RESUMEN
Recessive dystrophic epidermolysis bullosa is a rare genodermatosis caused by a mutation of the Col7a1 gene. The Col7a1 gene codes for collagen type VII protein, a major component of anchoring fibrils. Mutations of the Col7a1 gene can cause aberrant collagen type VII formation, causing an associated lack or absence of anchoring fibrils. This presents clinically as chronic blistering, scarring, and fibrosis, often leading to the development of cutaneous squamous cell carcinoma. Patients also experience persistent pain and pruritus. Pain management and supportive bandaging remain the primary treatment options. The pathology of recessive dystrophic epidermolysis bullosa was first described in the 1980s, and there has since been a multitude of encouraging treatment options developed. However, in vivo research has been hindered by inadequate models of the disease. The various mouse models in existence possess longevity and surface area constraints, or do not adequately model a normal human disease state. In this paper, we describe a novel rat model of recessive dystrophic epidermolysis bullosa that offers an alternative to previous murine models. An 8-base pair deletion was induced in the Col7a1 gene of Lewis rats, which was subsequently found to cause a premature stop codon downstream. Homozygous mutants presented with a fragile and chronically blistered phenotype postnatally. Further histological analysis revealed subepidermal clefting and the absence of anchoring fibrils. The generation of this novel model offers researchers an easily maintained organism that possesses a larger surface area for experimental topical and transfused therapies to be tested, which may provide great utility in the future study of this debilitating disease.
Asunto(s)
Colágeno Tipo VII , Modelos Animales de Enfermedad , Epidermólisis Ampollosa Distrófica , Mutación del Sistema de Lectura , Fenotipo , Colágeno Tipo VII/genética , Animales , Epidermólisis Ampollosa Distrófica/genética , Epidermólisis Ampollosa Distrófica/patología , Ratas , Genes Recesivos , Ratas Endogámicas Lew , Vesícula/genética , Vesícula/patología , Piel/patología , MasculinoRESUMEN
The biological mechanisms regulating tenocyte differentiation and morphological maturation have not been well-established, partly due to the lack of reliable in vitro systems that produce highly aligned collagenous tissues. In this study, we developed a scaffold-free, three-dimensional (3D) tendon culture system using mouse tendon cells in a differentially adherent growth channel. Transforming Growth Factor-ß (TGFß) signaling is involved in various biological processes in the tendon, regulating tendon cell fate, recruitment and maintenance of tenocytes, and matrix organization. This known function of TGFß signaling in tendon prompted us to utilize TGFß1 to induce tendon-like structures in 3D tendon constructs. TGFß1 treatment promoted a tendon-like structure in the peripheral layer of the constructs characterized by increased thickness with a gradual decrease in cell density and highly aligned collagen matrix. TGFß1 also enhanced cell proliferation, matrix production, and morphological maturation of cells in the peripheral layer compared to vehicle treatment. TGFß1 treatment also induced early tenogenic differentiation and resulted in sufficient mechanical integrity, allowing biomechanical testing. The current study suggests that this scaffold-free 3D tendon cell culture system could be an in vitro platform to investigate underlying biological mechanisms that regulate tenogenic cell differentiation and matrix organization.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Tendones , Tenocitos , Factor de Crecimiento Transformador beta1 , Animales , Factor de Crecimiento Transformador beta1/farmacología , Factor de Crecimiento Transformador beta1/metabolismo , Tendones/citología , Tendones/metabolismo , Ratones , Diferenciación Celular/efectos de los fármacos , Tenocitos/metabolismo , Tenocitos/citología , Proliferación Celular/efectos de los fármacos , Técnicas de Cultivo Tridimensional de Células/métodos , Células Cultivadas , Técnicas de Cultivo de Célula/métodos , Matriz Extracelular/metabolismo , Colágeno/metabolismo , Ingeniería de Tejidos/métodosRESUMEN
Although abnormal TGFß signaling is observed in several heritable forms of thoracic aortic aneurysms and dissections including Marfan syndrome, its precise role in aortic disease progression is still disputed. Using a mouse genetic approach and quantitative isobaric labeling proteomics, we sought to elucidate the role of TGFß signaling in three Fbn1 mutant mouse models representing a range of aortic disease from microdissection (without aneurysm) to aneurysm (without rupture) to aneurysm and rupture. Results indicated that reduced TGFß signaling and increased mast cell proteases were associated with microdissection. In contrast, increased abundance of extracellular matrix proteins, which could be reporters for positive TGFß signaling, were associated with aneurysm. Marked reductions in collagens and fibrillins, and increased TGFß signaling, were associated with aortic rupture. Our data indicate that TGFß signaling performs context-dependent roles in the pathogenesis of thoracic aortic disease.
Asunto(s)
Aneurisma de la Aorta Torácica , Síndrome de Marfan , Humanos , Aneurisma de la Aorta Torácica/genética , Fibrilina-1/genética , Fibrilinas , Síndrome de Marfan/genética , Síndrome de Marfan/patología , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
AMACO (VWA2 protein), secreted by epithelial cells, is strongly expressed at basement membranes when budding or invagination occurs in embryos. In skin, AMACO associates with proteins of the Fraser complex, which form anchoring cords. These, during development, temporally stabilize the dermal-epidermal junction, pending the formation of collagen VII-containing anchoring fibrils. Fraser syndrome in humans results if any of the core members of the Fraser complex (Fras1, Frem1, Frem2) are mutated. Fraser syndrome is characterized by subepidermal blistering, cryptophthalmos, and syndactyly. In an attempt to determine AMACO function, we generated and characterized AMACO-deficient mice. In contrast to Fraser complex mutant mice, AMACO-deficient animals lack an obvious phenotype. The mutually interdependent basement membrane deposition of the Fraser complex proteins, and the formation of anchoring cords, are not affected. Furthermore, hair follicle development in newborn AMACO-deficient mice showed no gross aberration. Surprisingly, it appears that, while AMACO is a component of the anchoring cords, it is not essential for their formation or function.
Asunto(s)
Proteínas de la Matriz Extracelular , Síndrome de Fraser , Animales , Humanos , Ratones , Células Epiteliales/metabolismo , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Síndrome de Fraser/metabolismo , Piel/metabolismoRESUMEN
EMILIN1 (elastin-microfibril-interface-located-protein-1) is a structural component of the elastic fiber network and localizes to the interface between the fibrillin microfibril scaffold and the elastin core. How EMILIN1 contributes to connective tissue integrity is not fully understood. Here, we report bi-allelic EMILIN1 loss-of-function variants causative for an entity combining cutis laxa, arterial tortuosity, aneurysm formation, and bone fragility, resembling autosomal-recessive cutis laxa type 1B, due to EFEMP2 (FBLN4) deficiency. In both humans and mice, absence of EMILIN1 impairs EFEMP2 extracellular matrix deposition and LOX activity resulting in impaired elastogenesis, reduced collagen crosslinking, and aberrant growth factor signaling. Collagen fiber ultrastructure and histopathology in EMILIN1- or EFEMP2-deficient skin and aorta corroborate these findings and murine Emilin1-/- femora show abnormal trabecular bone formation and strength. Altogether, EMILIN1 connects elastic fiber network with collagen fibril formation, relevant for both bone and vascular tissue homeostasis.
Asunto(s)
Enfermedades Óseas Metabólicas , Cutis Laxo , Animales , Humanos , Ratones , Colágeno/genética , Cutis Laxo/genética , Elastina/metabolismo , Proteínas de la Matriz Extracelular/metabolismoRESUMEN
Collagens are the most abundant proteins in the body and among the most biosynthetically complex. A molecular ensemble of over 20 endoplasmic reticulum resident proteins participates in collagen biosynthesis and contributes to heterogeneous post-translational modifications. Pathogenic variants in genes encoding collagens cause connective tissue disorders, including osteogenesis imperfecta, Ehlers-Danlos syndrome, and Gould syndrome (caused by mutations in COL4A1 and COL4A2), and pathogenic variants in genes encoding proteins required for collagen biosynthesis can cause similar but overlapping clinical phenotypes. Notably, pathogenic variants in lysyl hydroxylase 3 (LH3) cause a multisystem connective tissue disorder that exhibits pathophysiological features of collagen-related disorders. LH3 is a multifunctional collagen-modifying enzyme; however, its precise role(s) and substrate specificity during collagen biosynthesis has not been defined. To address this critical gap in knowledge, we generated LH3 KO cells and performed detailed quantitative and molecular analyses of collagen substrates. We found that LH3 deficiency severely impaired secretion of collagen α1α1α2(IV) but not collagens α1α1α2(I) or α1α1α1(III). Amino acid analysis revealed that LH3 is a selective LH for collagen α1α1α2(IV) but a general glucosyltransferase for collagens α1α1α2(IV), α1α1α2(I), and α1α1α1(III). Importantly, we identified rare variants that are predicted to be pathogenic in the gene encoding LH3 in two of 113 fetuses with intracranial hemorrhage-a cardinal feature of Gould syndrome. Collectively, our findings highlight a critical role of LH3 in α1α1α2(IV) biosynthesis and suggest that LH3 pathogenic variants might contribute to Gould syndrome.
Asunto(s)
Colágeno , Enfermedades del Tejido Conjuntivo , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa , Humanos , Colágeno/metabolismo , Glicosilación , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
AMACO (VWA2 protein) is a basement membrane-associated protein secreted by epithelial cells. It is strongly expressed when invagination or budding occurs during development. AMACO associates with the Fraser complex, which when mutated causes Fraser syndrome, characterized by subepidermal blistering, cryptophthalmos, and syndactyly. The core Fraser complex proteins FRAS1, FREM1, and FREM2 localize at the dermalâepidermal junction and mediate adhesion to the underlying dermis during embryonic development. Earlier transmission electron microscopy studies of adult mouse skin showed clustered AMACO deposition below the lamina densa. In this study, we report a distinct cord-like suprastructure in the neonate dermis to which AMACO- and Fraser complexâassociated proteins contribute. We propose anchoring cords to designate the suprastructure. Anchoring cords have a diameter of 60 nm when immunolabeled, originate from the basement membrane, and extend several microns into the dermis. In normal skin, they are evident after immunogold electron microscopy and are strikingly appreciated in thicker sections. In recessive dystrophic epidermolysis bullosa skin, they are directly visible where collagen VII anchoring fibrils are ablated. Immunofluorescence and coimmunoprecipitation of skin extracts identify a direct interaction of FREM2 and AMACO.
Asunto(s)
Epidermólisis Ampollosa Distrófica , Proteínas de la Matriz Extracelular , Ratones , Animales , Embarazo , Femenino , Proteínas de la Matriz Extracelular/metabolismo , Piel/metabolismo , Membrana Basal/metabolismo , Epidermólisis Ampollosa Distrófica/metabolismo , Colágeno/metabolismo , Proteínas de la Membrana/metabolismoRESUMEN
Ocular anterior segment dysgenesis (ASD) refers to a collection of developmental disorders affecting the anterior structures of the eye. Although a number of genes have been implicated in the etiology of ASD, the underlying pathogenetic mechanisms remain unclear. Mutations in genes encoding collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) cause Gould syndrome, a multi-system disorder that often includes ocular manifestations such as ASD and glaucoma. COL4A1 and COL4A2 are abundant basement membrane proteins that provide structural support to tissues and modulate signaling through interactions with other extracellular matrix proteins, growth factors, and cell surface receptors. In this study, we used a combination of histological, molecular, genetic and pharmacological approaches to demonstrate that altered TGFß signaling contributes to ASD in mouse models of Gould syndrome. We show that TGFß signaling was elevated in anterior segments from Col4a1 mutant mice and that genetically reducing TGFß signaling partially prevented ASD. Notably, we identified distinct roles for TGFß1 and TGFß2 in ocular defects observed in Col4a1 mutant mice. Importantly, we show that pharmacologically promoting type IV collagen secretion or reducing TGFß signaling ameliorated ocular pathology in Col4a1 mutant mice. Overall, our findings demonstrate that altered TGFß signaling contributes to COL4A1-related ocular dysgenesis and implicate this pathway as a potential therapeutic target for the treatment of Gould syndrome.
Asunto(s)
Colágeno Tipo IV/metabolismo , Anomalías del Ojo , Animales , Membrana Basal/metabolismo , Colágeno Tipo IV/genética , Ojo/metabolismo , Anomalías del Ojo/metabolismo , Ratones , Mutación , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Recessive dystrophic epidermolysis bullosa (RDEB) is a lifelong genodermatosis associated with blistering, wounding, and scarring caused by mutations in COL7A1, the gene encoding the anchoring fibril component, collagen VII (C7). Here, we evaluated beremagene geperpavec (B-VEC), an engineered, non-replicating COL7A1 containing herpes simplex virus type 1 (HSV-1) vector, to treat RDEB skin. B-VEC restored C7 expression in RDEB keratinocytes, fibroblasts, RDEB mice and human RDEB xenografts. Subsequently, a randomized, placebo-controlled, phase 1 and 2 clinical trial (NCT03536143) evaluated matched wounds from nine RDEB patients receiving topical B-VEC or placebo repeatedly over 12 weeks. No grade 2 or above B-VEC-related adverse events or vector shedding or tissue-bound skin immunoreactants were noted. HSV-1 and C7 antibodies sometimes presented at baseline or increased after B-VEC treatment without an apparent impact on safety or efficacy. Primary and secondary objectives of C7 expression, anchoring fibril assembly, wound surface area reduction, duration of wound closure, and time to wound closure following B-VEC treatment were met. A patient-reported pain-severity secondary outcome was not assessed given the small proportion of wounds treated. A global assessment secondary endpoint was not pursued due to redundancy with regard to other endpoints. These studies show that B-VEC is an easily administered, safely tolerated, topical molecular corrective therapy promoting wound healing in patients with RDEB.
Asunto(s)
Epidermólisis Ampollosa Distrófica , Animales , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismo , Epidermólisis Ampollosa Distrófica/genética , Epidermólisis Ampollosa Distrófica/metabolismo , Epidermólisis Ampollosa Distrófica/terapia , Terapia Genética , Humanos , Queratinocitos/metabolismo , Ratones , Piel/metabolismoRESUMEN
Barth syndrome is an inherited X-linked disorder that leads to cardiomyopathy, skeletal myopathy, and neutropenia. These symptoms result from the loss of function of the enzyme TAFAZZIN, a transacylase located in the inner mitochondrial membrane that is responsible for the final steps of cardiolipin production. The link between defective cardiolipin maturation and neutropenia remains unclear. To address potential mechanisms of neutropenia, we examined myeloid progenitor development within the fetal liver of TAFAZZIN knockout (KO) animals as well as within the adult bone marrow of wild-type recipients transplanted with TAFAZZIN-KO hematopoietic stem cells. We also used the ER-Hoxb8 system (estrogen receptor fused to Hoxb8) of conditional immortalization to establish a new murine model system for the ex vivo study of TAFAZZIN-deficient neutrophils. The TAFAZZIN-KO cells demonstrated the expected dramatic differences in cardiolipin maturation that result from a lack of TAFAZZIN enzyme activity. Contrary to our hypothesis, we did not identify any significant differences in neutrophil development or neutrophil function across a variety of assays including phagocytosis and the production of cytokines or reactive oxygen species. However, transcriptomic analysis of the TAFAZZIN-deficient neutrophil progenitors demonstrated an upregulation of markers of endoplasmic reticulum stress and confirmatory testing demonstrated that the TAFAZZIN-deficient cells had increased sensitivity to certain ER stress-mediated and non-ER stress-mediated triggers of apoptosis. Although the link between increased sensitivity to apoptosis and the variably penetrant neutropenia phenotype seen in some patients with Barth syndrome remains to be clarified, our studies and new model system set a foundation for further investigation.
Asunto(s)
Aciltransferasas/metabolismo , Síndrome de Barth , Neutropenia , Animales , Animales Modificados Genéticamente , Apoptosis , Síndrome de Barth/genética , Cardiolipinas , Modelos Animales de Enfermedad , Humanos , Ratones , Receptores de Estrógenos , Factores de Transcripción/genéticaRESUMEN
PURPOSE: Primary open-angle glaucoma (POAG) is a complex heterogeneous disease. While several POAG genes have been identified, a high proportion of estimated heritability remains unexplained. Elevated intraocular pressure (IOP) is a leading POAG risk factor and dysfunctional extracellular matrix (ECM) in the trabecular meshwork (TM) contributes to elevated IOP. In this study, we sought to identify missense variants in ECM genes that correlate with ocular hypertensive POAG. METHODS: Whole-genome sequencing was used to identify genetic variants in five members of a large POAG family (n = 68) with elevated IOP. The remaining family members were screened by Sanger sequencing. Unrelated normal (NTM) and glaucomatous (GTM) cells were sequenced for the identified variants. The ECM protein levels were determined by Western immunoblotting and confocal and electron microscopy investigated ECM ultrastructural organization. RESULTS: Three ECM gene variants were significantly associated with POAG or elevated IOP in a large POAG pedigree. These included rs2228262 (N700S; thrombospondin-1 (THBS1, TSP1)), rs112913396 (D563 G; collagen type VI, alpha 3 (COL6A3)) and rs34759087 (E987K; laminin subunit beta 2 (LAMB2)). Screening of unrelated TM cells (n = 27) showed higher prevalence of the THBS1 variant but not the LAMB2 variant, in GTM cells (39%) than NTM cells (11%). The rare COL6A3 variant was not detected. TSP1 protein was upregulated and COL6A3 was down-regulated in TM cells with N700S subject to mechanical stretch, an in vitro method that mimics elevated IOP. Immunofluorescence showed increased TSP1 immunostaining in cell strains with N700S compared to wild-type TM cells. Ultrastructural studies showed ECM disorganization and altered collagen type VI distribution in GTM versus NTM cells. CONCLUSIONS: Our results suggest that missense variants in ECM genes may not cause catastrophic changes to the TM, but over many years, subtle changes in ECM may accumulate and cause structural disorganization of the outflow resistance leading to elevated IOP in POAG patients.
Asunto(s)
Humor Acuoso/metabolismo , ADN/genética , Proteínas de la Matriz Extracelular/genética , Glaucoma de Ángulo Abierto/genética , Mutación Missense , Trombospondina 1/genética , Malla Trabecular/metabolismo , Adulto , Anciano , Western Blotting , Células Cultivadas , Análisis Mutacional de ADN , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Glaucoma de Ángulo Abierto/metabolismo , Humanos , Presión Intraocular/fisiología , Masculino , Persona de Mediana Edad , Linaje , Trombospondina 1/metabolismo , Malla Trabecular/citologíaRESUMEN
Understanding cell recruitment in damaged tendons is critical for improvements in regenerative therapy. We recently reported that targeted disruption of transforming growth factor beta (TGFß) type II receptor in the tendon cell lineage (Tgfbr2ScxCre) resulted in resident tenocyte dedifferentiation and tendon deterioration in postnatal stages. Here we extend the analysis and identify direct recruitment of stem/progenitor cells into the degenerative mutant tendons. Cre-mediated lineage tracing indicates that these cells are not derived from tendon-ensheathing tissues or from a Scleraxis-expressing lineage, and they turned on tendon markers only upon entering the mutant tendons. Through immunohistochemistry and inducible gene deletion, we further find that the recruited cells originated from a Sox9-expressing lineage and their recruitment was dependent on cell autonomous TGFß signaling. The cells identified in this study thus differ from previous reports of cell recruitment into injured tendons and suggest a critical role for TGFß signaling in cell recruitment, providing insights that may support improvements in tendon repair.
Asunto(s)
Transducción de Señal , Células Madre/metabolismo , Tendones/patología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Biomarcadores/metabolismo , Células Cultivadas , Células Clonales , Proteínas Fluorescentes Verdes/metabolismo , Integrasas/metabolismo , Ratones , Modelos Biológicos , Mutación/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , Tendones/ultraestructura , Factores de TiempoRESUMEN
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.
Asunto(s)
Tendón Calcáneo/patología , Proteínas de la Matriz Extracelular/deficiencia , Actividad Motora , Osteogénesis Imperfecta/patología , Osteogénesis Imperfecta/fisiopatología , Ligamento Rotuliano/patología , Tendón Calcáneo/metabolismo , Actinas/metabolismo , Factores de Edad , Animales , Modelos Animales de Enfermedad , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Proteínas de la Matriz Extracelular/genética , Colágenos Fibrilares/genética , Colágenos Fibrilares/metabolismo , Genes Recesivos , Predisposición Genética a la Enfermedad , Fuerza de la Mano , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Chaperonas Moleculares/genética , FN-kappa B/metabolismo , Osteogénesis Imperfecta/genética , Osteogénesis Imperfecta/metabolismo , Ligamento Rotuliano/metabolismo , Fenotipo , Fosforilación , Resistencia Física , Células Madre/metabolismo , Células Madre/patologíaRESUMEN
An efficient musculoskeletal system depends on the precise assembly and coordinated growth and function of muscles, skeleton, and tendons. However, the mechanisms that drive integrated musculoskeletal development and coordinated growth and differentiation of each of these tissues are still being uncovered. Epigenetic modifiers have emerged as critical regulators of cell fate differentiation, but so far almost nothing is known about their roles in tendon biology. Previous studies have shown that epigenetic modifications driven by Enhancer of zeste homolog 2 (EZH2), a major histone methyltransferase, have significant roles in vertebrate development including skeletal patterning and bone formation. We now find that targeting Ezh2 through the limb mesenchyme also has significant effects on tendon and muscle patterning, likely reflecting the essential roles of early mesenchymal cues mediated by Ezh2 for coordinated patterning and development of all tissues of the musculoskeletal system. Conversely, loss of Ezh2 in the tendon cells did not disrupt overall tendon structure or collagen organization suggesting that tendon differentiation and maturation are independent of Ezh2 signaling.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2 , Osteogénesis , Diferenciación Celular/genética , Proteína Potenciadora del Homólogo Zeste 2/genética , Mesodermo , Osteogénesis/genética , TendonesRESUMEN
Collagen is the most abundant protein in humans. It has a characteristic triple-helix structure and is heavily posttranslationally modified. The complex biosynthesis of collagen involves processing by many enzymes and chaperones in the rough endoplasmic reticulum. Lysyl hydroxylase 1 (LH1) is required to hydroxylate lysine for cross-linking and carbohydrate attachment within collagen triple helical sequences. Additionally, a recent study of prolyl 3-hydroxylase 3 (P3H3) demonstrated that this enzyme may be critical for LH1 activity; however, the details surrounding its involvement remain unclear. If P3H3 is an LH1 chaperone that is critical for LH1 activity, P3H3 and LH1 null mice should display a similar deficiency in lysyl hydroxylation. To test this hypothesis, we compared the amount and location of hydroxylysine in the triple helical domains of type V and I collagen from P3H3 null, LH1 null, and wild-type mice. The amount of hydroxylysine in type V collagen was reduced in P3H3 null mice, but surprisingly type V collagen from LH1 null mice contained as much hydroxylysine as type V collagen from wild-type mice. In type I collagen, our results indicate that LH1 plays a global enzymatic role in lysyl hydroxylation. P3H3 is also involved in lysyl hydroxylation, particularly at cross-link formation sites, but is not required for all lysyl hydroxylation sites. In summary, our study suggests that LH1 and P3H3 likely have two distinct mechanisms to recognize different collagen types and to distinguish cross-link formation sites from other sites in type I collagen.
Asunto(s)
Colágeno Tipo I/metabolismo , Colágeno Tipo V/metabolismo , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Animales , Colágeno/genética , Colágeno/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo V/genética , Retículo Endoplásmico Rugoso/metabolismo , Hidroxilación , Hidroxilisina/metabolismo , Lisina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Procolágeno-Prolina Dioxigenasa/genética , Conformación Proteica , Procesamiento Proteico-Postraduccional/genéticaRESUMEN
Dystrophic epidermolysis bullosa (DEB) is a blistering skin disease caused by mutations in the gene COL7A1 encoding collagen VII. DEB can be inherited as recessive DEB (RDEB) or dominant DEB (DDEB) and is associated with a high wound burden. Perpetual cycles of wounding and healing drive fibrosis in DDEB and RDEB, as well as the formation of a tumor-permissive microenvironment. Prolonging wound-free episodes by improving the quality of wound healing would therefore confer substantial benefit for individuals with DEB. The collagenous domain of collagen VII is encoded by 82 in-frame exons, which makes splice-modulation therapies attractive for DEB. Indeed, antisense oligonucleotide-based exon skipping has shown promise for RDEB. However, the suitability of antisense oligonucleotides for treatment of DDEB remains unexplored. Here, we developed QR-313, a clinically applicable, potent antisense oligonucleotide specifically targeting exon 73. We show the feasibility of topical delivery of QR-313 in a carbomer-composed gel for treatment of wounds to restore collagen VII abundance in human RDEB skin. Our data reveal that QR-313 also shows direct benefit for DDEB caused by exon 73 mutations. Thus, the same topically applied therapeutic could be used to improve the wound healing quality in RDEB and DDEB.
Asunto(s)
Colágeno Tipo VII/genética , Epidermólisis Ampollosa Distrófica/terapia , Terapia Genética/métodos , Oligonucleótidos Antisentido/administración & dosificación , Cicatrización de Heridas/genética , Animales , Biopsia , Línea Celular , Modelos Animales de Enfermedad , Epidermólisis Ampollosa Distrófica/genética , Epidermólisis Ampollosa Distrófica/patología , Exones/genética , Fibroblastos , Fibrosis , Humanos , Queratinocitos , Ratones , Ratones Transgénicos , Mutación , Oligonucleótidos Antisentido/genética , Cultivo Primario de Células , Piel/efectos de los fármacosRESUMEN
Growth of the musculoskeletal system requires precise coordination between bone, muscle, and tendon during development. Insufficient elongation of the muscle-tendon unit relative to bone growth results in joint contracture, a condition characterized by reduction or complete loss of joint range of motion. Here we establish a novel murine model of joint contracture by targeting Smad4 for deletion in the tendon cell lineage using Scleraxis-Cre (ScxCre). Smad4ScxCre mutants develop a joint contracture shortly after birth. The contracture is stochastic in direction and increases in severity with age. Smad4ScxCre mutant tendons exhibited a stable reduction in cellularity and a progressive reduction in extracellular matrix volume. Collagen fibril diameters were reduced in the Smad4ScxCre mutants, suggesting a role for Smad4 signaling in the regulation of matrix accumulation. Although ScxCre also has sporadic activity in both cartilage and muscle, we demonstrate an essential role for Smad4 loss in tendons for the development of joint contractures. Disrupting the canonical TGFß-pathway in Smad2;3ScxCre mutants did not result in joint contractures. Conversely, disrupting the BMP pathway by targeting BMP receptors (Alk3ScxCre/Alk6null) recapitulated many features of the Smad4ScxCre contracture phenotype, suggesting that joint contracture in Smad4ScxCre mutants is caused by disruption of BMP signaling. Overall, these results establish a model of murine postnatal joint contracture and a role for BMP signaling in tendon elongation and extracellular matrix accumulation.
Asunto(s)
Contractura/metabolismo , Contractura/patología , Proteína Smad4/metabolismo , Tendones/crecimiento & desarrollo , Animales , Desarrollo Óseo , Proteínas Morfogenéticas Óseas/metabolismo , Cartílago/crecimiento & desarrollo , Cartílago/metabolismo , Linaje de la Célula , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Miembro Anterior , Ratones , Músculo Esquelético/metabolismo , Transducción de Señal , Proteína Smad4/genética , Tendones/citología , Tendones/embriología , Tendones/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Studies of cell fate focus on specification, but little is known about maintenance of the differentiated state. In this study, we find that the mouse tendon cell fate requires continuous maintenance in vivo and identify an essential role for TGFß signaling in maintenance of the tendon cell fate. To examine the role of TGFß signaling in tenocyte function the TGFß type II receptor (Tgfbr2) was targeted in the Scleraxis-expressing cell lineage using the ScxCre deletor. Tendon development was not disrupted in mutant embryos, but shortly after birth tenocytes lost differentiation markers and reverted to a more stem/progenitor state. Viral reintroduction of Tgfbr2 to mutants prevented and even rescued tenocyte dedifferentiation suggesting a continuous and cell autonomous role for TGFß signaling in cell fate maintenance. These results uncover the critical importance of molecular pathways that maintain the differentiated cell fate and a key role for TGFß signaling in these processes.
Asunto(s)
Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , Tendones/citología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Desdiferenciación Celular , Linaje de la Célula , Regulación de la Expresión Génica , Ratones , Mutación , Transducción de Señal , Células Madre/citología , Células Madre/metabolismo , Tendones/metabolismo , Tenocitos/citología , Tenocitos/metabolismoRESUMEN
It is generally agreed within the microscopy community that the quality of ultrastructure within the connective tissue matrix resulting from high-pressure freezing followed by freeze-substitution (HPF/FS) far exceeds that gained following the "conventional" preparation method, which includes aqueous fixation, dehydration, and embedding. Exposure to cryogen at high pressure is the only cryopreservation method capable of vitrifying tissue structure to a depth exceeding 200 µm. Cells within connective tissues prepared by HPF/FS are universally larger, filling the commonly seen void at the juncture between cell and matrix. Without significant shrinkage of cells and the coincident extraction of the cytosolic components, well-resolved organelles are less clustered within an expanded cytosol. Much of the artifact from "conventional" methods occurs as large space filling and also smaller fibril-associated proteoglycans are extracted during fixation. However, the visualization of some matrix features by electron microscopy is actually dependent on the collapse or extraction of these "masking" components. Herein, we argue that an impression of ultrastructure within commonly studied matrices, in particular skin, is best gained following the evaluation of both conventional preparations and tissue prepared by HPF/FS. Anat Rec, 2019. © 2019 American Association for Anatomy.
Asunto(s)
Aorta/ultraestructura , Tejido Conectivo/ultraestructura , Piel/ultraestructura , Tendones/ultraestructura , Adolescente , Animales , Preescolar , Criopreservación/métodos , Substitución por Congelación/métodos , Humanos , Lactante , Ratones , Manejo de Especímenes/métodosRESUMEN
Immunolocalization studies have shown that fibrillin-1 is distributed ubiquitously in the connective tissue space from early embryonic times through old age. When mutated, the gene for fibrillin-1 (FBN1) causes the Marfan syndrome, a common inherited disorder of connective tissue. The multiple manifestations of the Marfan syndrome reflect the known distribution of fibrillin-1 in cardiovascular, musculoskeletal, ocular, and dermal tissues. In this study, a mouse model of Marfan syndrome in which fibrillin-1 is truncated and tagged with green fluorescence was used to estimate the relative abundance of fibrillin-1 in developing tissues. In embryonic tissues, the aorta was the only tissue in which fibrillin-1 green fluorescence was detectable. Other arteries gained detectable fibrillin-1 green fluorescence just after birth. Fibrillin-1 fluorescence was observed at later postnatal times in the lung, skin, perichondrium, tendon, and ocular tissues, while other tissues remained negative. These results indicated that tissues most affected in the Marfan syndrome are the tissues in which fibrillin-1 is most abundant. Focus was placed on the aorta, since aortic disease is life threatening in the Marfan syndrome and fibrillin-1 green fluorescence was most abundant in this tissue. Fibrillin-1 green fluorescence and immunostaining showed that fibrillin-1 is within aortic medial elastic lamellae. Endothelial-specific compared to smooth muscle-specific fibrillin-1 green fluorescence, together with light microscopic analyses of fragmentation of aortic elastic lamellae, demonstrated that smooth muscle cell mutated fibrillin-1 contributed most to progressive aortic fragmentation. However, these studies also indicated that other cells, possibly endothelial cells, also contribute to this aortic pathology. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc.