RESUMEN
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.
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Enfermedad de Crohn , Enfermedades Inflamatorias del Intestino , Humanos , Intestinos , Enfermedad de Crohn/metabolismo , Fibrosis , Metaloproteinasas de la Matriz/metabolismoRESUMEN
Dupuytren's disease is a chronic, progressive fibroproliferative condition of the hand fascia which results in digital contraction. So far, treatments do not directly interfere with the (myo)fibroblasts that are responsible for the formation of the collagen-rich cords and its contraction. Here we investigated whether verteporfin (VP) is able to inhibit the activation and subsequent differentiation of DD nodular fibroblasts into myofibroblasts. Fibroblasts were isolated from nodules of 7 Dupuytren patients. Cells are treated (1) for 48 h with 5 ng/ml transforming growth factor ß1 (TGF-ß1) followed by 48 h with/without 250 nM VP in the absence of TGF-ß1, or treated (2) for 48 h with TGF-ß1 followed by 48 h with/without VP in the presence of TGF-ß1. mRNA levels were measured by means of Real-Time PCR, and proteins were visualized by means of Western blotting and/or immunofluorescence. Quantitative data were statistically analyzed with GraphPad Prism using the paired t-test. We found that fibroblasts activated for 48 h with TGF-ß1 show a decrease in mRNA levels of COL1A1, COL3A1, COL4A1, PLOD2, FN1EDA, CCN2 and SERPINE1 when exposed for another 48 h with VP, whereas no decrease is seen for ACTA2, YAP1, SMAD2 and SMAD3 mRNA levels. Cells exposed for an additional 48 h with TGF-ß1, but now in the presence of VP, are not further activated anymore, whereas in the absence of VP the cells continue to differentiate into myofibroblasts. Collagen type I, fibronectin-extra domain A, α-smooth muscle actin, YAP1, Smad2 and Smad3 protein levels were attenuated by both VP treatments. We conclude that VP has strong anti-fibrotic properties: it is able to halt the differentiation of fibroblasts into myofibroblasts, and is also able to reverse the activation status of fibroblasts. The decreased protein levels of YAP1, Smad2 and Smad3 in the presence of VP explain in part the strong anti-fibrotic properties of VP. Verteporfin is clinically used as a photosensitizer for photodynamic therapy to eliminate abnormal blood vessels in the eye to attenuate macular degeneration. The antifibrotic properties of VP do not rely on photo-activation, as we used the molecule in its non-photoinduced state.
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Contractura de Dupuytren , Actinas/genética , Actinas/metabolismo , Células Cultivadas , Contractura de Dupuytren/tratamiento farmacológico , Fibroblastos/metabolismo , Humanos , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Verteporfina/metabolismo , Verteporfina/farmacologíaRESUMEN
BACKGROUND AND AIMS: Crohn's disease (CD) can be complicated by intestinal fibrosis. Pharmacological therapies against intestinal fibrosis are not available. The aim of this study was to determine whether pathways involved in collagen metabolism are upregulated in intestinal fibrosis, and to discuss which drugs might be suitable to inhibit excessive extracellular matrix formation targeting these pathways. METHODS: Human fibrotic and non-fibrotic terminal ileum was obtained from patients with CD undergoing ileocecal resection due to stenosis. Genes involved in collagen metabolism were analyzed using a microfluidic low-density TaqMan array. A literature search was performed to find potential anti-fibrotic drugs that target proteins/enzymes involved in collagen synthesis, its degradation and its recognition. RESULTS: mRNA expression of collagen type I (COL1A1, 0.76 ± 0.28 versus 37.82 ± 49.85, p = 0.02) and III (COL3A1, 2.01 ± 2.61 versus 68.65 ± 84.07, p = 0.02) was increased in fibrotic CD compared with non-fibrotic CD. mRNA expression of proteins involved in both intra- and extracellular post-translational modification of collagens (prolyl- and lysyl hydroxylases, lysyl oxidases, chaperones), collagen-degrading enzymes (MMPs and cathepsin-K), and collagen receptors were upregulated in the fibrosis-affected part. A literature search on the upregulated genes revealed several potential anti-fibrotic drugs. CONCLUSION: Expression of genes involved in collagen metabolism in intestinal fibrosis affected terminal ileum of patients with CD reveals a plethora of drug targets. Inhibition of post-translational modification and altering collagen metabolism might attenuate fibrosis formation in the intestine in CD. Which compound has the highest potential depends on a combination anti-fibrotic efficacy and safety, especially since some of the enzymes play key roles in the physiology of collagen.
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Animal models are a valuable tool in preclinical research. However, limited predictivity of human biological responses in the conventional models has stimulated the search for reliable preclinical tools that show translational robustness. Here, we used precision-cut kidney slices (PCKS) as a model of renal fibrosis and investigated its predictive capacity for screening the effects of anti-fibrotics. Murine and human PCKS were exposed to TGFß or PDGF pathway inhibitors with established anti-fibrotic efficacy. For each treatment modality, we evaluated whether it affected: (1) culture-induced collagen type I gene expression and interstitial accumulation; (2) expression of markers of TGFß and PDGF signaling; and (3) expression of inflammatory markers. We summarized the outcomes of published in vivo animal and human studies testing the three inhibitors in renal fibrosis, and drew a parallel to the PCKS data. We showed that the responses of murine PCKS to anti-fibrotics highly corresponded with the known in vivo responses observed in various animal models of renal fibrosis. Moreover, our results suggested that human PCKS can be used to predict drug efficacy in clinical trials. In conclusion, our study demonstrated that the PCKS model is a powerful predictive tool for ex vivo screening of putative drugs for renal fibrosis.
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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.
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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
An unsolved therapeutic problem in fibrosis is the overproduction of collagen. In order to screen the effect of anti-fibrotic drugs on collagen deposition, the Scar-in-a-Jar approach has been introduced about a decade ago. With macromolecular crowding a rapid deposition of collagen is seen, resulting in a substantial decrease in culture time, but the system has never been tested in an adequate way. We therefore have compared six different macromolecular crowders [Ficoll PM 70 (Fc70), Ficoll PM 400 (Fc400), a mixture of Ficoll 70 and 400 (Fc70/400), polyvinylpyrrolidone 40 (PVP40), polyvinylpyrrolidone 360 (PVP360), neutral dextran 670 (ND670), dextran sulfate 500 (DxS500), and carrageenan (CR)] under profibrotic conditions (addition of TGFß1) with primary human adult dermal fibroblasts in the presence of 0.5 and 10% FBS. We found that (1) collagen deposition and myofibroblast formation was superior with 0.5% FBS, (2) DxS500 and CR results in an aberrant collagen deposition pattern, (3) ND670 does not increase collagen deposition, and (4) CR, DxS500, and Fc40/700 affected important phenotypical properties of the cells when cultured under pro-fibrotic conditions, whereas PVP40 and PVP360 did less or not. Because of viscosity problems with PVP360, we conclude that PVP40 is the most optimal crowder for the screening of anti-fibrotic drugs. Finally, the effect of various concentrations of Imatinib, Galunisertib, Omipalisib or Nintedanib on collagen deposition and myofibroblast formation was tested with PVP40 as the crowder.
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Fibrosis is the hallmark of pathologic tissue remodelling in most chronic diseases. Despite advances in our understanding of the mechanisms of fibrosis, it remains uncured. Fibrogenic processes share conserved core cellular and molecular pathways across organs. In this study, we aimed to elucidate shared and organ-specific features of fibrosis using murine precision-cut tissue slices (PCTS) prepared from small intestine, liver and kidneys. PCTS displayed substantial differences in their baseline gene expression profiles: 70% of the extracellular matrix (ECM)-related genes were differentially expressed across the organs. Culture for 48â¯h induced significant changes in ECM regulation and triggered the onset of fibrogenesis in all PCTS in organ-specific manner. TGFß signalling was activated during 48â¯h culture in all PCTS. However, the degree of its involvement varied: both canonical and non-canonical TGFß pathways were activated in liver and kidney slices, while only canonical, Smad-dependent, cascade was involved in intestinal slices. The treatment with galunisertib blocked the TGFßRI/SMAD2 signalling in all PCTS, but attenuated culture-induced dysregulation of ECM homeostasis and mitigated the onset of fibrogenesis with organ-specificity. In conclusion, regardless the many common features in pathophysiology of organ fibrosis, PCTS displayed diversity in culture-induced responses and in response to the treatment with TGFßRI kinase inhibitor galunisertib, even though it targets a core fibrosis pathway. A clear understanding of the common and organ-specific features of fibrosis is the basis for developing novel antifibrotic therapies.
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Fibrosis/patología , Cirrosis Hepática/patología , Hígado/patología , Animales , Fibrosis/tratamiento farmacológico , Fibrosis/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Hígado/efectos de los fármacos , Hígado/metabolismo , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/metabolismo , Ratones , Ratones Endogámicos C57BL , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Quinolinas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
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.
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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.
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Induración Peniana , Colágeno , Humanos , Masculino , PeneAsunto(s)
Materiales Biocompatibles , Fibrosis , Animales , Reacción a Cuerpo Extraño/inmunología , Primates , RoedoresRESUMEN
The hallmark of fibrosis is an excessive accumulation of collagen, ultimately leading to organ failure. It has become evident that the deposited collagen also exhibits qualitative modifications. A marked modification is the increased cross-linking, leading to a stabilization of the collagen network and limiting fibrosis reversibility. Not only the level of cross-linking is increased, but also the composition of cross-linking is altered: an increase is seen in hydroxyallysine-derived cross-links at the expense of allysine cross-links. This results in irreversible fibrosis, as collagen cross-linked by hydroxyallysine is more difficult to degrade. Hydroxyallysine is derived from a hydroxylysine in the telopeptides of collagen. The expression of lysyl hydroxylase (LH) 2 (LH2), the enzyme responsible for the formation of telopeptidyl hydroxylysine, is universally up-regulated in fibrosis. It is expected that inhibition of this enzyme will lead to reversible fibrosis without interfering with the normal repair process. In this review, we discuss the molecular basis of collagen modifications and cross-linking, with an emphasis on LH2-mediated hydroxyallysine cross-links, and their implications for the pathogenesis and treatment of fibrosis.
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Colágeno/metabolismo , Fibrosis/etiología , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Ácido 2-Aminoadípico/análogos & derivados , Ácido 2-Aminoadípico/química , Animales , Colágeno/química , Inhibidores Enzimáticos/farmacología , Fibrosis/tratamiento farmacológico , Humanos , Hidroxilación , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/antagonistas & inhibidores , Proteína-Lisina 6-Oxidasa/metabolismo , Proteínas de Unión a Tacrolimus/antagonistas & inhibidoresRESUMEN
INTRODUCTION: In Europe and the United States, verteporfin (Visudyne; VP) is registered and used in treating macular degeneration. Research showed that VP decreased expression of fibrotic genes in fibroblasts collected from nodules of patients suffering from Dupuytren's disease, plausibly by de-activating transcription in the Yes Activated Protein (YAP) pathway. AIM: To analyze the effect of VP on myofibroblasts cultured from Peyronie's disease (PD) plaques. METHODS: At surgery for PD we took biopsies from the plaques of 5 patients. By immunostaining, the presence of the pathologic myofibroblasts was determined. After culturing cells, VP was dispensed in starvation medium for 24 and 48 hours and messenger(m)RNA levels of COL1A1, ACTA2, COL5A1, EDA-FN, LOXL2, CCN2, SERPINH1, PLOD2, and YAP were quantified and compared with controls with real-time polymerase chain reaction. MAIN OUTCOME MEASURE: mRNA-levels of COL1A1, ACTA2, COL5A1, EDA-FN, LOXL2, CCN2, SERPINH1, PLOD2, and YAP. RESULTS: The pathologic phenotype of cells isolated from PD plaques was confirmed with baseline immunofluorescent stainings that showed considerable levels of α-smooth muscle actin, being a marker for the presence of myofibroblasts. The mRNA ratios of all the genes related to fibrosis (COL1A1, etc.) except YAP decreased significantly after treatment with VP within 24 and 48 hours. These results suggest inhibition of fibrosis in the YAP cascade, downstream of YAP. CONCLUSION: In our opinion, urologists must move the focus to disease before deformity, and the search for new oral or intralesional agents, well-tolerated and effective in both the acute and chronic phase of PD must continue. VP blocked the expression of genes related to fibrosis in the YAP cascade in myofibroblasts derived from PD plaque. Mohede DCJ, de Jong IJ, Bank RA, et al. Verteporfin as a medical treatment in Peyronie's disease. Sex Med 2018;6:302-308.
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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.
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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
STUDY DESIGN: An in vitro study using human degenerated nucleus pulposus cells. OBJECTIVE: To determine the effect of osmolality and different osmolytes on the regeneration by human nucleus pulposus cells through gene expression and extracellular matrix production. SUMMARY OF BACKGROUND DATA: Intervertebral disc (IVD) degeneration is a major problem in developed countries. Regeneration of the IVD can prevent pain and costs due to diminished work absence and health care, and improve quality of life. The osmotic value of a disc decreases during degeneration due to loss of proteoglycans and might increase degeneration. It is known that gene expression of matrix genes of nucleus pulposus (NP) cells increases when cultured in hyperosmotic medium. Thus, increasing the osmolality of the disc might be beneficial for disc regeneration. METHODS: In the current study, isolated degenerated human NP cells were used in regeneration culture with medium of different osmolalities, adjusted with different osmolytes. NaCl, urea and sucrose. The cells were cultured for 28 days and expression of matrix genes and production of glycosaminoglycans and collagen II were measured. RESULTS: Gene expression for both collagen II and aggrecan increased with increasing osmolality using NaCl or sucrose, but not urea. Protein production however, was not affected by increasing osmolality and was decreased when using urea and sucrose. Expression of genes for Col1A1, MMP13, and MMP14 decreased with increasing osmolality, whereas expression of LOXL2 and LOXL3 increased. Transient expression of TonEBP was found 6âhours after the start of culture, but not at later time points. CONCLUSION: Although expression of matrix genes is upregulated, hyperosmolality does not enhance matrix production by nucleus pulposus cells. Raising osmolality can potentially increase matrix production, but in itself is not sufficient to accomplish regeneration in the current in vitro culture system. LEVEL OF EVIDENCE: N /A.
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Medios de Cultivo/farmacología , Matriz Extracelular/metabolismo , Degeneración del Disco Intervertebral/metabolismo , Núcleo Pulposo/metabolismo , Regeneración/fisiología , Regulación hacia Arriba/fisiología , Adulto , Anciano , Agrecanos/biosíntesis , Agrecanos/genética , Aminoácido Oxidorreductasas/biosíntesis , Aminoácido Oxidorreductasas/genética , Células Cultivadas , Medios de Cultivo/química , Matriz Extracelular/genética , Femenino , Glicosaminoglicanos/biosíntesis , Glicosaminoglicanos/genética , Humanos , Disco Intervertebral/citología , Disco Intervertebral/efectos de los fármacos , Disco Intervertebral/metabolismo , Degeneración del Disco Intervertebral/genética , Degeneración del Disco Intervertebral/patología , Masculino , Persona de Mediana Edad , Núcleo Pulposo/citología , Núcleo Pulposo/efectos de los fármacos , Proteoglicanos/biosíntesis , Proteoglicanos/genética , Regeneración/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
l-Ascorbic acid (AA), generally known as vitamin C, is a crucial cofactor for a variety of enzymes, including prolyl-3-hydroxylase (P3H), prolyl-4-hydroxylase (P4H), and lysyl hydroxylase (LH)-mediated collagen maturation. Here, we investigated whether AA has additional functions in the regulation of the myofibroblast phenotype, besides its function in collagen biosynthesis. We found that AA positively influences TGFß1-induced expression of COL1A1, ACTA2, and COL4A1 Moreover, we demonstrated that AA promotes αSMA stress fiber formation as well as the synthesis and deposition of collagens type I and IV Additionally, AA amplified the contractile phenotype of the myofibroblasts, as seen by increased contraction of a 3D collagen lattice. Moreover, AA increased the expression of several TGFß1-induced genes, including DDR1 and CCN2 Finally, we demonstrated that the mechanism of AA action seems independent of Smad2/3 signaling.
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Ácido Ascórbico/farmacología , Miofibroblastos/efectos de los fármacos , Fenotipo , Vitaminas/farmacología , Actinas/genética , Actinas/metabolismo , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Receptor con Dominio Discoidina 1/genética , Receptor con Dominio Discoidina 1/metabolismo , Humanos , Miofibroblastos/citología , Miofibroblastos/metabolismo , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaAsunto(s)
Fibroblastos , Comunicación Paracrina , Expresión Génica , Fenotipo , Transducción de SeñalRESUMEN
Macrophages are pivotal cells during the foreign body reaction (FBR), as they orchestrate the proinflammatory microenvironment inside and around biomaterials by secretion of inflammatory mediators. Furthermore, they are responsible for the degradation of biomaterials and are thought to instruct the fibroblasts that generate a fibrous capsule around implanted biomaterials. In this study, we investigated the events during the FBR when macrophages are not present. Hexamethylenediisocyanate crosslinked collagen scaffolds were implanted in "Macrophage Fas-Induced Apoptosis" mice, which allow "on demand" macrophage depletion. We observed that macrophage depletion completely inhibited inflammatory ingrowth into the scaffolds and resulted in an increased capsule size. Quantitative polymerase chain reaction analysis revealed decreased expression levels of proinflammatory mediators such as TNFα and IL1ß, and increased expression levels of collagens and fibroblast-stimulating growth factors such as EGF, FGF1, FGF2, and TGFα. Our results indicate that macrophages are indeed crucial for the generation of a proinflammatory microenvironment inside implanted biomaterials, leading to inflammatory ingrowth. In contrast, macrophages do not appear to be important for the generation of a fibrous capsule around implanted biomaterials. In fact, our data suggest that the macrophages present in the capsule might instruct the surrounding fibroblasts to produce less fibroblast-stimulating factors and less collagens.
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Apoptosis/efectos de los fármacos , Materiales Biocompatibles/farmacología , Reacción a Cuerpo Extraño/patología , Macrófagos/metabolismo , Receptor fas/metabolismo , Animales , Citocinas/genética , Citocinas/metabolismo , Reacción a Cuerpo Extraño/genética , Regulación de la Expresión Génica/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Macrófagos/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Tamaño de los Órganos , Reproducibilidad de los Resultados , Andamios del Tejido/químicaRESUMEN
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.
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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
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.
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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
BACKGROUND: Dupuytren's disease is a fibroproliferative disease of the hand and fingers, which usually manifests as two different phenotypes within the same patient. The disease first causes a nodule in the palm of the hand, while later, a cord develops, causing contracture of the fingers. RESULTS: We set out to characterize the two phenotypes by comparing matched cord and nodule tissue from ten Dupuytren's patients. We found that nodule tissue contained more proliferating cells, CD68-positive macrophages and α-smooth muscle actin (α-SMA)-positive myofibroblastic cells. qPCR analysis showed an increased expression of COL1A1, COL1A2, COL5A1, and COL6A1 in nodule tissue compared to cord tissue. Immunohistochemistry showed less deposition of collagen type I in nodules, although they contained more fibronectin, collagen type V, and procollagen 1. Lower collagen levels in nodule were confirmed by HPLC measurements of the Hyp/Pro ratio. PCOLCE2, an activator of BMP1, the main enzyme cleaving the C-terminal pro-peptide from procollagen, was also reduced in nodule. Cord tissue not only contained more collagen I, but also higher levels of hydroxylysylpyridinoline and lysylpyridinoline residues per triple helix, indicating more crosslinks. CONCLUSIONS: Our results clearly show that in Dupuytren's disease, the nodule is the active disease unit, although it does not have the highest collagen protein levels. The difference in collagen type I deposition compared to mRNA levels and procollagen 1 levels may be connected to a decrease in procollagen processing.