RESUMEN
OBJECTIVE: SSc is a disease characterized by inflammation and fibrosis. Heme Oxygenase-1 (HO-1) is a haem-degrading enzyme that mediates resolution of inflammation and is induced upon mediators abundantly present in SSc. We aimed to assess whether HO-1 expression/function is disturbed in SSc patients and could therefore be contributing to the ongoing inflammation. METHODS: In total, 92 SSc patients and 48 healthy controls were included. By measuring total bilirubin in plasma in vivo, HO-activity was assessed. HO-1 expression levels were determined with western blot in monocytes before and after induction of HO-1 with cobalt protoporphyrin (CoPP) with or without CXCL4. Monocyte-derived dendritic cells (DCs) were stimulated with several Toll-like receptor (TLR) ligands with or without pre-stimulation with CoPP for 24 h. Cytokine levels were measured in the supernatants using the Luminex Bead Array. RESULTS: SSc patients have lower plasma levels of bilirubin, suggestive of an aberrant HO-1 function. We demonstrated low HO-1 expression in immune cells from SSc patients, whereas induction with CoPP was able to restore HO-1 levels in DCs from SSc patients, almost normalizing the increased TLR response observed in SSc. Co-exposure to CXCL4 completely abrogated CoPP-induced HO-1 expression, suggesting that the high CXCL4 levels present in SSc patients block the normal induction of HO-1 and its function. CONCLUSION: We demonstrate that HO activity in SSc patients is decreased and show its functional consequences. Since CXCL4 blocks the induction of HO-1 expression, neutralization of CXCL4 in SSc patients could have clinical benefits by diminishing overactivation of immune cells and other anti-inflammatory effects of HO-1.
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Hemo-Oxigenasa 1/deficiencia , Factor Plaquetario 4/fisiología , Esclerodermia Sistémica/enzimología , Receptores Toll-Like/fisiología , Adulto , Bilirrubina/metabolismo , Monóxido de Carbono/metabolismo , Estudios de Casos y Controles , Citocinas/metabolismo , Células Dendríticas/metabolismo , Femenino , Fibroblastos/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , MasculinoRESUMEN
Impaired wound healing can lead to scarring, and aesthetical and functional problems. The cytoprotective haem oxygenase (HO) enzymes degrade haem into iron, biliverdin and carbon monoxide. HO-1 deficient mice suffer from chronic inflammatory stress and delayed cutaneous wound healing, while corneal wound healing in HO-2 deficient mice is impaired with exorbitant inflammation and absence of HO-1 expression. This study addresses the role of HO-2 in cutaneous excisional wound healing using HO-2 knockout (KO) mice. Here, we show that HO-2 deficiency also delays cutaneous wound closure compared to WT controls. In addition, we detected reduced collagen deposition and vessel density in the wounds of HO-2 KO mice compared to WT controls. Surprisingly, wound closure in HO-2 KO mice was accompanied by an inflammatory response comparable to WT mice. HO-1 induction in HO-2 deficient skin was also similar to WT controls and may explain this protection against exaggerated cutaneous inflammation but not the delayed wound closure. Proliferation and myofibroblast differentiation were similar in both two genotypes. Next, we screened for candidate genes to explain the observed delayed wound closure, and detected delayed gene and protein expression profiles of the chemokine (C-X-C) ligand-11 (CXCL-11) in wounds of HO-2 KO mice. Abnormal regulation of CXCL-11 has been linked to delayed wound healing and disturbed angiogenesis. However, whether aberrant CXCL-11 expression in HO-2 KO mice is caused by or is causing delayed wound healing needs to be further investigated.
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Regulación Enzimológica de la Expresión Génica , Hemo Oxigenasa (Desciclizante)/genética , Hemo-Oxigenasa 1/genética , Cicatrización de Heridas/genética , Actinas/genética , Actinas/metabolismo , Animales , Vasos Sanguíneos/metabolismo , Western Blotting , Proliferación Celular/genética , Quimiocina CXCL11/genética , Quimiocina CXCL11/metabolismo , Colágeno/metabolismo , Ciclooxigenasa 2/metabolismo , Perfilación de la Expresión Génica , Hemo Oxigenasa (Desciclizante)/deficiencia , Hemo-Oxigenasa 1/metabolismo , Inmunohistoquímica , Mediadores de Inflamación/metabolismo , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/lesiones , Piel/metabolismo , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Objectives: To develop a reliable instrument to objectively assess feedback quality, to use it for assessment of the quality of students' narrative feedback and to be used as a self-assessment instrument for students in their learning process. Methods: In a retrospective cohort study, 635 feedback narratives, provided by small groups of Medicine and Biomedical Sciences undergraduate students, have been extracted from available quarterly curriculum evaluation surveys. A rubric was developed based on literature and contents of our feedback education. It consists of seven subitems and has a maximum score of 20 points (sufficient score: >10 points). Rubric reliability was evaluated using intra-class correlation. The rubric was tested by analysing the feedback narratives. To test progression, we compared rubric scores between study years with a Kruskal-Wallis analysis and Dunn's post-hoc testing with Bonferroni correction. Results: The rubric has an intra-class correlation of 0.894. First year students had a mean rubric score of 11.5 points (SD 3.6), second year students 12.4 (SD 3.4) and third year students 13.1 (SD 3.6). Kruskal-Wallis testing showed significant differences in feedback quality between study years (χ2(2, N=635) = 17.53, p<0.001). Dunn's post-hoc test revealed significant differences between study years one and two (p=0.012) and one and three (p<0.001). Conclusions: The developed rubric is a reliable instrument to assess narrative feedback quality. Students were able to provide feedback of sufficient quality and quality improved across study years. The instrument will allow students to assess themselves and learn where there is still room for improvement.
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Evaluación Educacional , Estudiantes , Humanos , Retroalimentación , Reproducibilidad de los Resultados , Estudios RetrospectivosRESUMEN
Osteoarthritis (OA) is a multifactorial disease strongly correlated with history of joint trauma, joint dysplasia, and advanced age. Mesenchymal stem cells (MSCs) are promising cells for biological cartilage regeneration. Conflicting data have been published concerning the availability of MSCs from the iliac crest, depending on age and overall physical fitness. Here, we analyzed whether the availability and chondrogenic differentiation capacity of MSCs isolated from the femoral shaft as an alternative source is age- or OA etiology-dependent. MSCs were isolated from the bone marrow (BM) of 98 patients, categorized into three OA-etiology groups (age-related, joint trauma, joint dysplasia) at the time of total hip replacement. All BM samples were characterized for cell yield, proliferation capacity, and phenotype. Chondrogenic differentiation was studied using micromass culture and analyzed by histology, immunohistochemistry, and quantitative reverse transcriptase-polymerase chain reaction. Significant volumes of viable BM (up to 25 ml) could be harvested from the femoral shaft without observing donor-site morbidity, typically containing >10(7) mononuclear cells per milliliter. No correlation of age or OA etiology with the number of mononuclear cells in BM, MSC yield, or cell size was found. Proliferative capacity and cellular spectrum of the harvested cells were independent of age and cause of OA. From all tested donors, MSCs could be differentiated into the chondrogenic lineage. We conclude that, irrespective of age and OA etiology, sufficient numbers of MSCs can be isolated and that these cells possess an adequate chondrogenic differentiation potential. Therefore, a therapeutic application of MSCs for cartilage regeneration of OA lesions seems feasible. Disclosure of potential conflicts of interest is found at the end of this article.
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Células Madre Adultas/fisiología , Envejecimiento/fisiología , Condrocitos/fisiología , Células Madre Mesenquimatosas/fisiología , Osteoartritis/etiología , Osteoartritis/patología , Adulto , Células Madre Adultas/citología , Células Madre Adultas/patología , Anciano , Anciano de 80 o más Años , Envejecimiento/genética , Envejecimiento/patología , Células Cultivadas , Condrocitos/citología , Condrocitos/patología , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/patología , Persona de Mediana Edad , Osteoartritis/fisiopatologíaRESUMEN
Heme oxygenase (HO)-1 is the inducible isoform of the heme-degrading enzyme HO, which is upregulated by multiple stress stimuli. HO-1 has major immunomodulatory and anti-inflammatory effects via its cell-type-specific functions in mononuclear cells. Contradictory findings have been reported on HO-1 regulation by the Toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS) in these cells. Therefore, we reinvestigated the effects of LPS on HO-1 gene expression in human and murine mononuclear cells in vitro and in vivo. Remarkably, LPS downregulated HO-1 in primary human peripheral blood mononuclear cells (PBMCs), CD14(+) monocytes, macrophages, dendritic cells, and granulocytes, but upregulated this enzyme in primary murine macrophages and human monocytic leukemia cell lines. Furthermore, experiments with human CD14(+) monocytes revealed that activation of other TLRs including TLR1, -2, -5, -6, -8, and -9 decreased HO-1 mRNA expression. LPS-dependent downregulation of HO-1 was specific, because expression of cyclooxygenase-2, NADP(H)-quinone oxidoreductase-1, and peroxiredoxin-1 was increased under the same experimental conditions. Notably, LPS upregulated expression of Bach1, a critical transcriptional repressor of HO-1. Moreover, knockdown of this nuclear factor enhanced basal and LPS-dependent HO-1 expression in mononuclear cells. Finally, downregulation of HO-1 in response to LPS was confirmed in PBMCs from human individuals subjected to experimental endotoxemia. In conclusion, LPS downregulates HO-1 expression in primary human mononuclear cells via a Bach1-mediated pathway. As LPS-dependent HO-1 regulation is cell-type- and species-specific, experimental findings in cell lines and animal models need careful interpretation.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/metabolismo , Leucocitos Mononucleares/enzimología , Lipopolisacáridos/farmacología , Macrófagos/enzimología , Monocitos/enzimología , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Western Blotting , Regulación hacia Abajo , Endotoxemia/tratamiento farmacológico , Endotoxemia/enzimología , Endotoxemia/patología , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Hemo-Oxigenasa 1/genética , Humanos , Leucemia Monocítica Aguda/tratamiento farmacológico , Leucemia Monocítica Aguda/enzimología , Leucemia Monocítica Aguda/patología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Monocitos/citología , Monocitos/efectos de los fármacos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
To study the (patho)physiological role of transforming growth factor-beta (TGF-beta), potent and selective inhibitors are necessary. Since TGF-beta signaling is initiated by the high affinity binding to the type II receptor (RII), the extracellular part of RII (solRII) can function as a TGF-beta antagonist. SolRII was cloned and large-scale protein synthesis was performed in the yeast Pichia pastoris expression system. Our results indicate that via this system, high levels of pure concentrated solRII can be obtained. Moreover, purified solRII is an active protein as shown by ELISA and bioassay. In conclusion, our large-scale protein expression procedure results in high quantities of purified solRII, which is a powerful tool to study the natural role of TGF-beta.
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Pichia/genética , Receptores de Factores de Crecimiento Transformadores beta/biosíntesis , Factor de Crecimiento Transformador beta/metabolismo , Western Blotting , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Glicosilación , Focalización Isoeléctrica , Proteínas Serina-Treonina Quinasas , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteínas Recombinantes/biosíntesis , Transducción de Señal , Factor de Crecimiento Transformador beta2RESUMEN
Heme is the functional group of diverse hemoproteins and crucial for many cellular processes. However, heme is increasingly recognized as a culprit for a wide variety of pathologies, including sepsis, malaria, and kidney failure. Excess of free heme can be detrimental to tissues by mediating oxidative and inflammatory injury. Protective mechanisms against free heme are therefore pivotal for cellular survival. We postulated that overexpression of Heme Oxygenase-1 (HO-1) and Breast Cancer Resistance Protein (BCRP) would protect against heme-induced cytotoxicity. HO-1 is a heme-degrading enzyme generating carbon monoxide, iron, and biliverdin/bilirubin, while BCRP is a heme efflux transporter. Human embryonic kidney cells were transduced using a baculovirus system as a novel strategy to efficiently overexpress HO-1 and BCRP. Exposing cells to heme resulted in a dose-dependent increase in reactive oxygen species formation, DNA damage and cell death. Heme-induced cell death was significantly attenuated when cells overexpressed HO-1, BCRP, or both. The protective effects of HO-1 overexpression were most pronounced, while co-treatment with the HO-activity inhibitor tin mesoporphyrin reversed these protective effects. Also cells treated with the anti-oxidants N-acetylcysteine or HO-effector molecule bilirubin showed protection against heme insults, which may explain the increased protection by HO-1 compared to BCRP. In conclusion, both HO-1 and BCRP protect against heme-induced toxicity and may thus form novel therapeutic targets for heme-mediated pathologies.
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Transportadoras de Casetes de Unión a ATP/fisiología , Hemo-Oxigenasa 1/metabolismo , Hemo/toxicidad , Proteínas de Neoplasias/fisiología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Bencimidazoles/metabolismo , Western Blotting , Daño del ADN , Células HEK293 , Humanos , Oxidantes/toxicidad , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Wound healing is an intricate process requiring the concerted action of keratinocytes, fibroblasts, endothelial cells, and macrophages. Here, we review the literature on normal wound healing and the pathological forms of wound healing, such as hypertrophic or excessive scar formation, with special emphasis on the heme-heme oxygenase (HO) system and the versatile effector molecules that are formed after HO-mediated heme degradation. Excessive scar formation following wounding is thought to relate to prolonged oxidative and inflammatory stress in the skin. Evidence is accumulating that the heme-HO system forms a novel and important target in the control of wound healing. Heme-protein derived heme can act as a potent oxidative and inflammatory stress inducer, and excess levels of heme may thus contribute to delayed resolution of oxidative and inflammatory insults in the skin. This emphasizes the need for a timely reduction of the levels of heme. Heme-binding proteins, heme transporters, and the heme degrading protein, HO, form therefore a necessary defense. Deficiencies in these defense proteins or a disturbed redox status, as in diabetic patients, may render individuals more prone to heme-induced deleterious effects. A better understanding of the heme-heme oxygenase system as target during wound healing may result in novel strategies to reduce scar formation.
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Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo/metabolismo , Cicatrización de Heridas/fisiología , Animales , Apoptosis , Cicatriz/metabolismo , Cicatriz/fisiopatología , Cicatriz Hipertrófica/metabolismo , Cicatriz Hipertrófica/fisiopatología , Inducción Enzimática , Hemo-Oxigenasa 1/metabolismo , Hemoproteínas/metabolismo , Humanos , Inflamación/metabolismo , Miofibroblastos/metabolismo , Neovascularización Fisiológica , Estrés Oxidativo , Piel/metabolismo , Piel/fisiopatología , Piel/efectos de la radiación , Rayos Ultravioleta/efectos adversosRESUMEN
BACKGROUND: We previously identified curcumin as a potent inducer of fibroblast apoptosis, which could be used to treat hypertrophic scar formation. Here we investigated the underlying mechanism of this process. PRINCIPAL FINDINGS: Curcumin-induced apoptosis could not be blocked by caspase-inhibitors and we could not detect any caspase-3/7 activity. Curcumin predominantly induced mitochondria-mediated ROS formation and stimulated the expression of the redox-sensitive pro-apoptotic factor p53. Inhibition of the pro-apoptotic signaling enzyme glycogen synthase kinase-3beta (GSK-3beta) blocked curcumin-induced apoptosis. Apoptosis was associated with high molecular weight DNA damage, a possible indicator of apoptosis-inducing factor (AIF) activity. Indeed, curcumin caused nuclear translocation of AIF, which could be blocked by the antioxidant N-acetyl cysteine. We next investigated how AIF is effluxed from mitochondria in more detail. The permeability transition pore complex (PTPC), of which the voltage-dependent anion channel (VDAC) is a component, could be involved since the VDAC-inhibitor DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) efficiently blocked AIF translocation. However, PTPC is not involved in AIF release since cyclosporine A, a specific inhibitor of the complex did not block apoptosis. Alternatively, the pro-apoptotic protein Bax could have formed mitochondrial channels and interacted with VDAC. Curcumin caused mitochondrial translocation of Bax, which was blocked by DIDS, suggesting a Bax-VDAC interaction. Interestingly, ceramide channels can also release apoptogenic factors from mitochondria and we found that addition of ceramide induced caspase-independent apoptosis. Surprisingly, this process could also be blocked by DIDS, suggesting the concerted action of Bax, VDAC and ceramide in the efflux of AIF from the mitochondrion. CONCLUSIONS: Curcumin-induced fibroblast apoptosis is totally caspase-independent and relies on the mitochondrial formation of ROS and the subsequent nuclear translocation of AIF, which is released from a mitochondrial pore that involves VDAC, Bax and possibly ceramides. The composition of the AIF-releasing channel seems to be much more complex than previously thought.
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Factor Inductor de la Apoptosis/metabolismo , Apoptosis/efectos de los fármacos , Ceramidas/fisiología , Curcumina/farmacología , Mitocondrias/efectos de los fármacos , Canales Aniónicos Dependientes del Voltaje/fisiología , Proteína X Asociada a bcl-2/fisiología , Caspasas/metabolismo , ADN/química , ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Mitocondrias/metabolismo , Peso Molecular , Transporte de Proteínas , Especies Reactivas de Oxígeno/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
OBJECTIVE: Osteoarthritis (OA) is a joint disease characterized by osteophyte development, fibrosis, and articular cartilage damage. Effects of exogenous transforming growth factor beta (TGFbeta) isoforms and bone morphogenetic proteins (BMPs) suggest a role for these growth factors in the pathogenesis of OA. The aim of this study was to elucidate the role of endogenous TGFbeta and BMP during papain-induced OA-like changes in mice. METHODS: We used adenoviral overexpression of TGFbeta and BMP antagonists to block growth factor signaling. An adenovirus expressing a secreted, pan-specific TGFbeta antagonist called murine latency-associated peptide 1 (mLAP-1) was used. In addition, we used intracellular inhibitory Smad6 as a BMP antagonist and Smad7 as a TGFbeta/BMP inhibitor. Papain was injected into the knee joints of C57BL/6 mice to induce osteophyte development, synovial thickening, and articular cartilage proteoglycan (PG) loss. RESULTS: Intraarticular injection of papain caused increased protein expression of several TGFbeta and BMP isoforms in synovium and cartilage. Adenovirus transfection into the joint resulted in a strong expression of the transgenes in the synovial lining. Overexpression of mLAP-1, Smad6, and Smad7 led to a significant reduction in osteophyte formation compared with that in controls. Smad6 and Smad7 overexpression also significantly decreased synovial thickening. Furthermore, the secreted TGFbeta inhibitor mLAP-1 increased articular cartilage PG loss. CONCLUSION: Our results indicate a pivotal role of endogenous TGFbeta in the development of osteophytes and synovial thickening, implicating endogenous TGFbeta in the pathogenesis of OA. In contrast, the prevention of cartilage damage by endogenous TGFbeta signifies the protective role of TGFbeta in articular cartilage. This is the first study to demonstrate that endogenous BMPs are involved in osteophyte formation and synovial thickening in experimental OA.
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Osteoartritis de la Rodilla/fisiopatología , Fragmentos de Péptidos/genética , Precursores de Proteínas/genética , Factor de Crecimiento Transformador beta/genética , Adenoviridae/genética , Animales , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 4 , Proteína Morfogenética Ósea 6 , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Proteínas Morfogenéticas Óseas/genética , Células Cultivadas , Expresión Génica , Técnicas de Transferencia de Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Visón , Osteoartritis de la Rodilla/inducido químicamente , Osteoartritis de la Rodilla/patología , Papaína , Fragmentos de Péptidos/metabolismo , Precursores de Proteínas/metabolismo , Proteoglicanos/metabolismo , Mucosa Respiratoria/citología , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1 , Transgenes/fisiologíaRESUMEN
Osteoarthritis has as main characteristics the degradation of articular cartilage and the formation of new bone at the joint edges, so-called osteophytes. In this study enhanced expression of TGF-beta1 and -beta3 was detected in developing osteophytes and articular cartilage during murine experimental osteoarthritis. To determine the role of endogenous TGF-beta on osteophyte formation and articular cartilage, TGF-beta activity was blocked via a scavenging soluble TGF-beta-RII. Our results clearly show that inhibition of endogenous TGF-beta nearly completely prevented osteophyte formation. In contrast, treatment with recombinant soluble TGF-beta-RII markedly enhanced articular cartilage proteoglycan loss and reduced the thickness of articular cartilage. In conclusion, we show for the first time that endogenous TGF-beta is a crucial factor in the process of osteophyte formation and has an important function in protection against cartilage loss.