RESUMO
Scar tissue size following myocardial infarction is an independent predictor of cardiovascular outcomes, yet little is known about factors regulating scar size. We demonstrate that collagen V, a minor constituent of heart scars, regulates the size of heart scars after ischemic injury. Depletion of collagen V led to a paradoxical increase in post-infarction scar size with worsening of heart function. A systems genetics approach across 100 in-bred strains of mice demonstrated that collagen V is a critical driver of postinjury heart function. We show that collagen V deficiency alters the mechanical properties of scar tissue, and altered reciprocal feedback between matrix and cells induces expression of mechanosensitive integrins that drive fibroblast activation and increase scar size. Cilengitide, an inhibitor of specific integrins, rescues the phenotype of increased post-injury scarring in collagen-V-deficient mice. These observations demonstrate that collagen V regulates scar size in an integrin-dependent manner.
Assuntos
Cicatriz/metabolismo , Colágeno Tipo V/deficiência , Colágeno Tipo V/metabolismo , Traumatismos Cardíacos/metabolismo , Contração Miocárdica/genética , Miofibroblastos/metabolismo , Animais , Cicatriz/genética , Cicatriz/fisiopatologia , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Colágeno Tipo V/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Fibrose/genética , Fibrose/metabolismo , Regulação da Expressão Gênica/genética , Integrinas/antagonistas & inibidores , Integrinas/genética , Integrinas/metabolismo , Isoproterenol/farmacologia , Masculino , Mecanotransdução Celular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Força Atômica/instrumentação , Microscopia Eletrônica de Transmissão , Contração Miocárdica/efeitos dos fármacos , Miofibroblastos/citologia , Miofibroblastos/patologia , Miofibroblastos/ultraestrutura , Análise de Componente Principal , Proteômica , RNA-Seq , Análise de Célula ÚnicaRESUMO
The cell microenvironment, which is critical for stem cell maintenance, contains both cellular and non-cellular components, including secreted growth factors and the extracellular matrix1-3. Although Notch and other signalling pathways have previously been reported to regulate quiescence of stem cells4-9, the composition and source of molecules that maintain the stem cell niche remain largely unknown. Here we show that adult muscle satellite (stem) cells in mice produce extracellular matrix collagens to maintain quiescence in a cell-autonomous manner. Using chromatin immunoprecipitation followed by sequencing, we identified NOTCH1/RBPJ-bound regulatory elements adjacent to specific collagen genes, the expression of which is deregulated in Notch-mutant mice. Moreover, we show that Collagen V (COLV) produced by satellite cells is a critical component of the quiescent niche, as depletion of COLV by conditional deletion of the Col5a1 gene leads to anomalous cell cycle entry and gradual diminution of the stem cell pool. Notably, the interaction of COLV with satellite cells is mediated by the Calcitonin receptor, for which COLV acts as a surrogate local ligand. Systemic administration of a calcitonin derivative is sufficient to rescue the quiescence and self-renewal defects found in COLV-null satellite cells. This study reveals a Notch-COLV-Calcitonin receptor signalling cascade that maintains satellite cells in a quiescent state in a cell-autonomous fashion, and raises the possibility that similar reciprocal mechanisms act in diverse stem cell populations.
Assuntos
Proteína Semelhante a Receptor de Calcitonina/metabolismo , Colágeno/metabolismo , Músculo Esquelético/citologia , Receptores Notch/metabolismo , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Transdução de Sinais , Nicho de Células-Tronco , Animais , Diferenciação Celular , Proliferação de Células , Autorrenovação Celular , Colágeno/genética , Regulação da Expressão Gênica , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Camundongos , Transcrição GênicaRESUMO
No other tissue in the body depends more on the composition and organization of the extracellular matrix (ECM) for normal structure and function than the corneal stroma. The precise arrangement and orientation of collagen fibrils, lamellae and keratocytes that occurs during development and is needed in adults to maintain stromal function is dependent on the regulated interaction of multiple ECM components that contribute to attain the unique properties of the cornea: transparency, shape, mechanical strength, and avascularity. This review summarizes the contribution of different ECM components, their structure, regulation and function in modulating the properties of the corneal stroma. Fibril forming collagens (I, III, V), fibril associated collagens with interrupted triple helices (XII and XIV), network forming collagens (IV, VI and VIII) as well as small leucine-rich proteoglycans (SLRP) expressed in the stroma: decorin, biglycan, lumican, keratocan, and fibromodulin are some of the ECM components reviewed in this manuscript. There are spatial and temporal differences in the expression of these ECM components, as well as interactions among them that contribute to stromal function. Unique regions within the stroma like Bowman's layer and Descemet's layer are discussed. To define the complexity of corneal stroma composition and structure as well as the relationship to function is a daunting task. Our knowledge is expanding, and we expect that this review provides a comprehensive overview of current knowledge, definition of gaps and suggests future research directions.
Assuntos
Colágeno/metabolismo , Substância Própria/citologia , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Animais , Substância Própria/metabolismo , HumanosRESUMO
Purpose: Maintenance of a transparent corneal stroma is imperative for proper vision. The corneal stroma is composed of primarily collagen fibrils, small leucine-rich proteoglycans (SLRPs), as well as sparsely distributed cells called keratocytes. The lattice arrangement and spacing of the collagen fibrils that allows for transparency may be disrupted due to genetic mutations and injuries. The purpose of this study is to examine the therapeutic efficacy of human umbilical cord mesenchymal stem/stromal cells (UMSCs) in treating congenital and acquired corneal opacity associated with the loss of collagen V. Methods: Experimental mice, i.e., wild-type, Col5a1f/f and Kera-Cre/Col5a1f/f (Col5a1∆st/∆st , collagen V null in the corneal stroma) mice in a C57BL/6J genetic background, were subjected to a lamellar keratectomy, and treated with or without UMSC (104 cells/cornea) transplantation via an intrastromal injection or a fibrin plug. In vivo Heidelberg retinal tomograph (HRT II) confocal microscopy, second harmonic generated (SHG) confocal microscopy, histology, and immunofluorescence microscopy were used to assess the corneal transparency of the regenerated corneas. Results: Col5a1∆st/∆st mice display a cloudy cornea phenotype that is ameliorated following intrastromal transplantation of UMSCs. Loss of collagen V in Col5a1∆st/∆st corneas augments the formation of cornea scarring following the keratectomy. UMSC transplantation with a fibrin plug improves the healing of injured corneas and regeneration of transparent corneas, as determined with in vivo HRT II confocal microscopy. Second harmonic confocal microscopy revealed the improved collagen fibril lamellar architecture in the UMSC-transplanted cornea in comparison to the control keratectomized corneas. Conclusions: UMSC transplantation was successful in recovering some corneal transparency in injured corneas of wild-type, Col5a1f/f and Col5a1∆st/∆st mice. The production of collagen V by transplanted UMSCs may account for the regeneration of corneal transparency, as exemplified by better collagen fiber organization, as revealed with SHG signals.
Assuntos
Opacidade da Córnea/congênito , Opacidade da Córnea/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Animais , Colágeno Tipo V/metabolismo , Opacidade da Córnea/patologia , Substância Própria/patologia , Colágenos Fibrilares/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Resultado do Tratamento , Cordão Umbilical/citologiaRESUMO
Classic Ehlers-Danlos syndrome (cEDS) is characterized by fragile, hyperextensible skin and hypermobile joints. cEDS can be caused by heterozygosity for missense mutations in genes COL5A2 and COL5A1, which encode the α2(V) and α1(V) chains, respectively, of collagen V, and is most often caused by COL5A1 null alleles. However, COL5A2 null alleles have yet to be associated with cEDS or other human pathologies. We previously showed that mice homozygous null for the α2(V) gene Col5a2 are early embryonic lethal, whereas haploinsufficiency caused aberrancies of adult skin, but not a frank cEDS-like phenotype, as skin hyperextensibility at low strain and dermal cauliflower-contoured collagen fibril aggregates, two cEDS hallmarks, were absent. Herein, we show that ubiquitous postnatal Col5a2 knockdown results in pathognomonic dermal cauliflower-contoured collagen fibril aggregates, but absence of skin hyperextensibility, demonstrating these cEDS hallmarks to arise separately from loss of collagen V roles in control of collagen fibril growth and nucleation events, respectively. Col5a2 knockdown also led to loss of dermal white adipose tissue (WAT) and markedly decreased abdominal WAT that was characterized by miniadipocytes and increased collagen deposition, suggesting α2(V) to be important to WAT development/maintenance. More important, Col5a2 haploinsufficiency markedly increased the incidence and severity of abdominal aortic aneurysms, and caused aortic arch ruptures and dissections, indicating that α2(V) chain deficits may play roles in these pathologies in humans.
Assuntos
Tecido Adiposo/anormalidades , Aneurisma da Aorta Torácica/genética , Colágeno Tipo V/deficiência , Colágeno/deficiência , Predisposição Genética para Doença , Anormalidades da Pele/metabolismo , Pele/patologia , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/patologia , Animais , Aneurisma da Aorta Torácica/patologia , Colágeno/metabolismo , Colágeno Tipo V/metabolismo , Derme/patologia , Modelos Animais de Doenças , Síndrome de Ehlers-Danlos/patologia , Colágenos Fibrilares/metabolismo , Deleção de Genes , Técnicas de Silenciamento de Genes , Integrases/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reprodutibilidade dos Testes , Pele/efeitos dos fármacos , Pele/ultraestrutura , Anormalidades da Pele/patologia , Tamoxifeno/farmacologia , Cicatrização/efeitos dos fármacosRESUMO
Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause autosomal recessive cutis laxa (ARCL) 1B, characterized by loose skin, aortic aneurysm, arterial tortuosity, lung emphysema, and skeletal abnormalities. Homozygous missense mutations in FBLN4 are a prevalent cause of ARCL 1B. Here we generated a knock-in mouse strain bearing a recurrent fibulin-4 E57K homozygous missense mutation. The mutant mice survived into adulthood and displayed abnormalities in multiple organ systems, including loose skin, bent forelimb, aortic aneurysm, tortuous artery, and pulmonary emphysema. Biochemical studies of dermal fibroblasts showed that fibulin-4 E57K mutant protein was produced but was prone to dimer formation and inefficiently secreted, thereby triggering an endoplasmic reticulum stress response. Immunohistochemistry detected a low level of fibulin-4 E57K protein in the knock-in skin along with altered expression of selected elastic fiber components. Processing of a precursor to mature lysyl oxidase, an enzyme involved in cross-linking of elastin and collagen, was compromised. The knock-in skin had a reduced level of desmosine, an elastin-specific cross-link compound, and ultrastructurally abnormal elastic fibers. Surprisingly, structurally aberrant collagen fibrils and altered organization into fibers were characteristics of the knock-in dermis and forelimb tendons. Type I collagen extracted from the knock-in skin had decreased amounts of covalent intermolecular cross-links, which could contribute to the collagen fibril abnormalities. Our studies provide the first evidence that fibulin-4 plays a role in regulating collagen fibril assembly and offer a preclinical platform for developing treatments for ARCL 1B.
Assuntos
Vasos Sanguíneos/anormalidades , Osso e Ossos/anormalidades , Colágeno Tipo I/metabolismo , Cútis Laxa/patologia , Tecido Elástico/anormalidades , Proteínas da Matriz Extracelular/genética , Técnicas de Introdução de Genes , Pele/patologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Vasos Sanguíneos/patologia , Osso e Ossos/patologia , Colágeno Tipo I/ultraestrutura , Reagentes de Ligações Cruzadas/metabolismo , Cútis Laxa/metabolismo , Modelos Animais de Doenças , Tecido Elástico/patologia , Tecido Elástico/ultraestrutura , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/enzimologia , Fibroblastos/patologia , Membro Anterior/anormalidades , Membro Anterior/diagnóstico por imagem , Membro Anterior/patologia , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Biossíntese de Proteínas , Multimerização Proteica , Proteína-Lisina 6-Oxidase/metabolismo , Radiografia , Tendões/anormalidades , Tendões/patologia , Tendões/ultraestruturaRESUMO
Osteogenesis imperfecta (OI), or brittle bone disease, is most often caused by dominant mutations in the collagen I genes COL1A1/COL1A2, whereas rarer recessive OI is often caused by mutations in genes encoding collagen I-interacting proteins. Recently, mutations in the gene for the proteinase bone morphogenetic 1 (BMP1) were reported in two recessive OI families. BMP1 and the closely related proteinase mammalian tolloid-like 1 (mTLL1) are co-expressed in various tissues, including bone, and have overlapping activities that include biosynthetic processing of procollagen precursors into mature collagen monomers. However, early lethality of Bmp1- and Tll1-null mice has precluded use of such models for careful study of in vivo roles of their protein products. Here we employ novel mouse strains with floxed Bmp1 and Tll1 alleles to induce postnatal, simultaneous ablation of the two genes, thus avoiding barriers of Bmp1(-/-) and Tll1(-/-) lethality and issues of functional redundancy. Bones of the conditionally null mice are dramatically weakened and brittle, with spontaneous fractures-defining features of OI. Additional skeletal features include osteomalacia, thinned/porous cortical bone, reduced processing of procollagen and dentin matrix protein 1, remarkably high bone turnover and defective osteocyte maturation that is accompanied by decreased expression of the osteocyte marker and Wnt-signaling inhibitor sclerostin, and by marked induction of canonical Wnt signaling. The novel animal model presented here provides new opportunities for in-depth analyses of in vivo roles of BMP1-like proteinases in bone and other tissues, and for their roles, and for possible therapeutic interventions, in OI.
Assuntos
Proteína Morfogenética Óssea 1/genética , Fêmur/patologia , Técnicas de Silenciamento de Genes/métodos , Osteogênese Imperfeita/patologia , Metaloproteases Semelhantes a Toloide/genética , Animais , Proteína Morfogenética Óssea 1/metabolismo , Modelos Animais de Doenças , Fêmur/ultraestrutura , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Osteogênese Imperfeita/genética , Metaloproteases Semelhantes a Toloide/metabolismoRESUMO
Collagen VI-related myopathies are disorders of connective tissue presenting with an overlap phenotype combining clinical involvement from the muscle and from the connective tissue. Not all patients displaying related overlap phenotypes between muscle and connective tissue have mutations in collagen VI. Here, we report a homozygous recessive loss of function mutation and a de novo dominant mutation in collagen XII (COL12A1) as underlying a novel overlap syndrome involving muscle and connective tissue. Two siblings homozygous for a loss of function mutation showed widespread joint hyperlaxity combined with weakness precluding independent ambulation, while the patient with the de novo missense mutation was more mildly affected, showing improvement including the acquisition of walking. A mouse model with inactivation of the Col12a1 gene showed decreased grip strength, a delay in fiber-type transition and a deficiency in passive force generation while the muscle seems more resistant to eccentric contraction induced force drop, indicating a role for a matrix-based passive force-transducing elastic element in the generation of the weakness. This new muscle connective tissue overlap syndrome expands on the emerging importance of the muscle extracellular matrix in the pathogenesis of muscle disease.
Assuntos
Colágeno Tipo XII/genética , Doenças Musculares/genética , Mutação/genética , Animais , Pré-Escolar , Colágeno Tipo VI/genética , Colágeno Tipo VI/metabolismo , Colágeno Tipo XII/metabolismo , Modelos Animais de Doenças , Humanos , Lactente , Masculino , Camundongos , Músculo Esquelético/patologia , Doenças Musculares/metabolismo , Doenças Musculares/patologiaRESUMO
Collagen V mutations underlie classic Ehlers-Danlos syndrome, and joint hypermobility is an important clinical manifestation. We define the function of collagen V in tendons and ligaments, as well as the role of alterations in collagen V expression in the pathobiology in classic Ehlers-Danlos syndrome. A conditional Col5a1(flox/flox) mouse model was bred with Scleraxis-Cre mice to create a targeted tendon and ligament Col5a1-null mouse model, Col5a1(Δten/Δten). Targeting was specific, resulting in collagen V-null tendons and ligaments. Col5a1(Δten/Δten) mice demonstrated decreased body size, grip weakness, abnormal gait, joint laxity, and early-onset osteoarthritis. These gross changes were associated with abnormal fiber organization, as well as altered collagen fibril structure with increased fibril diameters and decreased fibril number that was more severe in a major joint stabilizing ligament, the anterior cruciate ligament (ACL), than in the flexor digitorum longus tendon. The ACL also had a higher collagen V content than did the flexor digitorum longus tendon. The collagen V-null ACL and flexor digitorum longus tendon both had significant alterations in mechanical properties, with ACL exhibiting more severe changes. The data demonstrate critical differential regulatory roles for collagen V in tendon and ligament structure and function and suggest that collagen V regulatory dysfunction is associated with an abnormal joint phenotype, similar to the hypermobility phenotype in classic Ehlers-Danlos syndrome.
Assuntos
Colágeno Tipo V/deficiência , Síndrome de Ehlers-Danlos/patologia , Síndrome de Ehlers-Danlos/fisiopatologia , Animais , Fenômenos Biomecânicos , Modelos Animais de Doenças , Marcha/fisiologia , Força da Mão/fisiologia , Immunoblotting , Imuno-Histoquímica , Articulações , Ligamentos/patologia , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Tendões/patologiaRESUMO
Null alleles for the COL5A1 gene and missense mutations for COL5A1 or the COL5A2 gene underlie cases of classic Ehlers-Danlos syndrome, characterized by fragile, hyperextensible skin and hypermobile joints. However, no classic Ehlers-Danlos syndrome case has yet been associated with COL5A2 null alleles, and phenotypes that might result from such alleles are unknown. We describe mice with null alleles for the Col5a2. Col5a2(-/-) homozygosity is embryonic lethal at approximately 12 days post conception. Unlike previously described mice null for Col5a1, which die at 10.5 days post conception and virtually lack collagen fibrils, Col5a2(-/-) embryos have readily detectable collagen fibrils, thicker than in wild-type controls. Differences in Col5a2(-/-) and Col5a1(-/-) fibril formation and embryonic survival suggest that α1(V)3 homotrimers, a rare collagen V isoform that occurs in the absence of sufficient levels of α2(V) chains, serve functional roles that partially compensate for loss of the most common collagen V isoform. Col5a2(+/-) adults have skin with marked hyperextensibility and reduced tensile strength at high strain but not at low strain. Col5a2(+/-) adults also have aortas with increased compliance and reduced tensile strength. Results thus suggest that COL5A2(+/-) humans, although unlikely to present with frank classic Ehlers-Danlos syndrome, are likely to have fragile connective tissues with increased susceptibility to trauma and certain chronic pathologic conditions.
Assuntos
Colágeno Tipo V/genética , Colágeno/genética , Síndrome de Ehlers-Danlos/genética , Adulto , Alelos , Animais , Colágeno/metabolismo , Colágeno Tipo V/metabolismo , Tecido Conjuntivo/anormalidades , Tecido Conjuntivo/patologia , Síndrome de Ehlers-Danlos/metabolismo , Síndrome de Ehlers-Danlos/patologia , Feminino , Heterozigoto , Homozigoto , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Fenótipo , Pele/patologiaRESUMO
Bone formation is precisely regulated by cell-cell communication in osteoblasts. We have previously demonstrated that genetic deletion of Col6a1 or Col12a1 impairs osteoblast connections and/or communication in mice, resulting in bone mass reduction and bone fragility. Mutations of the genes encoding collagen VI cause Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM), which have overlapping phenotypes involving connective tissue and muscle. Recent studies have identified COL12A1 gene mutations in patients with UCMD- and BM-like disorders harboring no COL6 mutations, indicating the shared functions of these collagens in connective tissue homeostasis. The purpose of this investigation has been to test the hypothesis that collagens VI and XII have coordinate regulatory role(s) during bone formation. We analyzed the localization of collagens VI and XII relative to primary osteoblasts during osteogenesis. Immunofluorescence analysis demonstrated that collagens VI and XII colocalized in matrix bridges between adjacent cells during periods when osteoblasts were establishing cell-cell connections. Quantification of cells harboring collagen bridges demonstrated that matrix bridges were composed of collagens VI and XII but not collagen I. Interestingly, matrix bridge formation was impaired in osteoblasts deficient in either Col6a1 or Col12a1, suggesting that both collagens were indispensable for matrix bridge formation. These data demonstrate, for the first time, a functional relationship between collagens VI and XII during osteogenesis and indicate that a complex containing collagens VI and XII is essential for the formation of a communicating cellular network during bone formation.
Assuntos
Comunicação Celular , Colágeno Tipo VI/metabolismo , Colágeno Tipo XII/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese , Animais , Células Cultivadas , Colágeno Tipo I/metabolismo , Meios de Cultura/farmacologia , Matriz Extracelular/metabolismo , Camundongos , Ligação ProteicaRESUMO
Fibulin-4 is an extracellular matrix glycoprotein essential for elastic fiber formation. Mice deficient in fibulin-4 die perinatally because of severe pulmonary and vascular defects associated with the lack of intact elastic fibers. Patients with fibulin-4 mutations demonstrate similar defects, and a significant number die shortly after birth or in early childhood from cardiopulmonary failure. The patients also demonstrate skeletal and other systemic connective tissue abnormalities, including joint laxity and flexion contractures of the wrist. A fibulin-4 null mouse strain was generated and used to analyze the roles of fibulin-4 in tendon fibrillogenesis. This mouse model displayed bilateral forelimb contractures, in addition to pulmonary and cardiovascular defects. The forelimb and hindlimb tendons exhibited disruption in collagen fibrillogenesis in the absence of fibulin-4 as analyzed by transmission electron microscopy. Fewer fibrils were assembled, and fibrils were disorganized compared with wild-type controls. The organization of developing tenocytes and compartmentalization of the extracellular space was also disrupted. Fibulin-4 was co-localized with fibrillin-1 and fibrillin-2 in limb tendons by using immunofluorescence microscopy. Thus, fibulin-4 seems to play a role in regulating tendon collagen fibrillogenesis, in addition to its essential function in elastogenesis.
Assuntos
Colágeno/metabolismo , Contratura/metabolismo , Contratura/patologia , Proteínas da Matriz Extracelular/deficiência , Membro Anterior/patologia , Tendões/anormalidades , Animais , Contratura/complicações , Proteínas da Matriz Extracelular/metabolismo , Fibrilinas/metabolismo , Hérnia/complicações , Hérnia/patologia , Fenótipo , Transporte Proteico , Tendões/metabolismo , Tendões/ultraestruturaRESUMO
Mutations in collagen V are associated with classic Ehlers-Danlos syndrome (EDS). A significant percentage of these mutations result in haploinsufficiency for collagen V. The purpose of this work was to determine if changes in collagen V expression are associated with altered dermal fibroblast behavior contributing to the poor wound healing response. A haploinsufficient Col5a1(+/-) mouse model of EDS was utilized. In vivo wound healing studies demonstrated that mutant mice healed significantly slower than Col5a1(+/+) mice. The basis for this difference was examined in vitro using dermal fibroblast strains isolated from Col5a1(+/-) and Col5a1(+/+) mice. Fibroblast proliferation was determined for each strain by counting cells at different time points after seeding as well as using the proliferation marker Ki-67. Fibroblast attachment to collagens I and III and fibronectin also was analyzed. In addition, in vitro scratch wounds were used to analyze fibroblast wound closure. Significantly decreased fibroblast proliferation was observed in Col5a1(+/-) compared to Col5a1(+/+) fibroblasts. Our data indicate that the decreased fibroblast number was not due to apoptosis. Wildtype Col5a1(+/+) fibroblasts attached significantly better to components of the wound matrix (collagens I and III and fibronectin) than Col5a1(+/-) fibroblasts. A significant difference in in vitro scratch wound closure rates also was observed. Col5a1(+/+) fibroblasts closed wounds in 22 h, while Col5a1(+/-) fibroblasts demonstrated ~80% closure. There were significant differences in closure at all time points analyzed. Our data suggest that decreased fibroblast proliferation, extracellular matrix attachment, and migration contribute to the decreased wound healing response in classic EDS.
Assuntos
Proliferação de Células/genética , Colágeno , Derme/metabolismo , Síndrome de Ehlers-Danlos , Fibroblastos/metabolismo , Haploinsuficiência , Animais , Apoptose/genética , Colágeno/genética , Colágeno/metabolismo , Derme/patologia , Modelos Animais de Doenças , Síndrome de Ehlers-Danlos/genética , Síndrome de Ehlers-Danlos/metabolismo , Fibroblastos/patologia , Camundongos , Camundongos Mutantes , Cicatrização/genéticaRESUMO
Dominant and recessive mutations in collagen VI genes, COL6A1, COL6A2, and COL6A3, cause a continuous spectrum of disorders characterized by muscle weakness and connective tissue abnormalities ranging from the severe Ullrich congenital muscular dystrophy to the mild Bethlem myopathy. Herein, we report the development of a mouse model for dominant collagen VI disorders by deleting exon 16 in the Col6a3 gene. The resulting heterozygous mouse, Col6a3(+/d16), produced comparable amounts of normal Col6a3 mRNA and a mutant transcript with an in-frame deletion of 54 bp of triple-helical coding sequences, thus mimicking the most common molecular defect found in dominant Ullrich congenital muscular dystrophy patients. Biosynthetic studies of mutant fibroblasts indicated that the mutant α3(VI) collagen protein was produced and exerted a dominant-negative effect on collagen VI microfibrillar assembly. The distribution of the α3(VI)-like chains of collagen VI was not altered in mutant mice during development. The Col6a3(+/d16) mice developed histopathologic signs of myopathy and showed ultrastructural alterations of mitochondria and sarcoplasmic reticulum in muscle and abnormal collagen fibrils in tendons. The Col6a3(+/d16) mice displayed compromised muscle contractile functions and thereby provide an essential preclinical platform for developing treatment strategies for dominant collagen VI disorders.
Assuntos
Colágeno Tipo VI/química , Colágeno Tipo VI/genética , Modelos Animais de Doenças , Doenças Musculares/fisiopatologia , Alelos , Animais , Éxons , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/metabolismo , Genes Dominantes , Heterozigoto , Camundongos , Camundongos Transgênicos , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Contração Muscular , Músculos/fisiopatologia , Doenças Musculares/genética , Distrofias Musculares/genética , Fenótipo , Retículo Sarcoplasmático/patologia , Deleção de Sequência , Tendões/patologiaRESUMO
The transparent cornea is the major refractive element of the eye. A finely controlled assembly of the stromal extracellular matrix is critical to corneal function, as well as in establishing the appropriate mechanical stability required to maintain corneal shape and curvature. In the stroma, homogeneous, small diameter collagen fibrils, regularly packed with a highly ordered hierarchical organization, are essential for function. This review focuses on corneal stroma assembly and the regulation of collagen fibrillogenesis. Corneal collagen fibrillogenesis involves multiple molecules interacting in sequential steps, as well as interactions between keratocytes and stroma matrix components. The stroma has the highest collagen V:I ratio in the body. Collagen V regulates the nucleation of protofibril assembly, thus controlling the number of fibrils and assembly of smaller diameter fibrils in the stroma. The corneal stroma is also enriched in small leucine-rich proteoglycans (SLRPs) that cooperate in a temporal and spatial manner to regulate linear and lateral collagen fibril growth. In addition, the fibril-associated collagens (FACITs) such as collagen XII and collagen XIV have roles in the regulation of fibril packing and inter-lamellar interactions. A communicating keratocyte network contributes to the overall and long-range regulation of stromal extracellular matrix assembly, by creating micro-domains where the sequential steps in stromal matrix assembly are controlled. Keratocytes control the synthesis of extracellular matrix components, which interact with the keratocytes dynamically to coordinate the regulatory steps into a cohesive process. Mutations or deficiencies in stromal regulatory molecules result in altered interactions and deficiencies in both transparency and refraction, leading to corneal stroma pathobiology such as stromal dystrophies, cornea plana and keratoconus.
Assuntos
Substância Própria/citologia , Substância Própria/metabolismo , Matriz Extracelular/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Colágeno/metabolismo , HumanosRESUMO
Collagen VI is a ubiquitously expressed extracellular microfibrillar protein. Its most common molecular form is composed of the α1(VI), α2(VI), and α3(VI) collagen α chains encoded by the COL6A1, COL6A2, and COL6A3 genes, respectively. Mutations in any of the three collagen VI genes cause congenital muscular dystrophy types Bethlem and Ullrich as well as intermediate phenotypes characterized by muscle weakness and connective tissue abnormalities. The α3(VI) collagen α chain has much larger N- and C-globular domains than the other two chains. Its most C-terminal domain can be cleaved off after assembly into microfibrils, and the cleavage product has been implicated in tumor angiogenesis and progression. Here we characterize a Col6a3 mutant mouse that expresses a very low level of a non-functional α3(VI) collagen chain. The mutant mice are deficient in extracellular collagen VI microfibrils and exhibit myopathic features, including decreased muscle mass and contractile force. Ultrastructurally abnormal collagen fibrils were observed in tendon, but not cornea, of the mutant mice, indicating a distinct tissue-specific effect of collagen VI on collagen I fibrillogenesis. Overall, the mice lacking normal α3(VI) collagen chains displayed mild musculoskeletal phenotypes similar to mice deficient in the α1(VI) collagen α chain, suggesting that the cleavage product of the α3(VI) collagen does not elicit essential functions in normal growth and development. The Col6a3 mouse mutant lacking functional α3(VI) collagen chains thus serves as an animal model for COL6A3-related muscular dystrophy.
Assuntos
Colágeno Tipo VI/deficiência , Colágeno Tipo VI/genética , Músculo Esquelético/metabolismo , Tendões/metabolismo , Animais , Colágeno Tipo VI/fisiologia , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Genótipo , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microfibrilas/metabolismo , Músculo Esquelético/fisiopatologia , Mutação , Fenótipo , Tendões/fisiopatologiaRESUMO
Decorin, a small leucine-rich proteoglycan (SLRP), is involved in the pathophysiology of human congenital stromal corneal dystrophy (CSCD). This disease is characterized by corneal opacities and vision impairment. In reported cases, the human gene encoding decorin contains point mutations in exon 10, generating a truncated form of decorin lacking the C-terminal 33 amino acid residues. We have previously described a transgenic mouse model carrying a similar mutation in the decorin gene that leads to an ocular phenotype characterized by corneal opacities identical to CSCD in humans. We have also identified abnormal synthesis and secretion of various SLRPs in mutant mouse corneas. In the present study, we found that mutant C-terminal truncated decorin was retained in the cytoplasm of mouse keratocytes in vivo and of transfected human embryonic kidney cells. This resulted in endoplasmic reticulum stress and an unfolded protein response. Thus, we propose a novel cell-based mechanism underlying CSCD in which a truncated SLRP protein core is retained intracellularly, its accumulation triggering endoplasmic reticulum stress that results in abnormal SLRP synthesis and secretion, which ultimately affects stromal structure and corneal transparency.
Assuntos
Distrofias Hereditárias da Córnea/genética , Decorina/genética , Retículo Endoplasmático/fisiologia , Mutação Puntual , Animais , Distrofias Hereditárias da Córnea/metabolismo , Decorina/metabolismo , Imunofluorescência , Marcadores Genéticos , Células HEK293 , Humanos , Immunoblotting , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase , Transporte Proteico , Estresse Fisiológico , Sequências Repetidas TerminaisRESUMO
Tendons and ligaments are connective tissues that guide motion, share loads, and transmit forces in a manner that is unique to each as well as the anatomical site and biomechanical stresses to which they are subjected. Collagens are the major molecular components of both tendons and ligaments. The hierarchical structure of tendon and its functional properties are determined by the collagens present, as well as their supramolecular organization. There are 28 different types of collagen that assemble into a variety of supramolecular structures. The assembly of specific supramolecular structures is dependent on the interaction with other matrix molecules as well as the cellular elements. Multiple suprastructural assemblies are integrated to form the functional tendon/ligament. This chapter begins with a discussion of collagen molecules. This is followed by a definition of the supramolecular structures assembled by different collagen types. The general principles involved in the assembly of collagen-containing suprastructures are presented focusing on the regulation of tendon collagen fibrillogenesis. Finally, site-specific differences are discussed. While generalizations can be made, differences exist between different tendons as well as between tendons and ligaments. Compositional differences will impact structure that in turn will determine functional differences. Elucidation of the unique physiology and pathophysiology of different tendons and ligaments will require an appreciation of the role compositional differences have on collagen suprastructural assembly, tissue organization, and function.
Assuntos
Colágeno/metabolismo , Colágeno/ultraestrutura , Ligamentos/metabolismo , Ligamentos/ultraestrutura , Tendões/metabolismo , Tendões/ultraestrutura , Fenômenos Biomecânicos , Colágeno/química , Colágeno/classificação , Humanos , Ligamentos/fisiopatologia , Especificidade de Órgãos , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Estresse Mecânico , Tendões/fisiopatologiaRESUMO
Mutant mouse models are valuable resources for the study of tendon and ligament biology. Many mutant mouse models are used because their manifested phenotypes mimic clinical pathobiology for several heritable disorders, such as Ehlers-Danlos Syndrome and Osteogenesis Imperfecta. Moreover, these models are helpful for discerning roles of specific genes in the development, maturation, and repair of musculoskeletal tissues. There are several categories of genes with essential roles in the synthesis and maintenance of tendon and ligament structures. The form and function of these tissues depend highly upon fibril-forming collagens, the primary extracellular macromolecules of tendons and ligaments. Models for these fibril-forming collagens, as well as for regulatory molecules like FACITs and SLRPs, are important for studying fibril assembly, growth, and maturation. Additionally, mouse models for growth factors and transcription factors are useful for defining regulation of cell proliferation, cell differentiation, and cues that stimulate matrix synthesis. Models for membrane-bound proteins assess the roles of cell-cell communication and cell-matrix interaction. In some cases, special considerations need to be given to spatio-temporal control of a gene in a model. Thus, conditional and inducible mouse models allow for specific regulation of genes of interest. Advances in mouse models have provided valuable tools for gaining insight into the form and function of tendons and ligaments.
Assuntos
Colágeno/genética , Síndrome de Ehlers-Danlos/genética , Colágenos Associados a Fibrilas/genética , Ligamentos/metabolismo , Osteogênese Imperfeita/genética , Tendões/metabolismo , Animais , Modelos Animais de Doenças , Síndrome de Ehlers-Danlos/metabolismo , Síndrome de Ehlers-Danlos/patologia , Fibrilinas , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Ligamentos/patologia , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Osteogênese Imperfeita/metabolismo , Osteogênese Imperfeita/patologia , Isoformas de Proteínas/genética , Tendões/patologia , Fatores de Transcrição/genéticaRESUMO
Collagen V (Col5) is a quantitatively minor component of collagen fibrils comprising tendon, however, plays a crucial role in regulation of development and dynamic healing processes. Clinically, patients with COL5a1 haploinsufficiency, known as classic Ehlers-Danlos Syndrome (cEDS), present with hyperextensible skin, joint instability and laxity, with females more likely to be affected. Previous studies in Col5-deficient mice indicated that reduced Col5a1 expression leads to a reduction in stiffness, fibril deposition, and altered fibril structure. Additionally, Col5-deficient male tendons demonstrated altered healing compared to wild-type tendons, however female mice have not yet been studied utilizing this model. Along with clinical differences between sexes in cEDS patient populations, differences in hormone physiology may be a factor influencing tendon health. Therefore, the objective of this study was to utilize a Col5a1+/ - female mouse model, to determine the effect of Col5 on tendon cell morphology, cell density, tissue composition, and mechanical properties throughout healing. We hypothesized that reduction in Col5 expression would result in an abnormal wound matrix post-injury, resulting in reduced mechanical properties compared to normal tendons. Following patellar tendon surgery, mice were euthanized at 1, 3, and 6-week post-injury. Col5-deficient tendons demonstrated altered and decreased healing compared to WT tendons. The lack of resolution in cellularity by 6-week post-injury in Col5-deficient tendons influenced the decreased mechanical properties. Stiffness did not increase post-injury in Col5-deficient mice, and collagen fiber realignment was delayed during mechanical loading. Therefore, increased Col5a1 expression post-injury is necessary to re-establish matrix engagement and cellularity throughout tendon healing.