RESUMEN
Liver fibrosis is characterized by the accumulation of extracellular matrix proteins, mainly composed of collagen. Hepatic stellate cells (HSCs) mediate liver fibrosis by secreting collagen. Vitamin C (ascorbic acid) is a cofactor of prolyl-hydroxylases that modify newly synthesized collagen on the route for secretion. Unlike most animals, humans cannot synthesize ascorbic acid and its role in liver fibrosis remains unclear. Here, we determined the effect of ascorbic acid and prolyl-hydroxylase inhibition on collagen production and secretion by human HSCs. Primary human HSCs (p-hHSCs) and the human HSCscell line LX-2 were treated with ascorbic acid, transforming growth factor-beta (TGFß) and/or the pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG). Expression of collagen-I was analyzed by RT-qPCR (COL1A1), Western blotting, and immunofluorescence microscopy. Collagen secretion was determined in the medium by Western blotting for collagen-I and by HPLC for hydroxyproline concentrations. Expression of solute carrier family 23 members 1 and 2 (SLC23A1/SLC23A2), encoding sodium-dependent vitamin C transporters 1 and 2 (SVCT1/SVCT2) was quantified in healthy and cirrhotic human tissue. In the absence of ascorbic acid, collagen-I accumulated intracellularly in p-hHSCs and LX-2 cells, which was potentiated by TGFß. Ascorbic acid co-treatment strongly promoted collagen-I excretion and enhanced extracellular hydroxyproline concentrations, without affecting collagen-I (COL1A1) mRNA levels. DMOG inhibited collagen-I release even in the presence of ascorbic acid and suppressed COL1A1 and alpha-smooth muscle actin (αSMA/ACTA2) mRNA levels, also under hypoxic conditions. Hepatocytes express both ascorbic acid transporters, while p-hHSCs and LX-2 express the only SVCT2, which is selectively enhanced in cirrhotic livers. Human HSCs rely on ascorbic acid for the efficient secretion of collagen-I, which can be effectively blocked by hydroxylase antagonists, revealing new therapeutic targets to treat liver fibrosis.
Asunto(s)
Aminoácidos Dicarboxílicos/farmacología , Ácido Ascórbico/metabolismo , Colágeno Tipo I/metabolismo , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/metabolismo , Inhibidores de Prolil-Hidroxilasa/farmacología , Actinas/genética , Actinas/metabolismo , Animales , Ácido Ascórbico/farmacología , Línea Celular , Células Cultivadas , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , Células Estrelladas Hepáticas/efectos de los fármacos , Humanos , Ratas , Transportadores de Sodio Acoplados a la Vitamina C/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Poor translation from animal studies to human clinical trials is one of the main hurdles in the development of new drugs. Here, we used precision-cut kidney slices (PCKS) as a translational model to study renal fibrosis and to investigate whether inhibition of tyrosine kinase receptors, with the selective inhibitor nintedanib, can halt fibrosis in murine and human PCKS. We used renal tissue of murine and human origins to obtain PCKS. Control slices and slices treated with nintedanib were studied to assess viability, activation of tyrosine kinase receptors, cell proliferation, collagen type I accumulation, and gene and protein regulation. During culture, PCKS spontaneously develop a fibrotic response that resembles in vivo fibrogenesis. Nintedanib blocked culture-induced phosphorylation of platelet-derived growth factor receptor and vascular endothelial growth factor receptor. Furthermore, nintedanib inhibited cell proliferation and reduced collagen type I accumulation and expression of fibrosis-related genes in healthy murine and human PCKS. Modulation of extracellular matrix homeostasis was achieved already at 0.1 µM, whereas high concentrations (1 and 5 µM) elicited possible nonselective effects. In PCKS from human diseased renal tissue, nintedanib showed limited capacity to reverse established fibrosis. In conclusion, nintedanib attenuated the onset of fibrosis in both murine and human PCKS by inhibiting the phosphorylation of tyrosine kinase receptors; however, the reversal of established fibrosis was not achieved.
Asunto(s)
Fibrosis/tratamiento farmacológico , Indoles/farmacología , Enfermedades Renales/tratamiento farmacológico , Riñón/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Progresión de la Enfermedad , Fibrosis/patología , Humanos , Indoles/uso terapéutico , Riñón/patología , Enfermedades Renales/patología , Ratones , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Transducción de Señal/efectos de los fármacosRESUMEN
Peyronie's disease (PD) is a fibroproliferative disease of the penis. Since little is known about the molecular pathogenesis of PD, we compared the biochemical make-up of PD plaques with normal tunica albuginea to clarify pathological processes in the scarred tissue. Protein and mRNA levels were measured in plaques and in unaffected pieces of the tunica albuginea. We investigated the presence of myofibroblasts, the deposition of collagens, and some key elements of Wnt and YAP1 signaling at protein level. The expression of 45 genes, all related to collagen homeostasis and extracellular matrix proteins, was quantified. In plaques, more myofibroblasts were present, and we observed an activation of Wnt signaling and YAP1 signaling. Increased levels of the collagens types I and III confirm the fibrotic nature of plaques. The mRNA ratio of collagen types III, IV, and VI to type I was increased. The expression of lysyl hydroxylase 3 was higher, whereas a decreased expression level was seen for fibronectin and cathepsin K. The biochemical composition of plaques was different from unaffected tunica albuginea: the relative and absolute abundance of various extracellular matrix proteins were changed, as well as the quality of collagen and the level of the collagen-degrading enzyme cathepsin K.
Asunto(s)
Induración Peniana , Colágeno , Humanos , Masculino , PeneRESUMEN
Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained transcriptional modulation in several in vivo models, further studies are needed to develop this approach into a straightforward technology for effective and specific interventions. Important goals of current research efforts are understanding the context-dependency of successful epigenetic editing and finding the most effective epigenetic effector(s) for specific tasks. Here we tested whether the fibrosis- and cancer-associated PLOD2 gene can be repressed by the DNA methyltransferase M.SssI, or by the non-catalytic Krüppel associated box (KRAB) repressor directed to the PLOD2 promoter via zinc finger- or CRISPR-dCas9-mediated targeting. M.SssI fusions induced de novo DNA methylation, changed histone modifications in a context-dependent manner, and led to 50%-70% reduction in PLOD2 expression in fibrotic fibroblasts and in MDA-MB-231 cancer cells. Targeting KRAB to PLOD2 resulted in the deposition of repressive histone modifications without DNA methylation and in almost complete PLOD2 silencing. Interestingly, both long-term TGFß1-induced, as well as unstimulated PLOD2 expression, was completely repressed by KRAB, while M.SssI only prevented the TGFß1-induced PLOD2 expression. Targeting transiently expressed dCas9-KRAB resulted in sustained PLOD2 repression in HEK293T and MCF-7 cells. Together, these findings point to KRAB outperforming DNA methylation as a small potent targeting epigenetic effector for silencing TGFß1-induced and uninduced PLOD2 expression.
Asunto(s)
Silenciador del Gen , Heterocromatina/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Adulto , Células Cultivadas , ADN-Citosina Metilasas/genética , ADN-Citosina Metilasas/metabolismo , Epigénesis Genética , Células HEK293 , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Células MCF-7 , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Regiones Promotoras Genéticas , Activación Transcripcional , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Collagen is a macromolecule that has versatile roles in physiology, ranging from structural support to mediating cell signaling. Formation of mature collagen fibrils out of procollagen α-chains requires a variety of enzymes and chaperones in a complex process spanning both intracellular and extracellular post-translational modifications. These processes include modifications of amino acids, folding of procollagen α-chains into a triple-helical configuration and subsequent stabilization, facilitation of transportation out of the cell, cleavage of propeptides, aggregation, cross-link formation, and finally the formation of mature fibrils. Disruption of any of the proteins involved in these biosynthesis steps potentially result in a variety of connective tissue diseases because of a destabilized extracellular matrix. In this review, we give a revised overview of the enzymes and chaperones currently known to be relevant to the conversion of lysine and proline into hydroxyproline and hydroxylysine, respectively, and the O-glycosylation of hydroxylysine and give insights into the consequences when these steps are disrupted.
Asunto(s)
Colágenos Fibrilares/metabolismo , Animales , Artrogriposis/metabolismo , Artrogriposis/patología , Enfermedades del Tejido Conjuntivo/metabolismo , Enfermedades del Tejido Conjuntivo/patología , Síndrome de Ehlers-Danlos/metabolismo , Síndrome de Ehlers-Danlos/patología , Colágenos Fibrilares/análisis , Glicosilación , Humanos , Hidroxilación , Hidroxilisina/análisis , Hidroxilisina/metabolismo , Hidroxiprolina/análisis , Hidroxiprolina/metabolismo , Lisina/análisis , Lisina/metabolismo , Osteogénesis Imperfecta/metabolismo , Osteogénesis Imperfecta/patología , Prolina/análisis , Prolina/metabolismo , Pliegue de ProteínaRESUMEN
Collagens are subjected to extensive posttranslational modifications, such as lysine hydroxylation. Bruck syndrome (BS) is a connective tissue disorder characterized at the molecular level by a loss of telopeptide lysine hydroxylation, resulting in reduced collagen pyridinoline cross-linking. BS results from mutations in the genes coding for lysyl hydroxylase (LH) 2 or peptidyl-prolyl cis-trans isomerase (PPIase) FKBP65. Given that the immunophilin FKBP65 does not exhibit LH activity, it is likely that LH2 activity is somehow dependent on FKPB65. In this report, we provide insights regarding the interplay between LH2 and FKBP65. We found that FKBP65 forms complexes with LH2 splice variants LH2A and LH2B but not with LH1 and LH3. Ablating the catalytic activity of FKBP65 or LH2 did not affect complex formation. Both depletion of FKBP65 and inhibition of FKBP65 PPIase activity reduced the dimeric (active) form of LH2 but did not affect the binding of monomeric (inactive) LH2 to procollagen Iα1. Furthermore, we show that LH2A and LH2B cannot form heterodimers with each other but are able to form heterodimers with LH1 and LH3. Collectively, our results indicate that FKBP65 is linked to pyridinoline cross-linking by specifically mediating the dimerization of LH2. Moreover, FKBP65 does not interact with LH1 and LH3, explaining why in BS triple-helical hydroxylysines are not affected. Our results provide a mechanistic link between FKBP65 and the loss of pyridinolines and may hold the key to future treatments for diseases related to collagen cross-linking anomalies, such as fibrosis and cancer.
Asunto(s)
Aminoácidos/química , Artrogriposis/metabolismo , Colágeno Tipo I/química , Colágeno/química , Reactivos de Enlaces Cruzados/química , Osteogénesis Imperfecta/metabolismo , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo , Aminoácidos/metabolismo , Artrogriposis/enzimología , Artrogriposis/genética , Colágeno/genética , Colágeno/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Dimerización , Humanos , Osteogénesis Imperfecta/enzimología , Osteogénesis Imperfecta/genética , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas de Unión a Tacrolimus/genéticaRESUMEN
Mechanosensing of fibroblasts plays a key role in the development of fibrosis. So far, no effective treatments are available to treat this devastating disorder. Spectrins regulate cell morphology and are potential mechanosensors in a variety of non-erythroid cells, but little is known about the role of spectrins in fibroblasts. We investigate whether αII- and ßII-spectrin are required for the phenotypic properties of adult human dermal (myo)fibroblasts. Knockdown of αII- or ßII-spectrin in fibroblasts did not affect cell adhesion, cell size and YAP nuclear/cytosolic localization. We further investigated whether αII- and ßII-spectrin play a role in the phenotypical switch from fibroblasts to myofibroblasts under the influence of the pro-fibrotic cytokine TGFß1. Knockdown of spectrins did not affect myofibroblast formation, nor did we observe changes in the organization of αSMA stress fibers. Focal adhesion assembly was unaffected by spectrin deficiency, as was collagen type I mRNA expression and protein deposition. Wound closure was unaffected as well, showing that important functional properties of myofibroblasts are unchanged without αII- or ßII-spectrin. In fact, fibroblasts stimulated with TGFß1 demonstrated significantly lower endogenous mRNA levels of αII- and ßII-spectrin. Taken together, despite the diverse roles of spectrins in a variety of other cells, αII- and ßII-spectrin do not regulate cell adhesion, cell size and YAP localization in human dermal fibroblasts and are not required for the dermal myofibroblast phenotypical switch.
Asunto(s)
Miofibroblastos/metabolismo , Espectrina/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Cultivadas , Colágeno Tipo I/metabolismo , Humanos , Miofibroblastos/citología , Miofibroblastos/efectos de los fármacos , Fosfoproteínas/metabolismo , Piel/citología , Piel/efectos de los fármacos , Piel/metabolismo , Factores de Transcripción , Cicatrización de Heridas/efectos de los fármacos , Proteínas Señalizadoras YAPRESUMEN
PURPOSE: To explore factors associated with pentosidine accumulation in the human vitreous. METHODS: Vitreous samples were obtained during trans pars plana vitrectomy for macular hole or rhegmatogenous retinal detachment. Patient characteristics included age, gender, and diabetes mellitus. Ocular characteristics included pseudophakia, posterior vitreous detachment, and presence of intraocular fibrosis (epiretinal membrane, proliferative vitreoretinopathy, or both). Pentosidine concentration as a measure of accumulation of advanced glycation end products was determined by high performance liquid chromatography. RESULTS: Pentosidine concentrations were measured in 222 vitrectomy samples (118 female and 104 male patients [median age 66 years], treated for macular hole [n = 105] or rhegmatogenous retinal detachment [n = 117]). Pentosidine was found to accumulate significantly with age (P < 0.001). After correction for age, a multivariable linear regression model revealed significantly higher pentosidine values in eyes with intraocular fibrosis (P = 0.001), in phakic as compared with pseudophakic eyes (P = 0.02), and in the absence of a complete posterior vitreous detachment (P = 0.018). The authors found no association with diabetes mellitus or gender. CONCLUSION: This study confirmed an age-related pentosidine accumulation in the vitreous and found new factors relating to pentosidine levels. Findings support the hypothesis of enzyme-induced vitreous liquefaction and the hypothesis of pentosidine as a pro-fibrotic factor.
Asunto(s)
Envejecimiento/metabolismo , Arginina/análogos & derivados , Lisina/análogos & derivados , Cuerpo Vítreo/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Arginina/metabolismo , Femenino , Humanos , Lisina/metabolismo , Masculino , Persona de Mediana Edad , Seudofaquia/metabolismo , Análisis de Regresión , Factores Sexuales , Desprendimiento del Vítreo/metabolismo , Adulto JovenRESUMEN
Implantation of biomaterials into the body, e.g. for tissue engineering purposes, induces a material-dependent inflammatory response called the foreign body reaction (FBR). A hallmark feature of this response is the formation of large multinucleated cells: foreign body giant cells (FBGCs). Biomaterials like cross-linked and non-cross-linked collagen often induce the formation of FBGCs. It is unknown whether different biomaterials result in the formation of different FBGCs. To investigate this, we implanted cross-linked and non-cross-linked dermal sheep collagen subcutaneously in mice. After 21 days the implanted material was collected and prepared for ultrastructural analysis. More FBGCs formed on and between implants of cross-linked collagen compared to non-cross-linked material. The ultrastructural aspects of the FBGCs present on the two types of implants proved to be similar. On both materials, they formed long slender protrusions on the basolateral membrane, they were very rich in mitochondria, contained numerous nuclei, and showed signs of the presence of a clear zone facing the implanted material. Similar clear zones, that resemble osteoclastic features, were also seen in FBGCs generated in vitro on bone slices, but these cells did not form a ruffled border. However, similarities in ultrastructure such as the occurrence of slender protrusions and high mitochondrion content were also found in the FBGCs generated in vitro. These data indicate that FBGCs formed on different substrates share many morphological characteristics. The formation of long finger-like protrusions seemed typical for the FBGCs, in vivo as well as in vitro, however the function of these structures needs further analysis.
Asunto(s)
Materiales Biocompatibles/farmacología , Células Gigantes de Cuerpo Extraño/ultraestructura , Implantes Experimentales , Animales , Adhesión Celular , Forma de la Célula , Reacción a Cuerpo Extraño , Células Gigantes de Cuerpo Extraño/citología , Ratones , Mitocondrias , Osteoclastos , OvinosRESUMEN
PLOD2 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2) hydroxylates lysine residues in collagen telopeptides and is essential for collagen pyridinoline cross-link formation. PLOD2 expression and subsequent pyridinoline cross-links are increased in fibrotic pathologies by transforming growth factor ß-1 (TGFß1). In this report we examined the molecular processes underlying TGFß1-induced PLOD2 expression. We found that binding of the TGFß1 pathway related transcription factors SMAD3 and SP1-mediated TGFß1 enhanced PLOD2 expression and could be correlated to an increase of acetylated histone H3 and H4 at the PLOD2 promoter. Interestingly, the classical co-activators of SMAD3 complexes, p300 and CBP, were not responsible for the enhanced H3 and H4 acetylation. Depletion of SMAD3 reduced PLOD2 acetylated H3 and H4, indicating that another as of yet unidentified histone acetyltransferase binds to SMAD3 at PLOD2. Assessing histone methylation marks at the PLOD2 promoter depicted an increase of the active histone mark H3K79me2, a decrease of the repressive H4K20me3 mark, but no role for the generally strong transcription-related modifications: H3K4me3, H3K9me3 and H3K27me3. Collectively, our findings reveal that TGFß1 induces a SP1- and SMAD3-dependent recruitment of histone modifying enzymes to the PLOD2 promoter other than the currently known TGFß1 downstream co-activators and epigenetic modifications. This also suggests that additional activation strategies are used downstream of the TGFß1 pathway, and hence their unraveling could be of great importance to fully understand TGFß1 activation of genes.
Asunto(s)
Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Acetilación , Células Cultivadas , Histonas/metabolismo , Humanos , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteína smad3/metabolismo , Factor de Transcripción Sp1/metabolismoRESUMEN
Dupuytren disease is a fibrotic disorder characterized by contraction of myofibroblast-rich cords and nodules in the hands. The Hippo member Yes-associated protein 1 (YAP1) is activated by tissue stiffness and the profibrotic transforming growth factor-ß1, but its role in cell fibrogenesis is yet unclear. We hypothesized that YAP1 regulates the differentiation of dermal fibroblasts into highly contractile myofibroblasts and that YAP1 governs the maintenance of a myofibroblast phenotype in primary Dupuytren cells. Knockdown of YAP1 in transforming growth factor-ß1-stimulated dermal fibroblasts decreased the formation of contractile smooth muscle α-actin stress fibers and the deposition of collagen type I, which are hallmark features of myofibroblasts. Translating our findings to a clinically relevant model, we found that YAP1 deficiency in Dupuytren disease myofibroblasts resulted in decreased expression of ACTA2, COL1A1, and CCN2 mRNA, but this did not result in decreased protein levels. YAP1-deficient Dupuytren myofibroblasts showed decreased contraction of a collagen hydrogel. Finally, we showed that YAP1 levels and nuclear localization were elevated in affected Dupuytren disease tissue compared with matched control tissue and partly co-localized with smooth muscle α-actin-positive cells. In conclusion, our data show that YAP1 is a regulator of myofibroblast differentiation and contributes to the maintenance of a synthetic and contractile phenotype, in both transforming growth factor-ß1-induced myofibroblast differentiation and primary Dupuytren myofibroblasts.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Contractura de Dupuytren/patología , Miofibroblastos/patología , Fosfoproteínas/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Colágeno/metabolismo , Contractura de Dupuytren/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Miofibroblastos/efectos de los fármacos , Fosfoproteínas/genética , Factores de Transcripción , Factor de Crecimiento Transformador beta1/farmacología , Proteínas Señalizadoras YAPRESUMEN
Fibrosis is a chronic disorder affecting many organs. A universal process in fibrosis is the formation of myofibroblasts and the subsequent collagen deposition by these cells. Transforming growth factor beta1 (TGFß1) plays a major role in the formation of myofibroblasts, e.g. by activating fibroblasts. Currently, no treatments are available to circumvent fibrosis. Caffeic acid phenethyl ester (CAPE) shows a broad spectrum of biological activities, including anti-fibrotic properties in vivo in mice and rats. However, little is known about the direct effects of CAPE on fibroblasts. We have tested whether CAPE is able to suppress myofibroblast formation and collagen formation of human dermal and lung fibroblasts exposed to TGFß1, and found that this was indeed the case. In fact, the formation of myofibroblasts by TGFß1 and subsequent collagen formation was completely abolished by CAPE. The same was observed for fibronectin and tenascin C. The lack of myofibroblast formation is likely due to the suppression of GLI1 and GLI2 expression by CAPE because of diminished nuclear SMAD2/3 levels. Post-treatment with CAPE after myofibroblast formation even resulted in a partial reversal of myofibroblasts into fibroblasts and/or reduction in collagen formation. Major discrepancies were seen between mRNA levels of collagen type I and cells stained positive for collagen, underlining the need for protein data in fibrosis studies to make reliable conclusions.
Asunto(s)
Ácidos Cafeicos/farmacología , Colágeno Tipo I/biosíntesis , Fibroblastos/patología , Miofibroblastos/patología , Alcohol Feniletílico/análogos & derivados , Factor de Crecimiento Transformador beta1/farmacología , Actinas/metabolismo , Adulto , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Colágeno Tipo I/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Fibrosis , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miofibroblastos/efectos de los fármacos , Miofibroblastos/metabolismo , Alcohol Feniletílico/farmacología , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Smad/metabolismo , Tenascina/genética , Tenascina/metabolismo , Factores de Transcripción/metabolismo , Adulto JovenRESUMEN
Excessive accumulation of a collagen-rich extracellular matrix (ECM) by myofibroblasts is a characteristic feature of fibrosis, a pathological state leading to serious organ dysfunction. Transforming growth factor beta1 (TGFß1) is a strong inducer of myofibroblast formation and subsequent collagen production. Currently, there are no remedies for the treatment of fibrosis. Activation of the nuclear factor kappa B (NF-κB) pathway by phosphorylating IκB with the enzyme IκB kinase (IKK) plays a major role in the induction of fibrosis. ACHP {2-Amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-(4-piperidinyl)-3 pyridinecarbonitrile}, a selective inhibitor of IKK, prohibits the activation of the NF-κB pathway. It is not known whether ACHP has potential anti-fibrotic properties. Using adult human dermal and lung fibroblasts we have investigated whether ACHP has the ability to inhibit the TGFß1-induced transition of fibroblasts into myofibroblasts and its excessive synthesis of ECM. The presence of ACHP strongly suppressed the induction of the myofibroblast markers alpha-smooth muscle actin (αSMA) and SM22α, as well as the deposition of the ECM components collagen type I and fibronectin. Furthermore, post-treatment with ACHP partly reversed the expression of αSMA and collagen type I production. Finally, ACHP suppressed the expression of the three collagen-modifying enzymes lysyl hydroxylase (PLOD1, PLOD2 and PLOD3) in dermal fibroblasts, but did not do so in lung fibroblasts. We conclude that the IKK inhibitor ACHP has potent antifibrotic properties, and that the NF-κB pathway plays an important role in myofibroblast biology.
Asunto(s)
Colágeno Tipo I/biosíntesis , Quinasa I-kappa B/antagonistas & inhibidores , Miofibroblastos/efectos de los fármacos , Ácidos Nicotínicos/farmacología , Nitrilos/farmacología , Factor de Crecimiento Transformador beta1/farmacología , Actinas/genética , Actinas/metabolismo , Adulto , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Colágeno Tipo I/genética , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Quinasa I-kappa B/metabolismo , Pulmón/citología , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Microscopía Confocal , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miofibroblastos/citología , Miofibroblastos/metabolismo , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/citología , Proteínas Smad/metabolismo , Factor de Transcripción ReIA/metabolismoRESUMEN
Although biallelic mutations in non-collagen genes account for <10% of individuals with osteogenesis imperfecta, the characterization of these genes has identified new pathways and potential interventions that could benefit even those with mutations in type I collagen genes. We identified mutations in FKBP10, which encodes the 65 kDa prolyl cis-trans isomerase, FKBP65, in 38 members of 21 families with OI. These include 10 families from the Samoan Islands who share a founder mutation. Of the mutations, three are missense; the remainder either introduce premature termination codons or create frameshifts both of which result in mRNA instability. In four families missense mutations result in loss of most of the protein. The clinical effects of these mutations are short stature, a high incidence of joint contractures at birth and progressive scoliosis and fractures, but there is remarkable variability in phenotype even within families. The loss of the activity of FKBP65 has several effects: type I procollagen secretion is slightly delayed, the stabilization of the intact trimer is incomplete and there is diminished hydroxylation of the telopeptide lysyl residues involved in intermolecular cross-link formation in bone. The phenotype overlaps with that seen with mutations in PLOD2 (Bruck syndrome II), which encodes LH2, the enzyme that hydroxylates the telopeptide lysyl residues. These findings define a set of genes, FKBP10, PLOD2 and SERPINH1, that act during procollagen maturation to contribute to molecular stability and post-translational modification of type I procollagen, without which bone mass and quality are abnormal and fractures and contractures result.
Asunto(s)
Artrogriposis/genética , Colágeno Tipo I/metabolismo , Genes Recesivos , Lisina/metabolismo , Mutación , Osteogénesis Imperfecta/genética , Proteínas de Unión a Tacrolimus/genética , Femenino , Humanos , Hidroxilación , Masculino , Procesamiento Proteico-PostraduccionalAsunto(s)
Materiales Biocompatibles , Fibrosis , Animales , Reacción a Cuerpo Extraño/inmunología , Primates , RoedoresRESUMEN
PURPOSE: The increasing prevalence and treatment costs of kidney diseases call for innovative therapeutic strategies that prevent disease progression at an early stage. We studied a novel method of subcapsular injection of monodisperse microspheres, to use as a local delivery system of drugs to the kidney. METHODS: We generated placebo- and rapamycin monodisperse microspheres to investigate subcapsular delivery of drugs. Using a rat model of acute kidney injury, subcapsular injection of placebo and rapamycin monodisperse microspheres (monospheres) was compared to subcutaneous injection, mimicking systemic administration. RESULTS: We did not find any adverse effects related to the delivery method. Irrespective of the injection site, a similar low dose of rapamycin was present in the circulation. However, only local intrarenal delivery of rapamycin from monospheres led to decreased macrophage infiltration and a significantly lower amount of myofibroblasts in the kidney, where systemic administration did not. Local delivery of rapamycin did cause a transient increase in the deposition of collagen I, but not of collagen III. CONCLUSIONS: We conclude that therapeutic effects can be increased when rapamycin is delivered subcapsularly by monospheres, which, combined with low systemic concentrations, may lead to an effective intrarenal delivery method.
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Enfermedades Renales/tratamiento farmacológico , Daño por Reperfusión/tratamiento farmacológico , Sirolimus/farmacología , Animales , Sistemas de Liberación de Medicamentos/métodos , Riñón/efectos de los fármacos , Masculino , Microesferas , Ratas , Ratas Endogámicas F344RESUMEN
PURPOSE: To assess the feasibility of a one-step surgical concept, employing adipose stem cells (ASCs) and a novel degradable radiolucent cage filler (poly-L-lactide-co-caprolactone; PLCL), within polyetheretherketone cages in a stand-alone caprine spinal fusion model. METHODS: A double-level fusion study was performed in 36 goats. Four cage filler groups were defined: (i) acellular PLCL, (ii) PLCL + SVF (freshly harvested stromal vascular fraction highly enriched in ASCs); (iii) PLCL + ASCs (cultured to homogeneity); and (iv) autologous iliac crest bone graft (ABG). Fusion was assessed after 3 and 6 months by radiography, micro-CT, biomechanics, and biochemical analysis of tissue formed inside the cage after 6 months. RESULTS: No adverse effects were observed in all groups. After 3 months, similar and low fusion rates were found. Segmental stability did not differ between groups in all tested directions. Micro-CT imaging revealed significantly higher amounts of mineralized tissue in the ABG group compared to all others. After 6 months, interbody fusion rates were: PLCL 53%, SVF 30%, ASC 43% and ABG 63%. A trend towards higher mineralized tissue content was found for the ABG group. Biochemical and biomechanical analyses revealed equal maturity of collagen cross-links and similar segmental stability between all groups. CONCLUSIONS: This study demonstrates the technical feasibility and safety of the one-step surgical procedure for spinal fusion for the first time. The radiolucent PLCL scaffold allowed in vivo monitoring of bone formation using plain radiography. Addition of stem cells to the PLCL scaffolds did not result in adverse effects, but did not enhance the rate and number of interbody fusions under the current conditions. A trend towards superior results with ABG was found. Further research is warranted to optimize the spinal fusion model for proper evaluation of both PLCL and stem cell therapy.
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Implantes Absorbibles , Tejido Adiposo/citología , Fusión Vertebral/instrumentación , Fusión Vertebral/métodos , Trasplante de Células Madre , Ingeniería de Tejidos , Animales , Estudios de Factibilidad , Cabras , Ilion/trasplante , Vértebras Lumbares/cirugía , Modelos Animales , Oseointegración , Poliésteres , Células del Estroma/trasplanteRESUMEN
Lysyl hydroxylase 2b (LH2b) is known to increase pyridinoline cross-links, making collagen less susceptible to enzymatic degradation. Previously, we observed a relationship between LH2b and osteoarthritis-related fibrosis in murine knee joint. For this study, we investigate if transforming growth factor-beta (TGF-ß) and connective tissue growth factor (CTGF) regulate procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) (gene encoding LH2b) and LH2b expression differently in osteoarthritic human synovial fibroblasts (hSF). Furthermore, we investigate via which TGF-ß route (Smad2/3P or Smad1/5/8P) LH2b is regulated, to explore options to inhibit LH2b during fibrosis. To answer these questions, fibroblasts were isolated from knee joints of osteoarthritis patients. The hSF were stimulated with TGF-ß with or without a kinase inhibitor of ALK4/5/7 (SB-505124) or ALK1/2/3/6 (dorsomorphin). TGF-ß, CTGF, constitutively active (ca)ALK1 and caALK5 were adenovirally overexpressed in hSF. The gene expression levels of PLOD1/2/3, CTGF and COL1A1 were analyzed with Q-PCR. LH2 protein levels were determined with western blot. As expected, TGF-ß induced PLOD2/LH2 expression in hSF, whereas CTGF did not. PLOD1 and PLOD3 were not affected by either TGF-ß or CTGF. SB-505124 prevented the induction of TGF-ß-induced PLOD2, CTGF and COL1A1. Surprisingly, dorsomorphin completely blocked the induction of CTGF and COL1A1, whereas TGF-ß-induced PLOD2 was only slightly reduced. Overexpression of caALK5 in osteoarthritic hSF significantly induced PLOD2/LH2 expression, whereas caALK1 had no effect. We showed, in osteoarthritic hSF, that TGF-ß induced PLOD2/LH2 via ALK5 Smad2/3P. This elevation of LH2b in osteoarthritic hSF makes LH2b an interesting target to interfere with osteoarthritis-related persistent fibrosis.
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Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Fibroblastos/metabolismo , Osteoartritis/metabolismo , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Células Cultivadas , Fibroblastos/patología , Fibrosis/genética , Fibrosis/metabolismo , Fibrosis/patología , Regulación de la Expresión Génica , Humanos , Osteoartritis/genética , Osteoartritis/patología , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/genética , Transducción de Señal , Proteína Smad1/metabolismo , Proteína Smad2/metabolismoRESUMEN
The hallmark of fibrosis is an accumulation of fibrillar collagens, especially of collagen type I. There is considerable debate whether in vivo type II epithelial-to-mesenchymal transition (EMT) is involved in organ fibrosis. Lineage tracing experiments by various groups show opposing data concerning the relative contribution of epithelial cells to the pool of myofibroblasts. We hypothesized that EMT-derived cells might directly contribute to collagen deposition. To study this, EMT was induced in human epithelial lung and renal cell lines in vitro by means of TGF-ß1 stimulation, and we compared the collagen type I (COL1A1) expression levels of transdifferentiated cells with that of myofibroblasts obtained by TGF-ß1 stimulation of human dermal and lung fibroblasts. COL1A1 expression levels of transdifferentiated epithelial cells appeared to be at least one to two orders of magnitude lower than that of myofibroblasts. This was confirmed at immunohistochemical level: in contrast to myofibroblasts, collagen type I deposition by EMT-derived cells was not or hardly detectable. We postulate that, even when type II EMT occurs in vivo, the direct contribution of EMT-derived cells to collagen accumulation is rather limited.
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Colágeno Tipo I/metabolismo , Células Epiteliales/citología , Transición Epitelial-Mesenquimal/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Colágeno Tipo I/genética , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Fibroblastos/citología , Fibrosis/metabolismo , Humanos , Factor de Crecimiento Transformador beta1/farmacología , Regulación hacia ArribaRESUMEN
Intestinal fibrosis is a common complication in patients with inflammatory bowel disease [IBD], in particular Crohn's disease [CD]. Unfortunately, at present intestinal fibrosis is not yet preventable, and cannot be treated by interventions other than surgical removal. Intestinal fibrosis is characterized by excessive accumulation of extracellular matrix [ECM], which is caused by activated fibroblasts and smooth muscle cells. Accumulation of ECM results from an imbalanced production and degradation of ECM. ECM degradation is mainly performed by matrix metalloproteinases [MMPs], enzymes that are counteracted by tissue inhibitors of MMPs [TIMPs]. In IBD patients, MMP activity [together with other protease activities] is increased. At the same time, CD patients have a generally lower MMP activity compared to ulcerative colitis patients, who usually do not develop intestinal strictures or fibrosis. The exact regulation and role[s] of these MMPs in fibrosis are far from understood. Here, we review the current literature about ECM remodelling by MMPs in intestinal fibrosis and their potential role as biomarkers for disease progression or druggable targets.