Objective: This study investigated a large number of families in which at least two individuals w... more Objective: This study investigated a large number of families in which at least two individuals were diagnosed with abdominal aortic aneurysms to identify the relationship of the affected relatives to the proband. Subjects and Methods: Families for the study were recruited through various vascular surgery centers in the United States,
The pathophysiology of aortic aneurysms (AA) is far from being understood. One reason for this la... more The pathophysiology of aortic aneurysms (AA) is far from being understood. One reason for this lack of understanding is basic research being constrained to fixated cells or isolated cell cultures, by which cell-to-cell and cell-to-matrix communications are missed. We present a new, in vitro method for extended preservation of aortic wall sections to study pathophysiological processes. Intraoperatively harvested, live aortic specimens were cut into 150 μm sections and cultured. Viability was quantified up to 92 days using immunofluorescence. Cell types were characterized using immunostaining. After 14 days, individual cells of enzymatically digested tissues were examined for cell type and viability. Analysis of AA sections (N = 8) showed a viability of 40% at 7 days and smooth muscle cells, leukocytes, and macrophages were observed. Protocol optimization (N = 4) showed higher stable viability at day 62 and proliferation of new cells at day 92. Digested tissues showed different cell types and a viability up to 75% at day 14. Aortic tissue viability can be preserved until at least 62 days after harvesting. Cultured tissues can be digested into viable single cells for additional techniques. Present protocol provides an appropriate ex vivo setting to discover and study pathways and mechanisms in cultured human aneurysmal aortic tissue. Aortic aneurysm (AA) is a common health problem, which is associated with high mortality rates in the event of a rupture. This unpredictable life-threatening rupture leads to rapid aortic exsanguination into thorax, abdominal cavity or retroperitoneum 1-3. To date, studies investigating the pathophysiology of AA have produced inconclusive results concerning the underlying mechanisms. Although different meaningful disease models exist, the lack of a functionally and structurally intact live aortic human tissue model which can bioactively be stimulated ex vivo, has posed severe limitations to the study of AA in understanding the pathophysiological mechanism which has restricted the identification of therapeutic targets and the development of efficient therapy 4. It has been observed previously that an increase of AA diameter occurs in relation to the transformation of the aortic vessel wall composition. Part of this transformation is a combination of vascular smooth muscle cell (SMC) loss and inflammation and degeneration of extracellular matrix (ECM). These changes are suggested to play a role in the pathogenesis of AA, but mechanisms contributing to these processes, are still poorly understood 5,6. Although numerous in vivo animal models to study AA exist, investigation of human material is limited to the use of fixated tissue or isolated cell cultures 7-12. Such approaches, however, have failed to retain the complex organ function of the aorta, which is based on cell-cell interactions, communication with ECM, immune cell infiltration and constant tissue remodelling 13-16. Unfortunately, fixated aortic tissue only provides a snapshot into the microstructural characteristics of the diseased tissue, and isolated cultured aortic cells cannot reproduce the complexity of cell-ECM interactions and live pathways. Additionally, usage of animal models is costly, ethically and technically challenging and animal
Using [ 18 F] Sodium Fuoride (NaF) Positron Emission Tomography (PET) it is not only possible to ... more Using [ 18 F] Sodium Fuoride (NaF) Positron Emission Tomography (PET) it is not only possible to identify the ossifying potency of a flare-up, but also to identify an asymptomatic chronic stage of fibrodysplasia ossificans progressiva (FOP). The purpose of this study was to investigate the diagnostic role of a more widely available imaging modality, Magnetic Resonance Imaging (MRI), which is of special interest for studies in pediatric FOP patients. MRI and [ 18 F]NaF PET/CT images at time of inclusion and subsequent follow-up CT scans of 4 patients were analyzed retrospectively. Presence, location, and intensity of edema identified by MRI were compared with activity on [ 18 F]NaF PET. Occurrence or progression of heterotopic ossification (HO) was examined on the follow-up CT images. Thirteen different lesions in various muscle groups were identified: five lesions with only edema, five lesions with both edema and increased [ 18 F]NaF uptake, one lesion with only increased [ 18 F]NaF uptake, and two lesions with neither edema nor uptake of [ 18 F]NaF. Mild edema, found in three lesions, was present at asymptomatic sites, which did not show increased [ 18 F] NaF uptake or progression of HO on consecutive CT images. Moderate edema was found in three symptomatic lesions, with increased [ 18 F]NaF on PET and progression of HO on CT. Severe edema was identified in four lesions. Interestingly, two of these lesions did not develop HO during follow-up; one of these two even gave obvious symptoms of a flare-up. MRI can identify whether symptoms are the result of an acute flare-up by the presence of moderate to severe edema. The occurrence of severe edema on MRI was not always related to an ossifying lesion. The additional diagnostic value of MRI requires further investigation, but MRI does not seem to fully replace the diagnostic characteristics of [ 18 F]NaF PET/CT in FOP.
Inherited bone disorders account for about 10% of documented Mendelian disorders and are associat... more Inherited bone disorders account for about 10% of documented Mendelian disorders and are associated with high financial burden. Their study requires osteoblasts which play a critical role in regulating the development and maintenance of bone tissue. However, bone tissue is not always available from patients. We developed a highly efficient platelet lysate-based approach to directly transdifferentiate skin-derived human fibroblasts to osteoblast-like cells. We extensively characterized our in vitro model by examining the expression of osteoblast-specific markers during the transdifferentiation process both at the mRNA and protein level. The transdifferentiated osteoblast-like cells showed significantly increased expression of a panel of osteogenic markers. Mineral deposition and ALP activity were also shown, confirming their osteogenic properties. RNAseq analysis allowed the global study of changes in the transcriptome of the transdifferentiated cells. The transdifferentiated cells clustered separately from the primary fibroblasts with regard to the significantly upregulated genes indicating a distinct transcriptome profile; transdifferentiated osteoblasts also showed significant enrichment in gene expression related to skeletal development and bone mineralization. Our presented in vitro model may potentially contribute to the prospect of studying osteoblast-dependent disorders in patient-derived cells. Bone disorders encompass a spectrum of etiology which extends from inherited bone disorders such as skeletal dysplasia, with an established monogenic cause, to disorders with a less defined genetic contribution such as the highly prevalent postmenopausal osteoporosis 1,2. In order to build knowledge on the underlying disease mechanism and identify points for therapeutic intervention, relevant cell models are necessary in which the pathological condition is reflected and can be studied. The acquisition of bone tissue from affected individuals is not always possible so suitable alternatives are being sought for diagnostic and research purposes. The development of a method to noninvasively produce bone cells from skin fibroblasts of a larger group of human donors may help to address this need. Bone homeostasis is based on bone remodeling which includes formation and development throughout life in combination with adaptation to mechanical demands 3. Bone tissue is maintained by osteoblasts, osteoclasts and osteocytes 4. Osteoblasts are responsible for the formation of bone tissue through the deposition of bone extracellular matrix which becomes mineralized by calcium hydroxyapatite to confer bone its strength and rigidity 5. Osteoclasts serve an opposing role by mediating bone resorption. Osteoblasts differentiate terminally to osteocytes which respond to mechanical stimuli by activating osteoblasts or osteoclasts to induce an anabolic or catabolic effect respectively. The function of these three types of cells is crucial in preserving the bone structure
UvA-DARE (Digital Academic Repository) Flare-up after maxillofacial surgery in a patient with fib... more UvA-DARE (Digital Academic Repository) Flare-up after maxillofacial surgery in a patient with fibrodysplasia ossificans progressiva An [18F]-NaF PET/CT study and a systematic review
Osteogenesis Imperfecta (OI) comprises a heterogeneous group of patients who share bone fragility... more 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.
Biochemical and Biophysical Research Communications, 2020
An in vitro model to evaluate the properties of matrices produced by fibroblasts from osteogenesi... more An in vitro model to evaluate the properties of matrices produced by fibroblasts from osteogenesis imperfecta and Ehlers-Danlos Syndrome patients.
Molecular Genetics & Genomic Medicine, Jun 17, 2019
This is an open access article under the terms of the Creat ive Commo ns Attri bution License, wh... more This is an open access article under the terms of the Creat ive Commo ns Attri bution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Effects of TGF-[beta] inhibition on osteogenesis and osteoclastogenesis by periodontal ligament fibroblasts from patients with fibrodysplasia ossificans progressiva
Osteogenesis imperfecta (OI) is a heritable connective tissue disorder that causes bone fragility... more Osteogenesis imperfecta (OI) is a heritable connective tissue disorder that causes bone fragility due to pathogenic variants in genes responsible for the synthesis of type I collagen. Efforts to classify the high clinical variability in OI led to the Sillence classification. However, this classification only partially takes into account extraskeletal manifestations and the high genetic variability. Little is known about the relation between genetic variants and phenotype as of yet. The aim of the study was to create a clinically relevant genetic stratification of a cohort of 675 Dutch OI patients based on their pathogenic variant types and to provide an overview of their respective medical care demands. The clinical records of 675 OI patients were extracted from the Amsterdam UMC Genome Database and matched with the records from Statistics Netherlands (CBS). The patients were categorized based on their harbored pathogenic variant. The information on hospital admissions, outpatient c...
To optimize care for children with Marfan syndrome (MFS) in the Netherlands, Dutch MFS growth cha... more To optimize care for children with Marfan syndrome (MFS) in the Netherlands, Dutch MFS growth charts were constructed. Additionally, we aimed to investigate the effect of FBN1 variant type (haploinsufficiency [HI]/dominant negative [DN]) on growth, and compare MFS‐related height increase across populations. Height and weight data of individuals with MFS aged 0–21 years were retrospectively collected. Generalized Additive Models for Location, Scale and Shape (GAMLSS) was used for growth chart modeling. To investigate genotype–phenotype relationships, FBN1 variant type was included as an independent variable in height‐for‐age and BMI‐for‐age models. MFS‐related height increase was compared with that of previous MFS growth studies from the United States, Korea, and France. Height and weight data of 389 individuals with MFS were included (210 males). Height‐for‐age, BMI‐for‐age, and weight‐for‐height charts reflected the tall and slender MFS habitus throughout childhood. Mean increase i...
Epidermolysis bullosa (EB) is a group of rare genetic diseases that exhibit mechanical fragility ... more Epidermolysis bullosa (EB) is a group of rare genetic diseases that exhibit mechanical fragility of the skin. This condition will result in the occurrence of skin blisters, skin erosions, and skin ulcerations when the skin is subjected to trauma. In this case report, we present a case of EB and multiple skeletal deformities in a 21-year-old female. She came to our clinic with recurrent skin exfoliations and blisters that occurred since she was 4 years old and multiple bones bowing since she was 9 years old. On physical examinations, we found generalized hypopigmentation macule with erythematous skin. There were numerous bullae and crusted lesions, with erosion and excoriations on the lesions. Laboratory examinations identified low vitamin D 25-OH (8.6 ng/mL). Bone densitometry measurement found low bone density, and X-ray examination found osteopenia and bone bowing. Using whole-exome sequencing, no causative pathogenic sequence or copy number variants in the genes associated with M...
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