (hCECs, hCFS, hCEnCs). Analysis of the genes specifically differentially regulated for a given cell-type in the microarray data using the Ingenuity Pathway Analysis (IPA) software revealed that the mean gene expression profile of hCECs cultured in the presence of exosomes would likely promote cell proliferation and migration whereas it would reduce differentiation when compared to control cells. Collectively, our findings represent a conceptual advance in understanding the mechanisms of corneal wound repair that may ultimately open new avenues for the development of novel therapeutic approaches to improve closure of corneal wounds.
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Lesões da Córnea , Exossomos , Humanos , Exossomos/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Células Endoteliais/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Proteômica , Cicatrização/fisiologia , Córnea/metabolismo , Lesões da Córnea/metabolismo , Células Epiteliais/metabolismo , Movimento CelularRESUMO
Several studies have revealed either self-reported chemosensory alterations in large groups or objective quantified chemosensory impairments in smaller populations of patients diagnosed with COVID-19. However, due to the great variability in published results regarding COVID-19-induced chemosensory impairments and their follow-up, prognosis for chemosensory functions in patients with such complaints remains unclear. Our objective is to describe the various chemosensory alterations associated with COVID-19 and their prevalence and evolution after infection. A cross-sectional study of 704 healthcare workers with a RT-PCR-confirmed SARS-CoV-2 infection between 2020 February 28 and 2020 June 14 was conducted 3-7 months after onset of symptoms. Data were collected with an online questionnaire. Outcomes included differences in reported chemosensory self-assessment of olfactory, gustatory, and trigeminal functions across time points and Chemosensory Perception Test scores from an easy-to-use at-home self-administered chemosensory test. Among the 704 participants, 593 (84.2%) were women, the mean (SD) age was 42 (12) years, and the questionnaire was answered on average 4.8 (0.8) months after COVID-19. During COVID-19, a decrease in olfactory, gustatory, and trigeminal sensitivities was reported by 81.3%, 81.5%, and 48.0%, respectively. Three to 7 months later, reduced sensitivity was still reported by 52.0%, 41.9%, and 23.3%, respectively. Chemosensory Perception Test scores indicate that 19.5% of participants had objective olfactory impairment. These data suggest a significant proportion of COVID-19 cases have persistent chemosensory impairments at 3-7 months after their infection, but the majority of those who had completely lost their olfactory, gustatory, and trigeminal sensitivities have improved.
Assuntos
COVID-19/complicações , Transtornos do Olfato/etiologia , Distúrbios do Paladar/etiologia , Adolescente , Adulto , Idoso , COVID-19/epidemiologia , COVID-19/etiologia , Pessoal de Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos do Olfato/diagnóstico , Transtornos do Olfato/epidemiologia , Prevalência , Autorrelato , Inquéritos e Questionários , Distúrbios do Paladar/epidemiologia , Fatores de TempoRESUMO
One of the great challenges in identifying effective therapy in many neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), is the lack of reliable biomarkers. In this study, we applied infrared imaging microspectroscopy, a valuable technique to investigate biomolecule fingerprints and secondary structure of proteins within biological tissue. We hypothesized that, since skin and CNS have the same embryonic origin, spectral differences associated with ALS-specific pathological events will be readily detectable through skin testing using this technique. Cells from healthy individuals and ALS patients were isolated from skin biopsies in order to generate tissue-engineered in vitro skin (TES). Infrared spectra of the generated TES were recorded using a focal-plane-array Fourier transform infrared (FPA-FTIR) spectrometer, and hierarchical cluster analysis of the spectral data was performed in order to establish clear differences between the tested TES specimens. Interestingly, our analyses showed that it was readily possible to discriminate ALS- and control-TES solely based on differences in associated FTIR spectra, mainly located between 1149 and 1473 cm-1, attributed to disruption of phospholipid cell membranes, extracellular matrix remodeling or cholesterol accumulation. Spectral differences within the TES samples may therefore be associated with disease state, paving the way for the identification of biomarkers in ALS.
Assuntos
Esclerose Lateral Amiotrófica/diagnóstico , Esclerose Lateral Amiotrófica/metabolismo , Pele/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Estudos de Casos e Controles , HumanosRESUMO
Recent genetic studies yielded conflicting results regarding a role for the variant chromogranin B (CHGB)P413L allele as a disease modifier in ALS. Moreover, potential deleterious effects of the CHGBP413L variant in ALS pathology have not been investigated. Here we report that in transfected cultured cells, the variant CHGBL413 protein exhibited aberrant properties including mislocalization, failure to interact with mutant superoxide dismutase 1 (SOD1) and defective secretion. The CHGBL413 transgene in SOD1G37R mice precipitated disease onset and pathological changes related to misfolded SOD1 specifically in female mice. However, the CHGBL413 variant also slowed down disease progression in SOD1G37R mice, which is in line with a very slow disease progression that we report for a Swedish woman with ALS who is carrier of two mutant SOD1D90A alleles and two variant CHGBP413L and CHGBR458Q alleles. In contrast, overexpression of the common CHGBP413 allele in SOD1G37R mice did not affect disease onset but significantly accelerated disease progression and pathological changes. As in transgenic mice, the CHGBP413L allele conferred an earlier ALS disease onset in women of Japanese and French Canadian origins with less effect in men. Evidence is presented that the sex-dependent effects of CHGBL413 allelic variant in ALS may arise from enhanced neuronal expression of CHGB in females because of a sex-determining region Y element in the gene promoter. Thus, our results suggest that CHGB variants may act as modifiers of onset and progression in some ALS populations and especially in females because of higher expression levels compared to males.
Assuntos
Esclerose Lateral Amiotrófica/genética , Cromogranina B/genética , Cromogranina B/metabolismo , Alelos , Animais , Técnicas de Cultura de Células , Modelos Animais de Doenças , Progressão da Doença , Feminino , Frequência do Gene/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Mutação/genética , Polimorfismo de Nucleotídeo Único/genética , Fatores Sexuais , Medula Espinal/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismoRESUMO
The past decade has seen great advances in unraveling the biological basis of hereditary ataxias. Molecular studies of spinocerebellar ataxias (SCA) have extended our understanding of dominant ataxias. Causative genes have been identified for a few autosomal recessive ataxias: Friedreich's ataxia, ataxia with vitamin E deficiency, ataxia telangiectasia, recessive spastic ataxia of Charlevoix-Saguenay and ataxia with oculomotor apraxia type 1 (refs. 6,7) and type 2 (ref. 8). Nonetheless, genes remain unidentified for most recessive ataxias. Additionally, pure cerebellar ataxias, which represent up to 20% of all ataxias, remain poorly studied with only two causative dominant genes being described: CACNA1A (ref. 9) and SPTBN2 (ref. 10). Here, we report a newly discovered form of recessive ataxia in a French-Canadian cohort and show that SYNE1 mutations are causative in all of our kindreds, making SYNE1 the first identified gene responsible for a recessively inherited pure cerebellar ataxia.
Assuntos
Ataxia Cerebelar/genética , Genes Recessivos , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Adulto , Idoso , Sequência de Aminoácidos , Animais , Sequência de Bases , Cerebelo/metabolismo , Mapeamento Cromossômico , Proteínas do Citoesqueleto , Análise Mutacional de DNA , Feminino , Testes Genéticos , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , QuebequeRESUMO
The mutations P56S and T46I in the gene encoding vesicle-associated membrane protein-associated protein B/C (VAPB) cause ALS8, a familial form of amyotrophic lateral sclerosis (ALS). Overexpression of mutant forms of VAPB leads to cytosolic aggregates, suggesting a gain of function of the mutant protein. However, recent work suggested that the loss of VAPB function could be the major mechanism leading to ALS8. Here, we used multiple genetic and experimental approaches to study whether VAPB loss of function might be sufficient to trigger motor neuron degeneration. In order to identify additional ALS-associated VAPB mutations, we screened the entire VAPB gene in a cohort of ALS patients and detected two mutations (A145V and S160Δ). To directly address the contribution of VAPB loss of function in ALS, we generated zebrafish and mouse models with either a decreased or a complete loss of Vapb expression. Vapb knockdown in zebrafish led to swimming deficits. Mice knocked-out for Vapb showed mild motor deficits after 18 months of age yet had innervated neuromuscular junctions (NMJs). Importantly, overexpression of VAPB mutations were unable to rescue the motor deficit caused by Vapb knockdown in zebrafish and failed to cause a toxic gain-of-function defect on their own. Thus, Vapb loss of function weakens the motor system of vertebrate animal models but is on its own unable to lead to a complete ALS phenotype. Our findings are consistent with the notion that VAPB mutations constitute a risk factor for motor neuron disease through a loss of VAPB function.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Membrana/metabolismo , Mutação de Sentido Incorreto , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Esclerose Lateral Amiotrófica/genética , Animais , Sequência de Bases , Estudos de Coortes , Feminino , Humanos , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Alinhamento de Sequência , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Peixe-ZebraRESUMO
BACKGROUND: The upper respiratory mucosa plays a crucial role in both the physical integrity and immunological function of the respiratory tract. However, in certain situations such as infections, trauma, or surgery, it might sustain damage. Tissue engineering, a field of regenerative medicine, has found applications in various medical fields including but not limited to plastic surgery, ophthalmology, and urology. However, its application to the respiratory system remains somewhat difficult due to the complex morphology and histology of the upper respiratory tract. To date, a culture protocol for producing a handleable, well-differentiated nasal mucosa has yet to be developed. The objective of this review is to describe the current state of research pertaining to cell culture techniques used for producing autologous healthy human upper respiratory cells and mucosal tissues, as well as describe its clinical applications. METHODS: A search of the relevant literature was carried out with no time restriction across Embase, Cochrane, PubMed, and Medline Ovid databases. Keywords related to "respiratory mucosa" and "culture techniques of the human airway" were the focus of the search strategy for this review. The risk of bias in retained studies was assessed using the Joanna Briggs Institute's (JBI) critical appraisal tools for qualitative research. A narrative synthesis of our results was then conducted. RESULTS: A total of 33 studies were included in this review, and thirteen of these focused solely on developing a cell culture protocol without further use. The rest of the studies used their own developed protocol for various applications such as cystic fibrosis, pharmacological, and viral research. One study was able to develop a promising model for nasal mucosa that could be employed as a replacement in nasotracheal reconstructive surgery. CONCLUSIONS: This systematic review extensively explored the current state of research regarding cell culture techniques for producing tissue-engineered nasal mucosa. Bioengineering the nasal mucosa holds great potential for clinical use. However, further research on mechanical properties is essential, as the comparison of engineered tissues is currently focused on morphology rather than comprehensive mechanical assessments.
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Entirely biological human tissue-engineered blood vessels (TEBV) were previously developed for clinical use. Tissue-engineered models have also proven to be valuable tools in disease modelling. Moreover, there is a need for complex geometry TEBV for study of multifactorial vascular pathologies, such as intracranial aneurysms. The main goal of the work reported in this article was to produce an entirely human branched small-caliber TEBV. The use of a novel spherical rotary cell seeding system allows effective and uniform dynamic cell seeding for a viable in vitro tissue-engineered model. In this report, the design and fabrication of an innovative seeding system with random spherical 360° rotation is described. Custom made seeding chambers are placed inside the system and hold Y-shaped polyethylene terephthalate glycol (PETG) scaffolds. The seeding conditions, such as cell concentration, seeding speed and incubation time were optimized via count of cells adhered on the PETG scaffolds. This spheric seeding method was compared to other approaches, such as dynamic and static seeding, and clearly shows uniform cell distribution on PETG scaffolds. With this simple to use spherical system, fully biological branched TEBV constructs were also produced by seeding human fibroblasts directly on custom-made complex geometry PETG mandrels. The production of patient-derived small-caliber TEBVs with complex geometry and optimized cellular distribution all along the vascular reconstructed may be an innovative way to model various vascular diseases such as intracranial aneurysms.
Assuntos
Aneurisma Intracraniano , Humanos , Engenharia Tecidual/métodos , Alicerces Teciduais , Vasos Sanguíneos , Células CultivadasRESUMO
Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is an early-onset neurodegenerative disease mainly characterized by spasticity in the lower limbs and poor muscle control. The disease is caused by mutations in the SACS gene leading in most cases to a loss of function of the sacsin protein, which is highly expressed in motor neurons and Purkinje cells. To investigate the impact of the mutated sacsin protein in these cells in vitro, induced pluripotent stem cell- (iPSC-) derived motor neurons and iPSC-derived Purkinje cells were generated from three ARSACS patients. Both types of iPSC-derived neurons expressed the characteristic neuronal markers ß3-tubulin, neurofilaments M and H, as well as specific markers like Islet-1 for motor neurons, and parvalbumin or calbindin for Purkinje cells. Compared to controls, iPSC-derived mutated SACS neurons expressed lower amounts of sacsin. In addition, characteristic neurofilament aggregates were detected along the neurites of both iPSC-derived neurons. These results indicate that it is possible to recapitulate in vitro, at least in part, the ARSACS pathological signature in vitro using patient-derived motor neurons and Purkinje cells differentiated from iPSCs. Such an in vitro personalized model of the disease could be useful for the screening of new drugs for the treatment of ARSACS.
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The role of non-coding regulatory elements and how they might contribute to tissue type specificity of disease phenotypes is poorly understood. Autosomal Dominant Leukodystrophy (ADLD) is a fatal, adult-onset, neurological disorder that is characterized by extensive CNS demyelination. Most cases of ADLD are caused by tandem genomic duplications involving the lamin B1 gene ( LMNB1 ) while a small subset are caused by genomic deletions upstream of the gene. Utilizing data from recently identified families that carry LMNB1 gene duplications but do not exhibit demyelination, ADLD patient tissues, CRISPR modified cell lines and mouse models, we have identified a novel silencer element that is lost in ADLD patients and that specifically targets overexpression to oligodendrocytes. This element consists of CTCF binding sites that mediate three-dimensional chromatin looping involving the LMNB1 and the recruitment of the PRC2 repressor complex. Loss of the silencer element in ADLD identifies a previously unknown role for silencer elements in tissue specificity and disease causation.
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Recently, chromogranins were reported to interact specifically with mutant forms of superoxide dismutase that are linked to amyotrophic lateral sclerosis (ALS). This interaction led us to analyze the frequencies of sequence variants of the CHGB gene in ALS patients and matched controls from three different countries. Of particular interest was the finding of the P413L CHGB variant present in 10% of ALS patients (n = 705) as compared to 4.5% in controls (n = 751), conferring a 2.2-fold greater relative risk to develop the disease (P < 0.0001). This effect was mainly contributed by the samples of French origin that yielded a frequency of the P413L variation at 17% in ALS (n = 289) and 5% in controls (n = 448), conferring a 3.3-fold greater risk to develop ALS. Furthermore, the P413L CHGB variant is associated with an earlier age of onset by almost a decade in both sporadic ALS and familial ALS cases. Genetic variation influencing age of onset in ALS had not previously been reported. Expression of fusion CHGB-EGFP constructs in SHSY-5Y cells revealed that the P413L variation can cause defective sorting of CHGB into secretory granules. The finding that CHGB may act as a susceptibility gene and modifier of onset in ALS is consistent with the emerging view that dysfunction of the secretory pathway may contribute to increased vulnerability of motor neurons.
Assuntos
Esclerose Lateral Amiotrófica/genética , Cromogranina B/genética , Mutação , Adulto , Idade de Início , Éxons , Feminino , França , Humanos , Íntrons , Masculino , Pessoa de Meia-Idade , Quebeque , Fatores de RiscoRESUMO
Enhanced and aberrant angiogenesis is one of the main features of Moyamoya disease (MMD) pathogenesis. The ring finger protein 213 (RNF213) and the variant p.R4810K have been linked with higher risks of MMD and intracranial arterial occlusion development in east Asian populations. The role of RNF213 in diverse aspects of the angiogenic process, such as proliferation, migration and capillary-like formation, is well-known but has been difficult to model in vitro. To evaluate the effect of the RNF213 MMD-associated gene on the angiogenic activity, we have generated RNF213 knockout in human cerebral microvascular endothelial cells (hCMEC/D3-RNF213-/-) using the CRISPR-Cas9 system. Matrigel-based assay and a tri-dimensional (3D) vascularized model using the self-assembly approach of tissue engineering were used to assess the formation of capillary-like structures. Quite interestingly, this innovative in vitro model of MMD recapitulated, for the first time, disease-associated pathophysiological features such as significant increase in angiogenesis in confluent endothelial cells devoid of RNF213 expression. These cells, grown to confluence, also showed a pro-angiogenic signature, i.e., increased secretion of soluble pro-angiogenic factors, that could be eventually used as biomarkers. Interestingly, we demonstrated that that these MMD-associated phenotypes are dependent of the cellular state, as only noted in confluent cells and not in proliferative RNF213-deficient cells.
Assuntos
Arteriopatias Oclusivas , Doença de Moyamoya , Humanos , Células Endoteliais/patologia , Predisposição Genética para Doença , Doença de Moyamoya/genética , Adenosina Trifosfatases/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Extracellular matrix (ECM) secretion, deposition and assembly are part of a whole complex biological process influencing the microenvironment and other cellular behaviors. Emerging evidence is attributing a significant role to extracellular vesicles (EVs) and exosomes in a plethora of ECM-associated functions, but the role of dermal fibroblast-derived EVs in paracrine signalling is yet unclear. Herein, we investigated the effect of exosomes isolated from stimulated human dermal fibroblasts. We report that tridimensional (3D) cell culture of dermal fibroblasts promotes secretion of exosomes carrying a large quantity of proteins involved in the formation, organisation and remodelling of the ECM. In our 3D model, gene expression was highly modulated and linked to ECM, cellular migration and proliferation, as well as inflammatory response. Mass spectrometry analysis of exosomal proteins, isolated from 3D cultured fibroblast-conditioned media, revealed ECM protein enrichment, of which many were associated with the matrisome. We also show that the cytokine interleukin 6 (IL-6) is predicted to be central to the signalling pathways related to ECM formation and contributing to cell migration and proliferation. Overall, our data suggest that dermal fibroblast-derived EVs participate in many steps of the establishment of dermis's ECM.
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Exossomos , Humanos , Exossomos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Técnicas de Cultura de Células , Fibroblastos/metabolismo , Matriz Extracelular/metabolismoRESUMO
More than 107 pathogenic variations were identified in Keratin 14 gene (KRT14) in patients affected by epidermolysis bullosa simplex (EBS), a rare skin disease with still no curative treatment. Disease models as human induced pluripotent stem cells (hiPSCs) are promising tool for further advance the knowledge about this disorder and accelerate therapies development. Here, two hiPSC lines were reprogrammed from skin fibroblasts of two EBS patients carrying mutations within KRT14 by using CytoTune®Sendai virus. These iPSCs display pluripotent cell morphology, pluripotent markers expression, and the capability to differentiate into the three germ layers.