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1.
Proc Natl Acad Sci U S A ; 121(11): e2313162121, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38451946

RESUMO

Water is known to play an important role in collagen self-assembly, but it is still largely unclear how water-collagen interactions influence the assembly process and determine the fibril network properties. Here, we use the H[Formula: see text]O/D[Formula: see text]O isotope effect on the hydrogen-bond strength in water to investigate the role of hydration in collagen self-assembly. We dissolve collagen in H[Formula: see text]O and D[Formula: see text]O and compare the growth kinetics and the structure of the collagen assemblies formed in these water isotopomers. Surprisingly, collagen assembly occurs ten times faster in D[Formula: see text]O than in H[Formula: see text]O, and collagen in D[Formula: see text]O self-assembles into much thinner fibrils, that form a more inhomogeneous and softer network, with a fourfold reduction in elastic modulus when compared to H[Formula: see text]O. Combining spectroscopic measurements with atomistic simulations, we show that collagen in D[Formula: see text]O is less hydrated than in H[Formula: see text]O. This partial dehydration lowers the enthalpic penalty for water removal and reorganization at the collagen-water interface, increasing the self-assembly rate and the number of nucleation centers, leading to thinner fibrils and a softer network. Coarse-grained simulations show that the acceleration in the initial nucleation rate can be reproduced by the enhancement of electrostatic interactions. These results show that water acts as a mediator between collagen monomers, by modulating their interactions so as to optimize the assembly process and, thus, the final network properties. We believe that isotopically modulating the hydration of proteins can be a valuable method to investigate the role of water in protein structural dynamics and protein self-assembly.


Assuntos
Colágeno , Água , Água/química , Termodinâmica , Hidrogênio
2.
PLoS Genet ; 19(6): e1010796, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37315079

RESUMO

Motile and non-motile cilia play critical roles in mammalian development and health. These organelles are composed of a 1000 or more unique proteins, but their assembly depends entirely on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). In mammals, malfunction of non-motile cilia due to IFT dysfunction results in complex developmental phenotypes that affect most organs. In contrast, disruption of motile cilia function causes subfertility, disruption of the left-right body axis, and recurrent airway infections with progressive lung damage. In this work, we characterize allele specific phenotypes resulting from IFT74 dysfunction in human and mice. We identified two families carrying a deletion encompassing IFT74 exon 2, the first coding exon, resulting in a protein lacking the first 40 amino acids and two individuals carrying biallelic splice site mutations. Homozygous exon 2 deletion cases presented a ciliary chondrodysplasia with narrow thorax and progressive growth retardation along with a mucociliary clearance disorder phenotype with severely shorted cilia. Splice site variants resulted in a lethal skeletal chondrodysplasia phenotype. In mice, removal of the first 40 amino acids likewise results in a motile cilia phenotype but with little effect on primary cilia structure. Mice carrying this allele are born alive but are growth restricted and developed hydrocephaly in the first month of life. In contrast, a strong, likely null, allele of Ift74 in mouse completely blocks ciliary assembly and causes severe heart defects and midgestational lethality. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia resulting from increased mechanical stress and repair needs could account for the motile cilia phenotype observed in human and mice.


Assuntos
Cílios , Ciliopatias , Humanos , Animais , Camundongos , Cílios/genética , Cílios/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas/genética , Aminoácidos/metabolismo , Mamíferos/metabolismo , Proteínas do Citoesqueleto/genética
3.
Calcif Tissue Int ; 114(3): 210-221, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-38243143

RESUMO

Osteogenesis imperfecta (OI) is a rare genetic disorder caused by abnormal collagen type I production. While OI is primarily characterized by bone fragility and deformities, patients also have extraskeletal manifestations, including an increased risk of cardiovascular disease. This review provides a comprehensive overview of the literature on cardiovascular diseases in OI patients in order to raise awareness of this understudied clinical aspect of OI and support clinical guidelines. In accordance with the PRISMA guidelines, a systematic literature search in PubMed, Embase, Web of Science and Scopus was conducted that included articles from the inception of these databases to April 2023. Valvular disease, heart failure, atrial fibrillation, and hypertension appear to be more prevalent in OI than in control individuals. Moreover, a larger aortic root was observed in OI compared to controls. Various cardiovascular diseases appear to be more prevalent in OI than in controls. These cardiovascular abnormalities are observed in all types of OI and at all ages, including young children. As there are insufficient longitudinal studies, it is unknown whether these abnormalities are progressive in nature in OI patients. Based on these findings, we would recommend referring individuals with OI to a cardiologist with a low-threshold.


Assuntos
Anormalidades Cardiovasculares , Doenças Cardiovasculares , Osteogênese Imperfeita , Criança , Humanos , Pré-Escolar , Osteogênese Imperfeita/genética , Doenças Cardiovasculares/complicações , Anormalidades Cardiovasculares/complicações , Colágeno Tipo I , Estudos Longitudinais
4.
Am J Med Genet A ; : e63605, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38752492

RESUMO

Germline SMAD4 pathogenic variants (PVs) cause juvenile polyposis syndrome (JPS), which is known for an increased risk of gastrointestinal juvenile polyps and gastrointestinal cancer. Many patients with SMAD4 PV also show signs of hereditary hemorrhagic telangiectasia (HHT) and some patients have aneurysms and dissections of the thoracic aorta. Here we describe two patients with a germline SMAD4 PV and a remarkable clinical presentation including multiple medium-sized arterial aneurysms. More data are needed to confirm whether the more extensive vascular phenotype and the other described features in our patients are indeed part of a broader JPS spectrum.

5.
Int J Mol Sci ; 25(6)2024 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-38542391

RESUMO

(1) Mesenchymal stem cells (MSCs) are a valuable cell model to study the bone pathology of Osteogenesis Imperfecta (OI), a rare genetic collagen-related disorder characterized by bone fragility and skeletal dysplasia. We aimed to generate a novel OI induced mesenchymal stem cell (iMSC) model from induced pluripotent stem cells (iPSCs) derived from human dermal fibroblasts. For the first time, OI iMSCs generation was based on an intermediate neural crest cell (iNCC) stage. (2) Skin fibroblasts from healthy individuals and OI patients were reprogrammed into iPSCs and subsequently differentiated into iMSCs via iNCCs. (3) Successful generation of iPSCs from acquired fibroblasts was confirmed with changes in cell morphology, expression of iPSC markers SOX2, NANOG, and OCT4 and three germ-layer tests. Following differentiation into iNCCs, cells presented increased iNCC markers including P75NTR, TFAP2A, and HNK-1 and decreased iPSC markers, shown to reach the iNCC stage. Induction into iMSCs was confirmed by the presence of CD73, CD105, and CD90 markers, low expression of the hematopoietic, and reduced expression of the iNCC markers. iMSCs were trilineage differentiation-competent, confirmed using molecular analyses and staining for cell-type-specific osteoblast, adipocyte, and chondrocyte markers. (4) In the current study, we have developed a multipotent in vitro iMSC model of OI patients and healthy controls able to differentiate into osteoblast-like cells.


Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Mesenquimais , Osteogênese Imperfeita , Humanos , Osteogênese Imperfeita/genética , Osteogênese Imperfeita/metabolismo , Diferenciação Celular , Colágeno/metabolismo , Pele , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética
6.
Hum Mol Genet ; 30(23): 2286-2299, 2021 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-34244757

RESUMO

Aortic aneurysms (AAs) are pathological dilatations of the aorta. Pathogenic variants in genes encoding for proteins of the contractile machinery of vascular smooth muscle cells (VSMCs), genes encoding proteins of the transforming growth factor beta signaling pathway and extracellular matrix (ECM) homeostasis play a role in the weakening of the aortic wall. These variants affect the functioning of VSMC, the predominant cell type in the aorta. Many variants have unknown clinical significance, with unknown consequences on VSMC function and AA development. Our goal was to develop functional assays that show the effects of pathogenic variants in aneurysm-related genes. We used a previously developed fibroblast transdifferentiation protocol to induce VSMC-like cells, which are used for all assays. We compared transdifferentiated VSMC-like cells of patients with a pathogenic variant in genes encoding for components of VSMC contraction (ACTA2, MYH11), transforming growth factor beta (TGFß) signaling (SMAD3) and a dominant negative (DN) and two haploinsufficient variants in the ECM elastic laminae (FBN1) to those of healthy controls. The transdifferentiation efficiency, structural integrity of the cytoskeleton, TGFß signaling profile, migration velocity and maximum contraction were measured. Transdifferentiation efficiency was strongly reduced in SMAD3 and FBN1 DN patients. ACTA2 and FBN1 DN cells showed a decrease in SMAD2 phosphorylation. Migration velocity was impaired for ACTA2 and MYH11 cells. ACTA2 cells showed reduced contractility. In conclusion, these assays for showing effects of pathogenic variants may be promising tools to help reclassification of variants of unknown clinical significance in AA-related genes.


Assuntos
Actinas/genética , Aneurisma Aórtico/etiologia , Fibrilina-1/genética , Cadeias Pesadas de Miosina/genética , Proteína Smad3/genética , Aneurisma Aórtico/metabolismo , Aneurisma Aórtico/patologia , Diferenciação Celular/genética , Transdiferenciação Celular/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Modelos Biológicos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Proteína Smad2/metabolismo
7.
Am J Hum Genet ; 107(5): 989-999, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33053334

RESUMO

Osteogenesis imperfecta (OI) is characterized primarily by susceptibility to fractures with or without bone deformation. OI is genetically heterogeneous: over 20 genetic causes are recognized. We identified bi-allelic pathogenic KDELR2 variants as a cause of OI in four families. KDELR2 encodes KDEL endoplasmic reticulum protein retention receptor 2, which recycles ER-resident proteins with a KDEL-like peptide from the cis-Golgi to the ER through COPI retrograde transport. Analysis of patient primary fibroblasts showed intracellular decrease of HSP47 and FKBP65 along with reduced procollagen type I in culture media. Electron microscopy identified an abnormal quality of secreted collagen fibrils with increased amount of HSP47 bound to monomeric and multimeric collagen molecules. Mapping the identified KDELR2 variants onto the crystal structure of G. gallus KDELR2 indicated that these lead to an inactive receptor resulting in impaired KDELR2-mediated Golgi-ER transport. Therefore, in KDELR2-deficient individuals, OI most likely occurs because of the inability of HSP47 to bind KDELR2 and dissociate from collagen type I. Instead, HSP47 remains bound to collagen molecules extracellularly, disrupting fiber formation. This highlights the importance of intracellular recycling of ER-resident molecular chaperones for collagen type I and bone metabolism and a crucial role of HSP47 in the KDELR2-associated pathogenic mechanism leading to OI.


Assuntos
Osso e Ossos/metabolismo , Colágeno Tipo I/metabolismo , Proteínas de Choque Térmico HSP47/metabolismo , Osteogênese Imperfeita/genética , Proteínas de Transporte Vesicular/metabolismo , Adulto , Alelos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Osso e Ossos/patologia , Galinhas , Pré-Escolar , Colágeno Tipo I/química , Colágeno Tipo I/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Expressão Gênica , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Proteínas de Choque Térmico HSP47/química , Proteínas de Choque Térmico HSP47/genética , Humanos , Lactente , Masculino , Osteogênese Imperfeita/diagnóstico , Osteogênese Imperfeita/metabolismo , Osteogênese Imperfeita/patologia , Linhagem , Cultura Primária de Células , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Transporte Proteico , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética
8.
Calcif Tissue Int ; 112(5): 621-627, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36764958

RESUMO

Pregnancy- and lactation-associated osteoporosis (PLO) is a rare form of osteoporosis, of which the pathogenesis and best treatment options are unclear. In this report, we describe the case of a 34-year old woman diagnosed with severe osteoporosis and multiple vertebral fractures after her first pregnancy, who was subsequently treated with teriparatide (TPTD) and zoledronic acid (ZA). We describe the clinical features, imaging examination, and genetic analysis. Substantial improvements were observed in areal and volumetric bone mineral density (BMD), microarchitecture, and strength between 7 and 40 months postpartum as assessed by dual-energy X-ray absorptiometry at the total hip and spine and by high-resolution peripheral quantitative CT at the distal radius and tibiae. At the hip, spine, and distal radius, these improvements were mainly enabled by treatment with TPTD and ZA, while at the distal tibiae, physiological recovery and postpartum physiotherapy due to leg pain after stumbling may have played a major role. Additionally, the findings show that, despite the improvements, BMD, microarchitecture, and strength remained severely impaired in comparison with healthy age- and gender-matched controls at 40 months postpartum. Genetic analysis showed no monogenic cause for osteoporosis, and it is suggested that PLO in this woman could have a polygenic origin with possible susceptibility based on familiar occurrence of osteoporosis.


Assuntos
Conservadores da Densidade Óssea , Osteoporose , Humanos , Gravidez , Feminino , Adulto , Teriparatida/uso terapêutico , Ácido Zoledrônico/uso terapêutico , Conservadores da Densidade Óssea/uso terapêutico , Osteoporose/etiologia , Densidade Óssea , Lactação
9.
Int J Mol Sci ; 24(3)2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36768622

RESUMO

Fibrodysplasia ossificans progressiva (FOP) is a catastrophic, ultra-rare disease of heterotopic ossification caused by genetic defects in the ACVR1 gene. The mutant ACVR1 receptor, when triggered by an inflammatory process, leads to heterotopic ossification of the muscles and ligaments. Activin A has been discovered as the main osteogenic ligand of the FOP ACVR1 receptor. However, the source of Activin A itself and the trigger of its production in FOP individuals have remained elusive. We used primary dermal fibroblasts from five FOP patients to investigate Activin A production and how this is influenced by inflammatory cytokines in FOP. FOP fibroblasts showed elevated Activin A production compared to healthy controls, both in standard culture and osteogenic transdifferentiation conditions. We discovered TGFß1 to be an FOP-specific stimulant of Activin A, shown by the upregulation of the INHBA gene and protein expression. Activin A and TGFß1 were both induced by BMP4 in FOP and control fibroblasts. Treatment with TNFα and IL6 produced negligible levels of Activin A and TGFß1 in both cell groups. We present for the first time TGFß1 as a triggering factor of Activin A production in FOP. As TGFß1 can promote the induction of the main driver of FOP, TGFß1 could also be considered a possible therapeutic target in FOP treatment.


Assuntos
Miosite Ossificante , Ossificação Heterotópica , Humanos , Miosite Ossificante/genética , Miosite Ossificante/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Transdução de Sinais/genética , Ossificação Heterotópica/genética , Fibroblastos/metabolismo , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Mutação
10.
Am J Med Genet C Semin Med Genet ; 190(1): 89-101, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35343062

RESUMO

Primary ciliary dyskinesia (PCD) is a heterogeneous disease, with impaired mucociliary clearance causing respiratory tract infections. A founding CCDC114 mutation has led to a relatively homogeneous and large Dutch PCD population in Volendam. Our aim was to describe their phenotype. Therefore, all Volendam PCD patients seen at the Amsterdam UMC were included in this study. Data were collected on lung function, microbiology, radiology, and ear-nose-throat (ENT) symptoms. A mixed effects model estimated lung function decline in %point per year (95% confidence interval [CI]). Thirty-three (60%) out of approximately 56 Volendam PCD patients were treated at our center and included in this study. Only 30% of patients had situs inversus. FEV1 declined in children (-1.43%/year, CI: -1.80/-1.05), but not in adults (0.01%/year, CI: -0.36/0.38). Pseudomonas aeruginosa was cultured in 21% of children and 60% of adults, respectively. Patients who have been infected at some point with P. aeruginosa had a steeper decline in FEV1 as compared to patients that have never been infected. Neonatal symptoms (79%) and ENT problems (94%) were common; fertility issues however, were not (11%) common. Compared to other PCD cohorts, the Volendam/CCDC114 patients have a moderately severe phenotype with lung function decline predominantly occurring in childhood.


Assuntos
Transtornos da Motilidade Ciliar , Proteínas Associadas aos Microtúbulos , Transtornos da Motilidade Ciliar/diagnóstico , Transtornos da Motilidade Ciliar/genética , Humanos , Proteínas Associadas aos Microtúbulos/genética , Mutação , Países Baixos , Fenótipo
11.
Arterioscler Thromb Vasc Biol ; 41(11): 2693-2707, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34470477

RESUMO

Multiple layers of vascular smooth muscle cells (vSMCs) are present in blood vessels forming the media of the vessel wall. vSMCs provide a vessel wall structure, enabling it to contract and relax, thus modulating blood flow. They also play a crucial role in the development of vascular diseases, such as atherosclerosis and aortic aneurysm formation. vSMCs display a remarkable high degree of plasticity. At present, the number of different vSMC phenotypes has only partially been characterized. By mapping vSMC phenotypes in detail and identifying triggers for phenotype switching, the relevance of the different phenotypes in vascular disease may be identified. Up until recently, vSMCs were classified as either contractile or dedifferentiated (ie, synthetic). However, single-cell RNA sequencing studies revealed such dedifferentiated arterial vSMCs to be highly diverse. Currently, no consensus exist about the number of vSMC phenotypes. Therefore, we reviewed the data from relevant single-cell RNA sequencing studies, and classified a total of 6 vSMC phenotypes. The central dedifferentiated vSMC type that we classified is the mesenchymal-like phenotype. Mesenchymal-like vSMCs subsequently seem to differentiate into fibroblast-like, macrophage-like, osteogenic-like, and adipocyte-like vSMCs, which contribute differentially to vascular disease. This phenotype switching between vSMCs requires the transcription factor KLF4 (Kruppel-like factor 4). Here, we performed an integrated analysis of the data about the recently identified vSMC phenotypes, their associated gene expression profiles, and previous vSMC knowledge to better understand the role of vSMC phenotype transitions in vascular pathology.


Assuntos
Aterosclerose/metabolismo , Diferenciação Celular , Plasticidade Celular , Fatores de Transcrição Kruppel-Like/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/patologia , Proliferação de Células , Humanos , Fator 4 Semelhante a Kruppel , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Fenótipo , Placa Aterosclerótica , Transdução de Sinais
12.
Hum Genet ; 140(8): 1121-1141, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34169326

RESUMO

Osteogenesis Imperfecta (OI) comprises a heterogeneous group of patients who share bone fragility and deformities as the main characteristics, albeit with different degrees of severity. Phenotypic variation also exists in other connective tissue aspects of the disease, complicating disease classification and disease course prediction. Although collagen type I defects are long established as the primary cause of the bone pathology, we are still far from comprehending the complete mechanism. In the last years, the advent of next generation sequencing has triggered the discovery of many new genetic causes for OI, helping to draw its molecular landscape. It has become clear that, in addition to collagen type I genes, OI can be caused by multiple proteins connected to different parts of collagen biosynthesis. The production of collagen entails a complex process, starting from the production of the collagen Iα1 and collagen Iα2 chains in the endoplasmic reticulum, during and after which procollagen is subjected to a plethora of posttranslational modifications by chaperones. After reaching the Golgi organelle, procollagen is destined to the extracellular matrix where it forms collagen fibrils. Recently discovered mutations in components of the retrograde transport of chaperones highlight its emerging role as critical contributor of OI development. This review offers an overview of collagen regulation in the context of recent gene discoveries, emphasizing the significance of transport disruptions in the OI mechanism. We aim to motivate exploration of skeletal fragility in OI from the perspective of these pathways to identify regulatory points which can hint to therapeutic targets.


Assuntos
Osso e Ossos/metabolismo , Colágeno Tipo I/biossíntese , Osteoblastos/metabolismo , Osteogênese Imperfeita/metabolismo , Pró-Colágeno/biossíntese , Processamento de Proteína Pós-Traducional , Osso e Ossos/patologia , Colágeno Tipo I/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Osteoblastos/patologia , Osteogênese Imperfeita/genética , Osteogênese Imperfeita/patologia , Pró-Colágeno/genética , Biossíntese de Proteínas , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Transporte Proteico , Índice de Gravidade de Doença
13.
J Endovasc Ther ; 28(4): 604-613, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33902345

RESUMO

INTRODUCTION: Abdominal aortic aneurysms (AAAs) are associated with overall high mortality in case of rupture. Since the pathophysiology is unclear, no adequate pharmacological therapy exists. Smooth muscle cells (SMCs) dysfunction and extracellular matrix (ECM) degradation have been proposed as underlying causes. We investigated SMC spatial organization and SMC-ECM interactions in our novel 3-dimensional (3D) vascular model. We validated our model for future use by comparing it to existing 2-dimensional (2D) cell culture. Our model can be used for translational studies of SMC and their role in AAA pathophysiology. MATERIALS AND METHODS: SMC isolated from the medial layer of were the aortic wall of controls and AAA patients seeded on electrospun poly-lactide-co-glycolide scaffolds and cultured for 5 weeks, after which endothelial cells (EC) are added. Cell morphology, orientation, mechanical properties and ECM production were quantified for validation and comparison between controls and patients. RESULTS: We show that cultured SMC proliferate into multiple layers after 5 weeks in culture and produce ECM proteins, mimicking their behavior in the medial aortic layer. EC attach to multilayered SMC, mimicking layer interactions. The novel SMC model exhibits viscoelastic properties comparable to biological vessels; cytoskeletal organization increases during the 5 weeks in culture; increased cytoskeletal alignment and decreased ECM production indicate different organization of AAA patients' cells compared with control. CONCLUSION: We present a valuable preclinical model of AAA constructed with patient specific cells with applications in both translational research and therapeutic developments. We observed SMC spatial reorganization in a time course of 5 weeks in our robust, patient-specific model of SMC-EC organization and ECM production.


Assuntos
Aneurisma da Aorta Abdominal , Células Endoteliais , Matriz Extracelular , Humanos , Miócitos de Músculo Liso , Resultado do Tratamento
14.
Eur J Vasc Endovasc Surg ; 61(6): 1008-1016, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33858751

RESUMO

OBJECTIVE: Perivascular adipose tissue (PVAT) contributes to vascular homeostasis and is increasingly linked to vascular pathology. PVAT density and volume were associated with abdominal aortic aneurysm (AAA) presence and dimensions on imaging. However, mechanisms underlying the role of PVAT in AAA have not been clarified. This study aimed to explore differences in PVAT from AAA using gene expression and functional tests. METHODS: Human aortic PVAT and control subcutaneous adipose tissue were collected during open AAA surgery. Gene analyses and functional tests were performed. The control group consisted of healthy aorta from non-living renal transplant donors. Gene expression tests were performed to study genes potentially involved in various inflammatory processes and AAA related genes. Live PVAT and subcutaneous adipose tissue (SAT) from AAA were used for ex vivo co-culture with smooth muscle cells (SMCs) retrieved from non-pathological aortas. RESULTS: Adipose tissue was harvested from 27 AAA patients (n [gene expression] = 22, n [functional tests] = 5) and five control patients. An increased inflammatory gene expression of PTPRC (p = .008), CXCL8 (p = .033), LCK (p = .003), CCL5 (p = .004) and an increase in extracellular matrix breakdown marker MMP9 (p = .016) were found in AAA compared with controls. Also, there was a decreased anti-inflammatory gene expression of PPARG in AAA compared with controls (p = .040). SMC co-cultures from non-pathological aortas with PVAT from AAA showed increased MMP9 (p = .033) and SMTN (p = .008) expression and SAT increased SMTN expression in these SMC. CONCLUSION: The data revealed that PVAT from AAA shows an increased pro-inflammatory and matrix metallopeptidase gene expression and decreased anti-inflammatory gene expression. Furthermore, increased expression of genes involved in aneurysm formation was found in healthy SMC co-culture with PVAT of AAA patients. Therefore, PVAT from AAA might contribute to inflammation of the adjacent aortic wall and thereby plays a possible role in AAA pathophysiology. These proposed pathways of inflammatory induction could reveal new therapeutic targets in AAA treatment.


Assuntos
Aneurisma da Aorta Abdominal/genética , Quimiocina CCL5/genética , Interleucina-8/genética , Antígenos Comuns de Leucócito/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Metaloproteinase 9 da Matriz/genética , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Idoso , Idoso de 80 Anos ou mais , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Estudos de Casos e Controles , Quimiocina CCL5/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Feminino , Humanos , Interleucina-8/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , PPAR gama/genética , PPAR gama/metabolismo , RNA Mensageiro/metabolismo
15.
Am J Hum Genet ; 100(1): 160-168, 2017 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-28041644

RESUMO

Defects in motile cilia and sperm flagella cause primary ciliary dyskinesia (PCD), characterized by chronic airway disease, infertility, and left-right body axis disturbance. Here we report maternally inherited and de novo mutations in PIH1D3 in four men affected with PCD. PIH1D3 is located on the X chromosome and is involved in the preassembly of both outer (ODA) and inner (IDA) dynein arms of cilia and sperm flagella. Loss-of-function mutations in PIH1D3 lead to absent ODAs and reduced to absent IDAs, causing ciliary and flagellar immotility. Further, PIH1D3 interacts and co-precipitates with cytoplasmic ODA/IDA assembly factors DNAAF2 and DNAAF4. This result has clinical and genetic counseling implications for genetically unsolved male case subjects with a classic PCD phenotype that lack additional phenotypes such as intellectual disability or retinitis pigmentosa.


Assuntos
Cílios/patologia , Transtornos da Motilidade Ciliar/genética , Dineínas/metabolismo , Genes Ligados ao Cromossomo X , Mutação/genética , Cauda do Espermatozoide/patologia , Cílios/metabolismo , Transtornos da Motilidade Ciliar/metabolismo , Transtornos da Motilidade Ciliar/patologia , Citoplasma/metabolismo , Feminino , Humanos , Masculino , Linhagem , Fenótipo , Motilidade dos Espermatozoides/genética , Cauda do Espermatozoide/metabolismo
16.
Biochem Biophys Res Commun ; 521(2): 310-317, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31668813

RESUMO

AIM OF THE STUDY: Osteogenesis imperfecta and Ehlers Danlos syndrome are hereditary disorders caused primarily by defective collagen regulation. Osteogenesis imperfecta patients were divided to haploinsufficient and dominant negative depending on the effect of COL1A1 and COL1A2 mutations whereas Ehlers Danlos syndrome patients had a mutation in PLOD1. Although collagen abnormalities have been extensively studied in monolayer cultures, there are no reports about 3D in vitro models which may reflect more accurately the dynamic cell environment. This is the first study presenting the structural and mechanical characterization of a 3D cell-secreted model using primary patient fibroblasts. MATERIALS AND METHODS: Fibroblasts from patients with osteogenesis imperfecta and Ehlers Danlos syndrome were cultured with ascorbic acid for 5 weeks. The effect of mutations on cytosolic and secreted collagen was tested by electrophoresis following incubation with radiolabeled 14C proline. Extracellular matrix was studied in terms of collagen fiber orientation, stiffness, as well as glycosaminoglycan and collagen content. RESULTS AND CONCLUSIONS: Osteogenesis imperfecta patients with haploinsufficient mutations had higher percentage of anisotropic collagen fibers alignment compared to other patient groups; all patients had a lower percentage of anisotropic samples compared to healthy controls. This correlated with higher average stiffness in the control group. Glycosaminoglycan content was lower in the control and haploinsufficient groups. In cells with PLOD1 mutations, there were no differences in PLOD2 expression. This proof of concept study was able to show differences in collagen fiber orientation between different patient groups which can potentially pave the way towards the development of 3D models aiming at improved investigation of disease mechanisms.


Assuntos
Síndrome de Ehlers-Danlos/patologia , Matriz Extracelular/ultraestrutura , Fibroblastos/patologia , Osteogênese Imperfeita/patologia , Adulto , Anisotropia , Estudos de Casos e Controles , Técnicas de Cultura de Células , Células Cultivadas , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Feminino , Fibroblastos/ultraestrutura , Glicosaminoglicanos/análise , Humanos , Masculino , Mutação , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética
17.
J Med Genet ; 56(4): 220-227, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-29967133

RESUMO

BACKGROUND: Missense variants in SMAD2, encoding a key transcriptional regulator of transforming growth factor beta signalling, were recently reported to cause arterial aneurysmal disease. OBJECTIVES: The aims of the study were to identify the genetic disease cause in families with aortic/arterial aneurysmal disease and to further define SMAD2 genotype-phenotype correlations. METHODS AND RESULTS: Using gene panel sequencing, we identified a SMAD2 nonsense variant and four SMAD2 missense variants, all affecting highly conserved amino acids in the MH2 domain. The premature stop codon (c.612dup; p.(Asn205*)) was identified in a marfanoid patient with aortic root dilatation and in his affected father. A p.(Asn318Lys) missense variant was found in a Marfan syndrome (MFS)-like case who presented with aortic root aneurysm and in her affected daughter with marfanoid features and mild aortic dilatation. In a man clinically diagnosed with Loeys-Dietz syndrome (LDS) that presents with aortic root dilatation and marked tortuosity of the neck vessels, another missense variant, p.(Ser397Tyr), was identified. This variant was also found in his affected daughter with hypertelorism and arterial tortuosity, as well as his affected mother. The third missense variant, p.(Asn361Thr), was discovered in a man presenting with coronary artery dissection. Variant genotyping in three unaffected family members confirmed its absence. The last missense variant, p.(Ser467Leu), was identified in a man with significant cardiovascular and connective tissue involvement. CONCLUSION: Taken together, our data suggest that heterozygous loss-of-function SMAD2 variants can cause a wide spectrum of autosomal dominant aortic and arterial aneurysmal disease, combined with connective tissue findings reminiscent of MFS and LDS.


Assuntos
Aneurisma/etiologia , Dissecção Aórtica/etiologia , Dissecção Aórtica/patologia , Artérias/patologia , Variação Genética , Proteína Smad2/genética , Adulto , Idoso , Alelos , Substituição de Aminoácidos , Aneurisma/patologia , Criança , Fácies , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Masculino , Síndrome de Marfan/complicações , Síndrome de Marfan/genética , Pessoa de Meia-Idade , Mutação , Linhagem , Fenótipo , Proteína Smad2/metabolismo
18.
J Cell Physiol ; 234(7): 10238-10247, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30417373

RESUMO

Fibrodysplasia ossificans progressiva (FOP) is a genetic disease characterized by heterotopic ossification (HO). The disease is caused by a mutation in the activin receptor type 1 (ACVR1) gene that enhances this receptor's responsiveness to Activin-A. Binding of Activin-A to the mutated ACVR1 receptor induces osteogenic differentiation. Whether Activin-A also affects osteoclast formation in FOP is not known. Therefore we investigated its effect on the osteoclastogenesis-inducing potential of periodontal ligament fibroblasts (PLF) from teeth of healthy controls and patients with FOP. We used western blot analysis of phosphorylated SMAD3 (pSMAD3) and quantitative polymerase chain reaction to assess the effect of Activin-A on the PLF. PLF-induced osteoclast formation and gene expression were studied by coculturing control and FOP PLF with CD14-positive osteoclast precursor cells from healthy donors. Osteoclast formation was also assessed in control CD14 cultures stimulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANK-L). Although Activin-A increased activation of the pSMAD3 pathway in both control and FOP PLF, it increased ACVR1, FK binding protein 12 (FKBP12), an inhibitor of DNA binding 1 protein (ID-1) expression only in FOP PLF. Activin-A inhibited PLF mediated osteoclast formation albeit only significantly when induced by FOP PLF. In these cocultures, it reduced M-CSF and dendritic cell-specific transmembrane protein (DC-STAMP) expression. Activin-A also inhibited osteoclast formation in M-CSF and RANK-L mediated monocultures of CD14+ cells by inhibiting their proliferation. This study brings new insight on the role of Activin A in osteoclast formation, which may further add to understanding FOP pathophysiology; in addition to the known Activin-A-mediated HO, this study shows that Activin-A may also inhibit osteoclast formation, thereby further promoting HO formation.


Assuntos
Ativinas/farmacologia , Comunicação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Miosite Ossificante/metabolismo , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Receptores de Ativinas Tipo I/metabolismo , Adolescente , Adulto , Estudos de Casos e Controles , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Proteína 1 Inibidora de Diferenciação/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Miosite Ossificante/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Ligamento Periodontal/metabolismo , Ligamento Periodontal/patologia , Fosforilação , Transdução de Sinais , Proteína Smad3/metabolismo , Proteína 1A de Ligação a Tacrolimo/metabolismo , Adulto Jovem
19.
Hum Mutat ; 39(5): 653-665, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29363216

RESUMO

We aimed to determine the diagnostic yield of a targeted-exome panel in a cohort of 74 Dutch primary ciliary dyskinesia (PCD) patients. The panel consisted of 26 PCD-related and 284 candidate genes. To prioritize PCD candidate genes, we investigated the transcriptome of human airway cells of 12 healthy volunteers during in vitro ciliogenesis and hypothesized that PCD-related genes show significant upregulation. We compared gene expression in epithelial precursor cells grown as collagen monolayer and ciliated cells grown in suspension by RNA sequencing. All genes reported as PCD causative, except NME8, showed significant upregulation during in vitro ciliogenesis. We observed 67.6% diagnostic yield when testing the targeted-exome panel in our cohort. There was relatively high percentage of DNAI and HYDIN mutations compared to other countries. The latter may be due to our solution for the problem of the confounding HYDIN2 pseudogene. Candidate genes included two recently published PCD-related genes DNAJB13 and PIH1D3; identification of the latter was a direct result of this study. In conclusion, we demonstrate 67.6% diagnostic yield by targeted exome sequencing in a Dutch PCD population and present a highly sensitive and moderately specific approach for identification of PCD-related genes, based on significant upregulation during in vitro ciliogenesis.


Assuntos
Síndrome de Kartagener/diagnóstico , Síndrome de Kartagener/genética , Adulto , Alelos , Exoma/genética , Regulação da Expressão Gênica , Humanos , Mutação/genética , Análise de Sequência de RNA
20.
Am J Med Genet A ; 176(5): 1216-1221, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29681102

RESUMO

Spondylocostal dysostosis (SCD) is a rare disorder characterized by vertebral segmentation defects and malformations of the ribs. SCD patients have some degree of (kypho)scoliosis, short stature and suffer from respiratory impairment due to the reduced size of their thoracic cage. Mutations in DLL3, MESP2, LFNG, HES7, TBX6, and RIPPLY2 are known to cause different subtypes of SCD. Here, we report on a male neonate with an apparent distinct SCD-like phenotype only partly overlapping the previously described SCD subtypes. The proband presented with severe rib malformations (missing, fused, bifid, and hypoplastic ribs), vertebral malformations (intervertebral fusions of the laminae and irregular ossification of the vertebral bodies), and a mild scoliosis. Clear segmentation defects of the vertebral bodies were lacking. Other dysmorphic features were present as well. Severe respiratory insufficiency was present from birth. Whole exome sequencing identified a homozygous start-loss variant in DMRT2 (NM_006557.6: c.1A > T p.[Met1?]) being a likely cause of the SCD-like phenotype in the proband. Mutations in DMRT2 (OMIM#604935) have not been described in relation to SCD-related phenotypes in humans before. However, Dmrt2 knock-out mice exhibit severe rib and vertebral defects that strikingly overlap with the radiological phenotype of the proband reported here. Therefore, it seems plausible that mutations in DMRT2 are associated with a different (novel) subtype of SCD mainly characterized by severe rib anomalies but lacking clear segmentation defects of the vertebral bodies.


Assuntos
Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Proteínas de Ligação a DNA/genética , Hérnia Diafragmática/diagnóstico , Hérnia Diafragmática/genética , Homozigoto , Mutação , Fenótipo , Costelas/anormalidades , Coluna Vertebral/anormalidades , Fatores de Transcrição/genética , Alelos , Evolução Fatal , Estudos de Associação Genética , Predisposição Genética para Doença , Heterozigoto , Humanos , Recém-Nascido , Masculino , Radiografia , Costelas/diagnóstico por imagem , Coluna Vertebral/diagnóstico por imagem , Tomografia Computadorizada Espiral , Sequenciamento do Exoma
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