RESUMO
BACKGROUND: The axolotl is a key model to study appendicular regeneration. The limb complexity resembles that of humans in structure and tissue components; however, axolotl limbs develop postembryonically. In this work, we evaluated the postembryonic development of the appendicular skeleton and its changes with aging. RESULTS: The juvenile limb skeleton is formed mostly by Sox9/Col1a2 cartilage cells. Ossification of the appendicular skeleton starts when animals reach a length of 10 cm, and cartilage cells are replaced by a primary ossification center, consisting of cortical bone and an adipocyte-filled marrow cavity. Vascularization is associated with the ossification center and the marrow cavity formation. We identified the contribution of Col1a2-descendants to bone and adipocytes. Moreover, ossification progresses with age toward the epiphyses of long bones. Axolotls are neotenic salamanders, and still ossification remains responsive to l-thyroxine, increasing the rate of bone formation. CONCLUSIONS: In axolotls, bone maturation is a continuous process that extends throughout their life. Ossification of the appendicular bones is slow and continues until the complete element is ossified. The cellular components of the appendicular skeleton change accordingly during ossification, creating a heterogenous landscape in each element. The continuous maturation of the bone is accompanied by a continuous body growth.
Assuntos
Ambystoma mexicanum , Osso e Ossos , Envelhecimento , Animais , Desenvolvimento Ósseo , OsteogêneseRESUMO
Fibrosis is a common feature of several chronic diseases and is characterized by exacerbated accumulation of ECM. An understanding of the cellular and molecular mechanisms involved in the development of this condition is crucial for designing efficient treatments for those pathologies. Connective tissue growth factor (CTGF/CCN2) is a pleiotropic protein with strong profibrotic activity. In this report, we present experimental evidence showing that ECM stimulates the synthesis of CTGF in response to lysophosphatidic acid (LPA).The integrin/focal adhesion kinase (FAK) signaling pathway mediates this effect, since CTGF expression is abolished by the use of the Arg-Gly-Asp-Ser peptide and also by an inhibitor of FAK autophosphorylation at tyrosine 397. Cilengitide, a specific inhibitor of αv integrins, inhibits the expression of CTGF mediated by LPA or transforming growth factor ß1. We show that ECM obtained from decellularized myofibroblast cultures or derived from activated fibroblasts from muscles of the Duchenne muscular dystrophy mouse model ( mdx) induces the expression of CTGF. This effect is dependent on FAK phosphorylation in response to its activation by integrin. We also found that the fibrotic ECM inhibits skeletal muscle differentiation. This novel regulatory mechanism of CTGF expression could be acting as a positive profibrotic feedback between the ECM and CTGF, revealing a novel concept in the control of fibrosis under chronic damage.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Matriz Extracelular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Quinase 1 de Adesão Focal/metabolismo , Integrina alfaV/metabolismo , Lisofosfolipídeos/farmacologia , Músculo Esquelético/efeitos dos fármacos , Distrofia Muscular de Duchenne/enzimologia , Mioblastos/efeitos dos fármacos , Animais , Linhagem Celular , Fator de Crescimento do Tecido Conjuntivo/genética , Modelos Animais de Doenças , Matriz Extracelular/enzimologia , Matriz Extracelular/patologia , Fibroblastos/enzimologia , Fibroblastos/patologia , Fibrose , Integrina alfaV/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Mioblastos/enzimologia , Mioblastos/patologia , Fosforilação , Transdução de Sinais/efeitos dos fármacosRESUMO
Fibrotic disorders are characterized by an increase in extracellular matrix protein expression and deposition, Duchene Muscular Dystrophy being one of them. Among the factors that induce fibrosis are Transforming Growth Factor type ß (TGF-ß) and the matricellular protein Connective Tissue Growth Factor (CTGF/CCN2), the latter being a target of the TGF-ß/SMAD signaling pathway and is the responsible for the profibrotic effects of TGF-ß. Both CTGF and TGF are increased in tissues affected by fibrosis but little is known about the regulation of the expression of CTGF mediated by TGF-ß in muscle cells. By using luciferase reporter assays, site directed mutagenesis and specific inhibitors in C2C12 cells; we described a novel SMAD Binding Element (SBE) located in the 5' UTR region of the CTGF gene important for the TGF-ß-mediated expression of CTGF in myoblasts. In addition, our results suggest that additional transcription factor binding sites (TFBS) present in the 5' UTR of the CTGF gene are important for this expression and that SP1/SP3 factors are involved in TGF-ß-mediated CTGF expression.