RESUMEN
Latent transforming growth factor ß (TGFß)-binding proteins (LTBPs) are microfibril-associated proteins essential for anchoring TGFß in the extracellular matrix (ECM) as well as for correct assembly of ECM components. Variants in LTBP2, LTBP3, and LTBP4 have been identified in several autosomal recessive Mendelian disorders with skeletal abnormalities with or without impaired development of elastin-rich tissues. Thus far, the human phenotype associated with LTBP1 deficiency has remained enigmatic. In this study, we report homozygous premature truncating LTBP1 variants in eight affected individuals from four unrelated consanguineous families. Affected individuals present with connective tissue features (cutis laxa and inguinal hernia), craniofacial dysmorphology, variable heart defects, and prominent skeletal features (craniosynostosis, short stature, brachydactyly, and syndactyly). In vitro studies on proband-derived dermal fibroblasts indicate distinct molecular mechanisms depending on the position of the variant in LTBP1. C-terminal variants lead to an altered LTBP1 loosely anchored in the microfibrillar network and cause increased ECM deposition in cultured fibroblasts associated with excessive TGFß growth factor activation and signaling. In contrast, N-terminal truncation results in a loss of LTBP1 that does not alter TGFß levels or ECM assembly. In vivo validation with two independent zebrafish lines carrying mutations in ltbp1 induce abnormal collagen fibrillogenesis in skin and intervertebral ligaments and ectopic bone formation on the vertebrae. In addition, one of the mutant zebrafish lines shows voluminous and hypo-mineralized vertebrae. Overall, our findings in humans and zebrafish show that LTBP1 function is crucial for skin and bone ECM assembly and homeostasis.
Asunto(s)
Colágeno/metabolismo , Cutis Laxo/etiología , Variación Genética , Proteínas de Unión a TGF-beta Latente/genética , Adolescente , Alelos , Animales , Células Cultivadas , Niño , Preescolar , Cutis Laxo/patología , Matriz Extracelular/metabolismo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Lactante , Masculino , Linaje , Piel/metabolismo , Piel/patología , Pez CebraRESUMEN
In vivo micro-computed tomography (µCT) provides the ability to measure longitudinal changes to tibia microarchitecture, but the effect of this radiation is not well understood. The right proximal tibia of Sprague-Dawley rats (n = 12/group) randomized to Sham-control (Sham) or ovariectomy (OVX) surgery at 12 weeks of age was scanned using µCT at 13, 17, 21, and 25 weeks of age, at a resolution of 18 µm and a radiation dose of 603 mGy. The left proximal tibia was scanned only at 25 weeks of age to serve as an internal non-irradiated control. Repeated irradiation did not affect tibia microarchitecture in Sham or OVX groups, although there was an increase in cortical eccentricity (P < 0.05). All trabecular outcomes and cortical BMD were different (P < 0.05) between groups after only 1 week post-surgery and differences persisted to study endpoint. Characteristic changes to trabecular bone were observed in OVX rats over time. Interactions of time and hormone status were found for cortical BMD (P < 0.001), Ps. Pm., and Ec. Pm. (P < 0.05). Repeated irradiation of the tibia at 13, 17, 21, and 25 weeks does not cause adverse effects to microarchitecture, regardless of hormone status. This radiation dose can be applied over a typical 3-month study period to comprehensively understand how an intervention alters tibia microarchitecture without confounding effects of radiation.
Asunto(s)
Densidad Ósea/efectos de la radiación , Osteoporosis Posmenopáusica , Tibia/efectos de la radiación , Microtomografía por Rayos X/efectos adversos , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Estudios Longitudinales , Ovariectomía , Ratas , Ratas Sprague-DawleyRESUMEN
Proteoglycans are structurally and functionally diverse biomacromolecules found abundantly on cell membranes and in the extracellular matrix. They consist of a core protein linked to glycosaminoglycan chains via a tetrasaccharide linkage region. Here, we show that CRISPR/Cas9-mediated b3galt6 knock-out zebrafish, lacking galactosyltransferase II, which adds the third sugar in the linkage region, largely recapitulate the phenotypic abnormalities seen in human ß3GalT6-deficiency disorders. These comprise craniofacial dysmorphism, generalized skeletal dysplasia, skin involvement and indications for muscle hypotonia. In-depth TEM analysis revealed disturbed collagen fibril organization as the most consistent ultrastructural characteristic throughout different affected tissues. Strikingly, despite a strong reduction in glycosaminoglycan content, as demonstrated by anion-exchange HPLC, subsequent LC-MS/MS analysis revealed a small amount of proteoglycans containing a unique linkage region consisting of only three sugars. This implies that formation of glycosaminoglycans with an immature linkage region is possible in a pathogenic context. Our study, therefore unveils a novel rescue mechanism for proteoglycan production in the absence of galactosyltransferase II, hereby opening new avenues for therapeutic intervention.
RESUMEN
The parameters of a micro-computed tomography (µCT) scan, including whether a bone is imaged in vivo or ex vivo, determine the quality of the resulting image. In turn, this impacts the accuracy of the trabecular and cortical outcomes. The absolute impact of µCT scanning at different voxel sizes and whether the sample is imaged in vivo or ex vivo on the morphological outcomes of the proximal tibia in the rat is unknown. The right proximal tibia of 6-month-old Sham-control and ovariectomized (OVX) rats (n = 8/group) was scanned using µCT (SkyScan 1176, Bruker, Kontich, Belgium) using three sets of parameters (9 µm ex vivo, 18 µm ex vivo, 18 µm in vivo) to compare the trabecular and cortical outcomes. Regardless of scan protocols, differences between Sham and OVX groups were observed as expected. At a voxel size of 18 µm, scanning in vivo or ex vivo had no effect on any of the outcomes measured. However, compared to a 9 µm voxel size scan, imaging at 18 µm resulted in significant underestimation of the connectivity density (p < 0.05) of the trabecular bone and a significant overestimation (p < 0.05) of the trabecular indices (trabecular thickness, degree of anisotropy) and of the cortical indices (cortical bone area, cortical area fraction, cortical thickness) in both Sham and OVX rats. These results suggest the benefit to scanning the proximal tibia of rats at a voxel size as low as 9 µm, although considerations must be made for the increased acquisition time, anesthesia, animal welfare, and radiation exposure associated with lower voxel size in vivo scanning. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1690-1698, 2017.
Asunto(s)
Tibia/diagnóstico por imagen , Microtomografía por Rayos X/métodos , Animales , Femenino , Distribución Aleatoria , Ratas Sprague-DawleyRESUMEN
Long-term effects of repeated in vivo micro-computed tomography (µCT) scanning at key stages of growth and bone development (ages 2, 4 and 6 months) on trabecular and cortical bone structure, as well as developmental patterns, have not been studied. We determined the effect of repetitive µCT scanning at age 2, 4 and 6 months on tibia bone structure of male and female CD-1 mice and characterized developmental changes. At 2, 4 and 6 months of age, right tibias were scanned using in vivo µCT (Skyscan 1176) at one of three doses of radiation per scan: 222, 261 or 460 mGy. Left tibias of the same mice were scanned only at 6 months to serve as non-irradiated controls to determine whether recurrent radiation exposure alters trabecular and cortical bone structure at the proximal tibia. In males, eccentricity was lower (P<0.05) in irradiated compared with non-irradiated tibias (222 mGy group). Within each sex, all other structural outcomes were similar between irradiated and non-irradiated tibias regardless of dose. Trabecular bone loss occurred in all mice due to age while cortical development continued to age 6 months. In conclusion, repetitive µCT scans at various radiation doses did not damage trabecular or cortical bone structure of proximal tibia in male and female CD-1 mice. Moreover, scanning at 2, 4 and 6 months of age highlight the different developmental time course between trabecular and cortical bone. These scanning protocols can be used to investigate longitudinal responses of bone structures to an intervention.
RESUMEN
Structure model index (SMI) is widely used to measure rods and plates in trabecular bone. It exploits the change in surface curvature that occurs as a structure varies from spherical (SMI = 4), to cylindrical (SMI = 3) to planar (SMI = 0). The most important assumption underlying SMI is that the entire bone surface is convex and that the curvature differential is positive at all points on the surface. The intricate connections within the trabecular continuum suggest that a high proportion of the surface could be concave, violating the assumption of convexity and producing regions of negative differential. We implemented SMI in the BoneJ plugin and included the ability to measure the amounts of surface that increased or decreased in area after surface mesh dilation, and the ability to visualize concave and convex regions. We measured SMI and its positive (SMI(+)) and negative (SMI(-)) components, bone volume fraction (BV/TV), the fraction of the surface that is concave (CF), and mean ellipsoid factor (EF) in trabecular bone using 38 X-ray microtomography (XMT) images from a rat ovariectomy model of sex steroid rescue of bone loss, and 169 XMT images from a broad selection of 87 species' femora (mammals, birds, and a crocodile). We simulated bone resorption by eroding an image of elephant trabecule and recording SMI and BV/TV at each erosion step. Up to 70%, and rarely <20%, of the trabecular surface is concave (CF 0.155-0.700). SMI is unavoidably influenced by aberrations induced by SMI(-), which is strongly correlated with BV/TV and CF. The plate-to-rod transition in bone loss is an erroneous observation resulting from the close and artifactual relationship between SMI and BV/TV. SMI cannot discern between the distinctive trabecular geometries typical of mammalian and avian bone, whereas EF clearly detects birds' more plate-like trabecule. EF is free from confounding relationships with BV/TV and CF. SMI results reported in the literature should be treated with suspicion. We propose that EF should be used instead of SMI for measurements of rods and plates in trabecular bone.