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1.
Front Bioeng Biotechnol ; 11: 1179332, 2023.
Article in English | MEDLINE | ID: mdl-37346792

ABSTRACT

Tissue Engineering of cartilage has been hampered by the inability of engineered tissue to express native levels of type II collagen in vitro. Inadequate levels of type II collagen are, in part, due to a failure to recapitulate the physiological environment in culture. In this study, we engineered primary rabbit chondrocytes to express a secreted reporter, Gaussia Luciferase, driven by the type II collagen promoter, and applied a Design of Experiments approach to assess chondrogenic differentiation in micronutrient-supplemented medium. Using a Response Surface Model, 240 combinations of micronutrients absent in standard chondrogenic differentiation medium, were screened and assessed for type II collagen promoter-driven Gaussia luciferase expression. While the target of this study was to establish a combination of all micronutrients, alpha-linolenic acid, copper, cobalt, chromium, manganese, molybdenum, vitamins A, E, D and B7 were all found to have a significant effect on type II collagen promoter activity. Five conditions containing all micronutrients predicted to produce the greatest luciferase expression were selected for further study. Validation of these conditions in 3D aggregates identified an optimal condition for type II collagen promoter activity. Engineered cartilage grown in this condition, showed a 170% increase in type II collagen expression (Day 22 Luminescence) and in Young's tensile modulus compared to engineered cartilage in basal media alone.Collagen cross-linking analysis confirmed formation of type II-type II collagen and type II-type IX collagen cross-linked heteropolymeric fibrils, characteristic of mature native cartilage. Combining a Design of Experiments approach and secreted reporter cells in 3D aggregate culture enabled a high-throughput platform that can be used to identify more optimal physiological culture parameters for chondrogenesis.

2.
Matrix Biol Plus ; 12: 100070, 2021 Dec.
Article in English | MEDLINE | ID: mdl-34825162

ABSTRACT

Tendons and ligaments tend to be pooled into a single category as dense elastic bands of collagenous connective tissue. They do have many similar properties, for example both tissues are flexible cords of fibrous tissue that join bone to either muscle or bone. Tendons and ligaments are both prone to degenerate and rupture with only limited capacity to heal, although tendons tend to heal faster than ligaments. Type I collagen constitutes about 80% of the dry weight of tendons and ligaments and is principally responsible for the core strength of each tissue. Collagen synthesis is a complex process with multiple steps and numerous post-translational modifications including proline and lysine hydroxylation, hydroxylysine glycosylation and covalent cross-linking. The chemistry, placement and quantity of intramolecular and intermolecular cross-links are believed to be key contributors to the tissue-specific variations in material strength and biological properties of collagens. As tendons and ligaments grow and develop, the collagen cross-links are known to chemically mature, strengthen and change in profile. Accordingly, changes in cross-linking and other post-translational modifications are likely associated with tissue development and degeneration. Using mass spectrometry, we have compared tendon and ligaments from fetal and adult bovine knee joints to investigate changes in collagen post-translational properties. Although hydroxylation levels at the type I collagen helical cross-linking lysine residues were similar in all adult tissues, ligaments had significantly higher levels of glycosylation at these sites compared to tendon. Differences in lysine hydroxylation were also found between the tissues at the telopeptide cross-linking sites. Total collagen cross-linking analysis, including mature trivalent cross-links and immature divalent cross-links, revealed unique cross-linking profiles between tendon and ligament tissues. Tendons were found to have a significantly higher frequency of smaller diameter collagen fibrils compared with ligament, which we suspect is functionally associated with the unique cross-linking profile of each tissue. Understanding the specific molecular characteristics that define and distinguish these specialized tissues will be important to improving the design of orthopedic treatment approaches.

3.
Sci Rep ; 11(1): 10868, 2021 05 25.
Article in English | MEDLINE | ID: mdl-34035379

ABSTRACT

Tendon plays a critical role in the joint movement by transmitting force from muscle to bone. This transmission of force is facilitated by its specialized structure, which consists of highly aligned extracellular matrix consisting predominantly of type I collagen. Tenocytes, fibroblast-like tendon cells residing between the parallel collagen fibers, regulate this specialized tendon matrix. Despite the importance of collagen structure and tenocyte function, the biological mechanisms regulating fibrillogenesis and tenocyte maturation are not well understood. Here we examine the function of Reticulocalbin 3 (Rcn3) in collagen fibrillogenesis and tenocyte maturation during postnatal tendon development using a genetic mouse model. Loss of Rcn3 in tendon caused decreased tendon thickness, abnormal tendon cell maturation, and decreased mechanical properties. Interestingly, Rcn3 deficient mice exhibited a smaller collagen fibril distribution and over-hydroxylation in C-telopeptide cross-linking lysine from α1(1) chain. Additionally, the proline 3-hydroxylation sites in type I collagen were also over-hydroxylated in Rcn3 deficient mice. Our data collectively suggest that Rcn3 is a pivotal regulator of collagen fibrillogenesis and tenocyte maturation during postnatal tendon development.


Subject(s)
Calcium-Binding Proteins/genetics , Collagen/metabolism , Tendons/growth & development , Tendons/metabolism , Animals , Biomarkers , Cell Differentiation , Gene Knockdown Techniques , Hydrolysis , Immunohistochemistry , Mass Spectrometry , Mice , Mice, Knockout , Organogenesis/genetics , Tendons/embryology
4.
Elife ; 102021 05 26.
Article in English | MEDLINE | ID: mdl-34036937

ABSTRACT

Osteogenesis imperfecta (OI) is characterized by short stature, skeletal deformities, low bone mass, and motor deficits. A subset of OI patients also present with joint hypermobility; however, the role of tendon dysfunction in OI pathogenesis is largely unknown. Using the Crtap-/- mouse model of severe, recessive OI, we found that mutant Achilles and patellar tendons were thinner and weaker with increased collagen cross-links and reduced collagen fibril size at 1- and 4-months compared to wildtype. Patellar tendons from Crtap-/- mice also had altered numbers of CD146+CD200+ and CD146-CD200+ progenitor-like cells at skeletal maturity. RNA-seq analysis of Achilles and patellar tendons from 1-month Crtap-/- mice revealed dysregulation in matrix and tendon marker gene expression concomitant with predicted alterations in TGF-ß, inflammatory, and metabolic signaling. At 4-months, Crtap-/- mice showed increased αSMA, MMP2, and phospho-NFκB staining in the patellar tendon consistent with excess matrix remodeling and tissue inflammation. Finally, a series of behavioral tests showed severe motor impairments and reduced grip strength in 4-month Crtap-/- mice - a phenotype that correlates with the tendon pathology.


Subject(s)
Achilles Tendon/pathology , Extracellular Matrix Proteins/deficiency , Motor Activity , Osteogenesis Imperfecta/pathology , Osteogenesis Imperfecta/physiopathology , Patellar Ligament/pathology , Achilles Tendon/metabolism , Actins/metabolism , Age Factors , Animals , Disease Models, Animal , Extracellular Matrix/genetics , Extracellular Matrix/metabolism , Extracellular Matrix/pathology , Extracellular Matrix Proteins/genetics , Fibrillar Collagens/genetics , Fibrillar Collagens/metabolism , Genes, Recessive , Genetic Predisposition to Disease , Hand Strength , Matrix Metalloproteinase 2/metabolism , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Molecular Chaperones/genetics , NF-kappa B/metabolism , Osteogenesis Imperfecta/genetics , Osteogenesis Imperfecta/metabolism , Patellar Ligament/metabolism , Phenotype , Phosphorylation , Physical Endurance , Stem Cells/metabolism , Stem Cells/pathology
5.
Transl Psychiatry ; 9(1): 4, 2019 01 15.
Article in English | MEDLINE | ID: mdl-30664616

ABSTRACT

Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder with a strong genetic component. Although next-generation sequencing (NGS) technologies have been successfully applied to gene identification in de novo ASD, the genetic architecture of familial ASD remains largely unexplored. Our approach, which leverages the high specificity and sensitivity of NGS technology, has focused on rare variants in familial autism. We used NGS exome sequencing in 26 families with distantly related affected individuals to identify genes with private gene disrupting and missense variants of interest (VOI). We found that the genes carrying VOIs were enriched for biological processes related to cell projection organization and neuron development, which is consistent with the neurodevelopmental hypothesis of ASD. For a subset of genes carrying VOIs, we then used targeted NGS sequencing and gene-based variant burden case-control analysis to test for association with ASD. Missense variants in one gene, CEP41, associated significantly with ASD (p = 6.185e-05). Homozygous gene-disrupting variants in CEP41 were initially found to be responsible for recessive Joubert syndrome. Using a zebrafish model, we evaluated the mechanism by which the CEP41 variants might contribute to ASD. We found that CEP41 missense variants affect development of the axonal tract, cranial neural crest migration and social behavior phenotype. Our work demonstrates the involvement of CEP41 heterozygous missense variants in ASD and that biological processes involved in cell projection organization and neuron development are enriched in ASD families we have studied.


Subject(s)
Autism Spectrum Disorder/genetics , Genetic Predisposition to Disease , Mutation, Missense , Proteins/genetics , Animals , Behavior, Animal , Case-Control Studies , Disease Models, Animal , Exome , Family Health , Female , High-Throughput Nucleotide Sequencing , Humans , Male , Exome Sequencing , Zebrafish
6.
J Biol Chem ; 293(40): 15620-15627, 2018 10 05.
Article in English | MEDLINE | ID: mdl-30143533

ABSTRACT

Nonenzymatic glycation of collagen has long been associated with the progressive secondary complications of diabetes. How exactly such random glycations result in impaired tissues is still poorly understood. Because of the slow turnover rate of most fibrillar collagens, they are more susceptible to accumulate time-dependent glycations and subsequent advanced glycation end-products. The latter are believed to include cross-links that stiffen host tissues. However, diabetic animal models have also displayed weakened tendons with reduced stiffness. Strikingly, not a single experimentally identified specific molecular site of glycation in a collagen has been reported. Here, using targeted MS, we have identified partial fructosyl-hydroxylysine glycations at each of the helical domain cross-linking sites of type I collagen that are elevated in tissues from a diabetic mouse model. Glycation was not found at any other collagen lysine residues. Type I collagen in mouse tendons is cross-linked intermolecularly by acid-labile aldimine bonds formed by the addition of telopeptide lysine aldehydes to hydroxylysine residues at positions α1(I)Lys87, α1(I)Lys930, α2(I)Lys87, and α2(I)Lys933 of the triple helix. Our data reveal that site-specific glycations of these specific lysines may significantly impair normal lysyl oxidase-controlled cross-linking in diabetic tendons. We propose that such N-linked glycations can hinder the normal cross-linking process, thus altering the content and/or placement of mature cross-links with the potential to modify tissue material properties.


Subject(s)
Collagen Type I/chemistry , Diabetes Mellitus, Type 2/metabolism , Lysine/chemistry , Obesity/metabolism , Tendons/metabolism , Amino Acid Sequence , Amino Acids/chemistry , Animals , Blood Glucose/metabolism , Collagen Type I/metabolism , Cross-Linking Reagents/chemistry , Diabetes Mellitus, Type 2/pathology , Disease Models, Animal , Glycated Hemoglobin/metabolism , Glycation End Products, Advanced/chemistry , Glycation End Products, Advanced/metabolism , Glycosylation , Hydroxylation , Hydroxylysine/chemistry , Hydroxylysine/metabolism , Lysine/metabolism , Male , Mass Spectrometry , Mice , Obesity/pathology , Protein-Lysine 6-Oxidase/chemistry , Protein-Lysine 6-Oxidase/metabolism , Tail , Tendons/chemistry , Tendons/pathology
7.
Autism Res ; 10(8): 1338-1343, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28419775

ABSTRACT

Autism is a complex genetic disorder where both de-novo and inherited genetics factors play a role. Next generation sequencing approaches have been extensively used to identify rare variants associated with autism. To date, all such studies were focused on nuclear genome; thereby leaving the role of mitochondrial DNA (mtDNA) variation in autism unexplored. Recently, analytical tools have been developed to evaluate mtDNA in whole-exome data. We have analyzed the mtDNA sequence derived from whole-exome sequencing in 10 multiplex families. In one of the families we have identified two variants of interest in MT-ND5 gene that were previously determined to impair mitochondrial function. In addition in a second family we have identified two VOIs; mtDNA variant in MT-ATP6 and nuclear DNA variant in NDUFS4, where both VOIs are within mitochondrial Respiratory Chain Complex. Our findings provide further support for the role of mitochondria in ASD and confirm that whole-exome sequencing allows for analysis of mtDNA, which sets a stage for further comprehensive genetic investigations of the role of mitochondria in autism. Autism Res 2017, 10: 1338-1343. © 2017 International Society for Autism Research, Wiley Periodicals, Inc.


Subject(s)
Autism Spectrum Disorder/genetics , DNA, Mitochondrial/genetics , High-Throughput Nucleotide Sequencing/methods , Exome/genetics , Female , Genetic Predisposition to Disease/genetics , Humans , Registries
8.
Hum Genet ; 134(10): 1055-68, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26204995

ABSTRACT

Autism spectrum disorders (ASDs) are a group of neurodevelopmental disorders, characterized by impairment in communication and social interactions, and by repetitive behaviors. ASDs are highly heritable, and estimates of the number of risk loci range from hundreds to >1000. We considered 7 extended families (size 12-47 individuals), each with ≥3 individuals affected by ASD. All individuals were genotyped with dense SNP panels. A small subset of each family was typed with whole exome sequence (WES). We used a 3-step approach for variant identification. First, we used family-specific parametric linkage analysis of the SNP data to identify regions of interest. Second, we filtered variants in these regions based on frequency and function, obtaining exactly 200 candidates. Third, we compared two approaches to narrowing this list further. We used information from the SNP data to impute exome variant dosages into those without WES. We regressed affected status on variant allele dosage, using pedigree-based kinship matrices to account for relationships. The p value for the test of the null hypothesis that variant allele dosage is unrelated to phenotype was used to indicate strength of evidence supporting the variant. A cutoff of p = 0.05 gave 28 variants. As an alternative third filter, we required Mendelian inheritance in those with WES, resulting in 70 variants. The imputation- and association-based approach was effective. We identified four strong candidate genes for ASD (SEZ6L, HISPPD1, FEZF1, SAMD11), all of which have been previously implicated in other studies, or have a strong biological argument for their relevance.


Subject(s)
Autism Spectrum Disorder/genetics , Eye Proteins/genetics , Membrane Proteins/genetics , Phosphotransferases (Phosphate Group Acceptor)/genetics , Transcription Factors/genetics , Exome , Female , Gene Frequency , Genes, Dominant , Genetic Association Studies , Genetic Predisposition to Disease , Humans , Linkage Disequilibrium , Male , Models, Genetic , Polymorphism, Single Nucleotide , Repressor Proteins , Sequence Analysis, DNA
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