RESUMO
BACKGROUND: In recent decades, phytotherapy has remained as a key therapeutic option for the treatment of various cancers. Evodiamine, an excellent phytocompound from Evodia fructus, exerts anticancer activity in several cancers by modulating drug resistance. However, the role of evodiamine in cisplatin-resistant NSCLC cells is not clear till now. Therefore, we have used evodiamine as a chemosensitizer to overcome cisplatin resistance in NSCLC. METHODS: Here, we looked into SOX9 expression and how it affects the cisplatin sensitivity of cisplatin-resistant NSCLC cells. MTT and clonogenic assays were performed to check the cell proliferation. AO/EtBr and DAPI staining, ROS measurement assay, transfection, Western blot analysis, RT-PCR, Scratch & invasion, and comet assay were done to check the role of evodiamine in cisplatin-resistant NSCLC cells. RESULTS: SOX9 levels were observed to be higher in cisplatin-resistant A549 (A549CR) and NCI-H522 (NCI-H522CR) compared to parental A549 and NCI-H522. It was found that SOX9 promotes cisplatin resistance by regulating ß-catenin. Depletion of SOX9 restores cisplatin sensitivity by decreasing cell proliferation and cell migration and inducing apoptosis in A549CR and NCI-H522CR. After evodiamine treatment, it was revealed that evodiamine increases cisplatin-induced cytotoxicity in A549CR and NCI-H522CR cells through increasing intracellular ROS generation. The combination of both drugs also significantly inhibited cell migration by inhibiting epithelial to mesenchymal transition (EMT). Mechanistic investigation revealed that evodiamine resensitizes cisplatin-resistant cells toward cisplatin by decreasing the expression of SOX9 and ß-catenin. CONCLUSION: The combination of evodiamine and cisplatin may be a novel strategy for combating cisplatin resistance in NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Quinazolinas , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cisplatino/farmacologia , beta Catenina , Transição Epitelial-Mesenquimal , Espécies Reativas de Oxigênio , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Morte Celular , Fatores de Transcrição SOX9/genéticaRESUMO
Introduction Bushen Zhuangjin Decoction (BZD), a well-known formulation in Traditional Chinese Medicine, has been widely used for the treatment of osteoarthritis (OA). Due to the poor intrinsic repair capacity of chondrocytes, promoting the proliferation of chondrocytes is an efficient treatment to delay the progression of cartilage degradation.Hypothesis/Gap Statement Therefore, to explore the regulatory mechanism of Bushen Zhuangjin Decoction in chondrocytes will contribute to the repair of chondrocyte injury in OA, and may serve as a potential therapy for OA diseases.Aim To investigate the expression and distribution of SOX9 mediated by serum containing Bushen Zhuangjin Decoction (BZD) and its therapeutic effect on chondrocyte injury in rats.Methodology. The subcultured second-generation rat chondrocytes were randomly divided into four groups, and they were intervened with medium containing different serums, including: blank serum group, low-concentration BZD group, medium-concentration BZD group, and high-concentration BZD group. The viability, proliferation and apoptosis of chondrocytes were detected by MTT assay and flow cytometry. The gene and protein levels of SOX9, aggrecan and type II collagen genes were analysed by qRT-PCR and Western blot analysis. Immunofluorescence staining was used to analyse the expression and distribution of SOX9. Inflammatory factors in different culture mediums of chondrocytes were detected by ELISA.Results Compared with the control group, the activity of chondrocytes in the BZD drug-containing serum group was significantly enhanced, and the degree of apoptosis was significantly decreased. The gene and protein levels of SOX9, proteoglycan aggrecan and collagen II in chondrocytes increased significantly. The inflammatory factors in the culture medium also decreased significantly. And in the above experiments, the medium concentration group BZD drug-containing serum had the best effect.Conclusion Our research results show that BZD medicated serum can up-regulate the expression of SOX9, reduce the release of inflammatory factors, and promote changes in the phenotype of chondrocytes, which protects chondrocytes from damage.
Assuntos
Condrócitos , Fatores de Transcrição SOX9 , Ratos , Animais , Condrócitos/metabolismo , Agrecanas/genética , Agrecanas/metabolismo , Agrecanas/farmacologia , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/farmacologia , FenótipoRESUMO
BACKGROUND: 7-Hydroxycoumarin (7-HC), also known as umbelliferon, is commonly found in Chinese herbs (e.g. Eucommiae Cortex, Prunellae Spica, Radix Angelicae Biseratae). Previous laboratory studies have indicated that 7-HC has anti-inflammatory, anti-oxidative, and anti-tumor effects. Cisplatin is a widely used chemotherapeutic agent for cancer. Nephrotoxicity is one of the limiting side effects of cisplatin use. PURPOSE: This study aimed to evaluate the renoprotective effect of 7-HC in a cisplatin-induced acute kidney injury (AKI) mouse model. METHODS: AKI was induced in male C57BL/6 mice (aged 6-8 weeks) by a single intraperitoneal injection of cisplatin at 20 mg/kg. The mice received 7-HC at 30, 60, and 90 mg/kg intraperitoneally before or after cisplatin administration. Renal function, necroptosis, and cell proliferation were measured. Mechanisms underlying the reno-protective effect of 7-HC were explored in renal tubular epithelial cells treated with or without cisplatin. RESULTS: In-vivo experiments showed that 7-HC significantly improved the loss in kidney function induced by cisplatin, as indicated by lower levels of serum creatinine and blood urea nitrogen, in AKI mice. Consistent herewith, cisplatin-induced tubular damage was alleviated by 7-HC as shown by morphological (periodic acid-Schiff staining) and kidney injury marker (KIM-1) analyses. We found that 7-HC suppressed renal necroptosis via the RIPK1/RIPK3/MLKL pathway and accelerated renal repair as evidenced by the upregulation of cyclin D1 in cisplatin-induced nephropathy. In-vitro experiments showed that knockdown of Sox9 attenuated the suppressive effect of 7-HC on KIM-1 and reversed the stimulatory effect of 7-HC on cyclin D1 expression in cisplatin-treated HK-2 cells, indicating that 7-HC may protect against AKI via a Sox9-dependent mechanism. CONCLUSION: 7-HC inhibits cisplatin-induced AKI by suppressing RIPK1/RIPK3/MLKL-mediated necroptosis and promoting Sox9-mediated tubular epithelial cell proliferation. 7-HC may serve as a preventive and therapeutic agent for AKI.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/prevenção & controle , Cisplatino/efeitos adversos , Rim/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Umbeliferonas/farmacologia , Injúria Renal Aguda/tratamento farmacológico , Animais , Antineoplásicos/efeitos adversos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Rim/patologia , Testes de Função Renal , Masculino , Camundongos Endogâmicos C57BL , Necroptose/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismoRESUMO
BACKGROUND: Chondrogenesis represents a highly dynamic cellular process that leads to the establishment of various types of cartilage. However, when stress-related injuries occur, a rapid and efficient regeneration of the tissues is necessary to maintain cartilage integrity. Mesenchymal stem cells (MSCs) are known to exhibit high capacity for self-renewal and pluripotency effects, and thus play a pivotal role in the repair and regeneration of damaged cartilage. On the other hand, the influence of certain pathological conditions such as metabolic disorders on MSCs can seriously impair their regenerative properties and thus reduce their therapeutic potential. OBJECTIVES: In this investigation, we attempted to improve and potentiate the in vitro chondrogenic ability of adipose-derived mesenchymal stromal stem cells (ASCs) isolated from horses suffering from metabolic syndrome. METHODS: Cultured cells in chondrogenic-inductive medium supplemented with Cladophora glomerata methanolic extract were experimented for expression of the main genes and microRNAs involved in the differentiation process using RT-PCR, for their morphological changes through confocal and scanning electron microscopy and for their physiological homeostasis. RESULTS: The different added concentrations of C. glomerata extract to the basic chondrogenic inductive culture medium promoted the proliferation of equine metabolic syndrome ASCs (ASCsEMS) and resulted in chondrogenic phenotype differentiation and higher mRNA expression of collagen type II, aggrecan, cartilage oligomeric matrix protein, and Sox9 among others. The results reveal an obvious inhibitory effect of hypertrophy and a strong repression of miR-145-5p, miR-146-3p, and miR-34a and miR-449a largely involved in cartilage degradation. Treated cells additionally exhibited significant reduced apoptosis and oxidative stress, as well as promoted viability and mitochondrial potentiation. CONCLUSION: Chondrogenesis in EqASCsEMS was found to be prominent after chondrogenic induction in conditions containing C. glomerata extract, suggesting that the macroalgae could be considered for the enhancement of ASC cultures and their reparative properties.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Clorófitas/química , Condrogênese/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Síndrome Metabólica/patologia , Extratos Vegetais/farmacologia , Agrecanas/genética , Agrecanas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Clorófitas/metabolismo , Condrócitos/citologia , Condrócitos/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Cavalos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Síndrome Metabólica/metabolismo , MicroRNAs/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/fisiologia , Extratos Vegetais/química , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismoRESUMO
Retinoic acid modulates numerous cellular events, namely, proliferation, differentiation, apoptosis, and patterning, hence influencing both embryo development and adult homeostasis. In vitro explant culture is a valuable technique for studying the impact of growth factors and signaling molecules, such as retinoic acid, in organ development since tissue architecture is maintained. This technique allows controlled supplementation of culture medium and straightforward analysis of its effect on morphogenesis. This chapter describes the detailed protocol for culturing embryonic chick lung explants and testing the impact of retinoic acid in branching and patterning, based on morphometric and molecular analysis.
Assuntos
Pulmão/crescimento & desenvolvimento , Morfogênese/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Padronização Corporal/efeitos dos fármacos , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hibridização In Situ , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Técnicas de Cultura de Órgãos , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOXB1/genéticaRESUMO
OBJECTIVE: Juvenile ischemic osteonecrosis (JIO) of the femoral head is one of the most serious hip disorders causing a permanent deformity of the femoral head in childhood. We recently reported that interleukin 6 (IL6) is predominantly increased in the hip synovial fluid of patients with JIO and that articular chondrocytes are primary source of IL6. This study investigated whether an inhibition of IL6 receptor improves cartilage preservation and bone healing in JIO. METHOD: A small animal model (i.e., 6-week-old mouse) of JIO was treated with either saline or tocilizumab, an IL6 receptor blocker, for 6 weeks. RESULTS: TUNEL-positive chondrocytes in the articular cartilage were reduced by the tocilizumab treatment, concomitant with the increase in cartilage matrix. The levels of a cartilage anabolic marker Sox9 was significantly increased in the articular cartilage of mice treated with tocilizumab. Micro-CT assessment showed tocilizumab treatment significantly increased trabecular epiphyseal bone volume (P = 0.001, n = 10), thickness (P = 0.007) and number (P = 0.014) and decreased bone separation (P = 0.002) and its deformity (P = 0.003). A bone formation marker, BMP2, and an angiogenic marker, vascular endothelial growth factor (VEGF), were both significantly increased by tocilizumab treatment under hypoxia using human chondrocytes while the bone resorption marker, RANKL/OPG ratio, was reduced. CONCLUSION: Tocilizumab treatment following ischemic osteonecrosis has cartilage anabolic effect and increases bone volume in JIO mouse model. The findings lead to a possible application of tocilizumab for preclinical study using a large animal model of JIO and a clinical trial to validate this treatment.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Remodelação Óssea/efeitos dos fármacos , Cartilagem Articular/patologia , Osteonecrose/patologia , Receptores de Interleucina-6/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Fêmur/metabolismo , Fêmur/patologia , Interleucina-6/metabolismo , Metabolismo/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular/métodos , Osteonecrose/tratamento farmacológico , Receptores de Interleucina-6/fisiologia , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Regulação para Cima/efeitos dos fármacos , Microtomografia por Raio-XRESUMO
BACKGROUND & AIMS: Upon liver injury in which hepatocyte proliferation is compromised, liver progenitor cells (LPCs), derived from biliary epithelial cells (BECs), differentiate into hepatocytes. Little is known about the mechanisms of LPC differentiation. We used zebrafish and mouse models of liver injury to study the mechanisms. METHODS: We used transgenic zebrafish, Tg(fabp10a:CFP-NTR), to study the effects of compounds that alter epigenetic factors on BEC-mediated liver regeneration. We analyzed zebrafish with disruptions of the histone deacetylase 1 gene (hdac1) or exposed to MS-275 (an inhibitor of Hdac1, Hdac2, and Hdac3). We also analyzed zebrafish with mutations in sox9b, fbxw7, kdm1a, and notch3. Zebrafish larvae were collected and analyzed by whole-mount immunostaining and in situ hybridization; their liver tissues were collected for quantitative reverse transcription polymerase chain reaction. We studied mice in which hepatocyte-specific deletion of ß-catenin (Ctnnb1flox/flox mice injected with Adeno-associated virus serotype 8 [AAV8]-TBG-Cre) induces differentiation of LPCs into hepatocytes after a choline-deficient, ethionine-supplemented (CDE) diet. Liver tissues were collected and analyzed by immunohistochemistry and immunoblots. We performed immunohistochemical analyses of liver tissues from patients with compensated or decompensated cirrhosis or acute on chronic liver failure (n = 15). RESULTS: Loss of Hdac1 activity in zebrafish blocked differentiation of LPCs into hepatocytes by increasing levels of sox9b mRNA and reduced differentiation of LPCs into BECs by increasing levels of cdk8 mRNA, which encodes a negative regulator gene of Notch signaling. We identified Notch3 as the receptor that regulates differentiation of LPCs into BECs. Loss of activity of Kdm1a, a lysine demethylase that forms repressive complexes with Hdac1, produced the same defects in differentiation of LPCs into hepatocytes and BECs as observed in zebrafish with loss of Hdac1 activity. Administration of MS-275 to mice with hepatocyte-specific loss of ß-catenin impaired differentiation of LPCs into hepatocytes after the CDE diet. HDAC1 was expressed in reactive ducts and hepatocyte buds of liver tissues from patients with cirrhosis. CONCLUSIONS: Hdac1 regulates differentiation of LPCs into hepatocytes via Sox9b and differentiation of LPCs into BECs via Cdk8, Fbxw7, and Notch3 in zebrafish with severe hepatocyte loss. HDAC1 activity was also required for differentiation of LPCs into hepatocytes in mice with liver injury after the CDE diet. These pathways might be manipulated to induce LPC differentiation for treatment of patients with advanced liver diseases.
Assuntos
Ductos Biliares/enzimologia , Diferenciação Celular , Proliferação de Células , Quinase 8 Dependente de Ciclina/metabolismo , Hepatócitos/enzimologia , Histona Desacetilase 1/metabolismo , Regeneração Hepática , Fígado/enzimologia , Fatores de Transcrição SOX9/metabolismo , Células-Tronco/enzimologia , Proteínas de Peixe-Zebra/metabolismo , Insuficiência Hepática Crônica Agudizada/enzimologia , Insuficiência Hepática Crônica Agudizada/patologia , Animais , Ductos Biliares/patologia , Deficiência de Colina/genética , Deficiência de Colina/metabolismo , Deficiência de Colina/patologia , Quinase 8 Dependente de Ciclina/genética , Modelos Animais de Doenças , Proteína 7 com Repetições F-Box-WD/genética , Proteína 7 com Repetições F-Box-WD/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Hepatócitos/patologia , Histona Desacetilase 1/genética , Humanos , Fígado/patologia , Cirrose Hepática/enzimologia , Cirrose Hepática/patologia , Camundongos Knockout , Mutação , Receptor Notch3/genética , Receptor Notch3/metabolismo , Fatores de Transcrição SOX9/genética , Transdução de Sinais , Células-Tronco/patologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Although the regeneration of the adult liver depends on hepatic progenitor cells (HPCs), many uncertainties regarding hepatic regeneration in the injured liver remain. Trefoil factor family 1 (TFF1), a secretory protein predominantly expressed in the gastrointestinal tract, is responsible for mucosal restitution. Here, we investigated the role of TFF1 in liver regeneration using a mouse model of hepatic injury (choline-deficient ethionine-supplemented diet and carbon tetrachloride administration) and genetically engineered mice (TFF1 knockout (TFF1-/-)). Immunohistochemistry analysis of human liver samples revealed TFF1 expression in the hepatocytes close to ductular reaction and the regenerating biliary epithelium in injured liver. The number of cytokeratin 19 (CK19)-positive bile ducts was significantly decreased in the TFF1-/- mice after liver injury. Notch pathway in the TFF1-/- mice was also downregulated. HPCs in the control mice differentiated into biliary cells (CK19+/SRY HMG box 9 (SOX9)+) more frequently. In contrast, HPCs in the TFF1-/- mice more frequently differentiated into a hepatic lineage (alpha fetoprotein+/SOX9+) after acute liver damage. Hepatocyte proliferation was upregulated, and the liver weight was increased in TFF1-/- mice in response to chronic liver damage. Thus, TFF1 is responsible for liver regeneration after liver injury by promoting HPC differentiation into a biliary lineage and inhibiting HPC differentiation into a hepatic lineage.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/genética , Hepatócitos/metabolismo , Regeneração Hepática/genética , Células-Tronco/metabolismo , Fator Trefoil-1/genética , Animais , Ductos Biliares/citologia , Ductos Biliares/efeitos dos fármacos , Ductos Biliares/metabolismo , Tetracloreto de Carbono/administração & dosagem , Carcinógenos/administração & dosagem , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/secundário , Diferenciação Celular , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Deficiência de Colina/genética , Deficiência de Colina/metabolismo , Deficiência de Colina/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Dieta/efeitos adversos , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Etionina/administração & dosagem , Regulação da Expressão Gênica , Hepatite Crônica/genética , Hepatite Crônica/metabolismo , Hepatite Crônica/patologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Queratina-19/genética , Queratina-19/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Regeneração Hepática/efeitos dos fármacos , Camundongos , Camundongos Knockout , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Fator Trefoil-1/deficiênciaRESUMO
BACKGROUND/AIMS: Fibulin-3, an extracellular matrix glycoprotein, inhibits vascular oxidative stress and remodeling in hypertension. Oxidative stress is prevalent in chronic kidney disease (CKD) patients and is an important mediator of osteo-/chondrogenic transdifferentiation and calcification of vascular smooth muscle cells (VSMCs) during hyperphosphatemia. Therefore, the present study explored the effects of Fibulin-3 on phosphate-induced vascular calcification. METHODS: Experiments were performed in primary human aortic smooth muscle cells (HAoSMCs) treated with control or with phosphate without or with additional treatment with recombinant human Fibulin-3 protein or with hydrogen peroxide as an exogenous source of oxidative stress. RESULTS: Treatment with calcification medium significantly increased calcium deposition in HAoSMCs, an effect significantly blunted by additional treatment with Fibulin-3. Moreover, phosphate-induced alkaline phosphatase activity and mRNA expression of osteogenic and chondrogenic markers MSX2, CBFA1, SOX9 and ALPL were all significantly reduced by addition of Fibulin-3. These effects were paralleled by similar regulation of oxidative stress in HAoSMCs. Phosphate treatment significantly up-regulated mRNA expression of the oxidative stress markers NOX4 and CYBA, down-regulated total antioxidant capacity and increased the expression of downstream effectors of oxidative stress PAI-1, MMP2 and MMP9 as well as BAX/BLC2 ratio in HAoSMCs, all effects blocked by additional treatment with Fibulin-3. Furthermore, the protective effects of Fibulin-3 on phosphate-induced osteogenic and chondrogenic markers expression in HAoSMCs were reversed by additional treatment with hydrogen peroxide. CONCLUSIONS: Fibulin-3 attenuates phosphate-induced osteo-/ chondrogenic transdifferentiation and calcification of VSMCs, effects involving inhibition of oxidative stress. Up-regulation or supplementation of Fibulin-3 may be beneficial in reducing the progression of vascular calcification during hyperphosphatemic conditions such as CKD.
Assuntos
Calcificação Fisiológica/efeitos dos fármacos , Proteínas da Matriz Extracelular/farmacologia , Glicerofosfatos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Linhagem Celular , Transdiferenciação Celular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Osteogênese/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismoRESUMO
BACKGROUND: Spinacia oleracea is an important dietary vegetable in India and throughout the world and has many beneficial effects. It is cultivated globally. However, its effect on osteoarthritis that mainly targets the cartilage cells remains unknown. In this study we aimed to evaluate the anti-osteoarthritic and chondro-protective effects of SOE on chemically induced osteoarthritis (OA). METHODS: OA was induced by intra-patellar injection of monosodium iodoacetate (MIA) at the knee joint in rats. SOE was then given orally at 250 and 500 mg.kg- 1 day- 1 doses for 28 days to these rats. Anti-osteoarthritic potential of SOE was evaluated by micro-CT, mRNA and protein expression of pro-inflammatory and chondrogenic genes, clinically relevant biomarker's and behavioural experiments. RESULTS: In vitro cell free and cell based assays indicated that SOE acts as a strong anti-oxidant and an anti-inflammatory agent. Histological analysis of knee joints at the end of the experiment by safranin-o and toluidine blue staining established its protective effect. Radiological data corroborated the findings with improvement in the joint space and irregularity of the articular and atrophied femoral condyles and tibial plateau. Micro-CT analysis of sub-chondral bone indicated that SOE had the ability to mitigate OA effects by increasing bone volume to tissue volume (BV/TV) which resulted in decrease of trabecular pattern factor (Tb.Pf) by more than 200%. SOE stimulated chondrogenic marker gene expression with reduction in pro-inflammatory markers. Purified compounds isolated from SOE exhibited increased Sox-9 and Col-II protein expression in articular chondrocytes. Serum and urine analysis indicated that SOE had the potential to down-regulate glutathione S-transferase (GST) activity, clinical markers of osteoarthritis like cartilage oligometric matrix protein (COMP) and CTX-II. Overall, this led to a significant improvement in locomotion and balancing activity in rats as assessed by Open-field and Rota rod test. CONCLUSION: On the basis of in vitro and in vivo experiments performed with Spinacea oleracea extract we can deduce that SOE has the ability to alleviate the MIA induced deleterious effects.
Assuntos
Osteoartrite/tratamento farmacológico , Extratos Vegetais/administração & dosagem , Spinacia oleracea/química , Animais , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Índia , Iodoacetatos/efeitos adversos , Articulação do Joelho/efeitos dos fármacos , Articulação do Joelho/metabolismo , Articulação do Joelho/patologia , Osteoartrite/induzido quimicamente , Osteoartrite/genética , Osteoartrite/metabolismo , Extratos Vegetais/química , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Tíbia/efeitos dos fármacos , Tíbia/metabolismo , Tíbia/patologiaRESUMO
Chondrosarcoma is the second most malignant bone tumor with poor prognosis and limited treatment options. Thus, development of more effective treatments has become urgent. Recently, natural compounds derived from medicinal plants have emerged as promising therapeutic options via targeting multiple key cellular molecules. Andrographolide (Andro) is such a compound, which has previously been shown to induce cell cycle arrest and apoptosis in several human cancers. However, the molecular mechanism through which Andro exerts its anti-cancer effect on chondrosarcoma remains to be elucidated. In the present study, we showed that Andro-induced G2/M cell cycle arrest of chondrosarcoma by fine-tuning the expressions of several cell cycle regulators such as p21, p27, and Cyclins, and that prolonged treatment of cells with Andro caused pronounced cell apoptosis. Remarkably, we found that SOX9 was highly expressed in poor-differentiated chondrosarcoma, and that knockdown of SOX9 suppressed chondrosarcoma cell growth. Further, our results showed that Andro dose-dependently down-regulated SOX9 expression in chondrosarcoma cells. Concomitantly, an inhibition of T cell factor 1 (TCF-1) mRNA expression and an enhancement of TCF-1 protein degradation by Andro were observed. In contrast, the expression and subcellular localization of ß-catenin were not altered upon the treatment of Andro, suggesting that ß-catenin might not function as the primary target of Andro. Additionally, we provided evidence that there was a mutual regulation between TCF-1 and SOX9 in chondrosarcoma cells. In conclusion, these results highlight the potential therapeutic effects of Andro in treatment of chondrosarcoma via targeting the TCF-1/SOX9 axis. J. Cell. Biochem. 118: 4575-4586, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Ósseas/tratamento farmacológico , Condrossarcoma/tratamento farmacológico , Diterpenos/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fator 1 de Transcrição de Linfócitos T/metabolismo , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Condrossarcoma/genética , Condrossarcoma/metabolismo , Condrossarcoma/patologia , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/genética , Proteínas de Neoplasias/genética , Fatores de Transcrição SOX9/genética , Fator 1 de Transcrição de Linfócitos T/genéticaRESUMO
The aim of the present study was to develop chick-embryo chorioallantoic membrane (CAM) bioluminescent tumor models employing low passage cell cultures obtained from primary pancreatic ductal adenocarcinoma (PDAC) cells. Primary PDAC cells transduced with lentivirus expressing Firefly-luciferase (Fluc) were established and inoculated onto the CAM membrane, with >80% engraftment. Fluc signal reliably correlated with tumor growth. Tumor features were evaluated by immunohistochemistry and genetic analyses, including analysis of mutations and mRNA expression of PDAC pivotal genes, as well as microRNA (miRNA) profiling. These studies showed that CAM tumors had histopathological and genetic characteristic comparable to the original tumors. We subsequently tested the modulation of key miRNAs and the activity of gemcitabine and crizotinib on CAM tumors, showing that combination treatment resulted in 63% inhibition of tumor growth as compared to control (p < 0.01). These results were associated with reduced expression of miR-21 and increased expression of miR-155. Our study provides the first evidence that transduced primary PDAC cells can form tumors on the CAM, retaining several histopathological and (epi)genetic characteristics of original tumors. Moreover, our results support the use of these models for drug testing, providing insights on molecular mechanisms underlying antitumor activity of new drugs/combinations.
Assuntos
Membrana Corioalantoide/metabolismo , Avaliação Pré-Clínica de Medicamentos , Medições Luminescentes/métodos , Modelos Biológicos , Neoplasias Pancreáticas/patologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Carcinogênese/patologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Galinhas , Crizotinibe/farmacologia , Crizotinibe/uso terapêutico , Análise Mutacional de DNA , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Fator 6 Nuclear de Hepatócito/genética , Fator 6 Nuclear de Hepatócito/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Análise de Sequência de DNA , Células Tumorais Cultivadas , Gencitabina , Neoplasias PancreáticasRESUMO
Psoriasis is a chronic inflammatory skin disorder. The aim of this study was to determine a potential role of microRNA (miR)-130a in psoriasis, and underlying mechanism. Expression levels of miR-130a in psoriasis specimens and normal skin tissues were analyzed. MiR-130a mimic, inhibitor, miR-control, small interfering RNA (siRNA) specific serine/threonine kinase 40 (STK40), or sex-determining region Y chromosome-box 9 (SOX9) were transfected to human keratinocyte HaCaT cells, respectively. After transfection, the cell viability, apoptosis, and migration were determined. Luciferase reporter assay, quantitative reverse transcription-polymerase chain reaction, and western blot were performed to explore whether STK40 was a target of miR-130a. The effects of aberrant expressions of miR-130a, STK40, or SOX9 on key proteins of NF-κB and c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase (MAPK) pathway were assessed. The miR-130a levels were significantly higher in patients with psoriasis compared to the healthy controls (p < 0.01). Overexpressing miR-130a strikingly promoted HaCaT cell viability and migration and inhibited apoptosis (p < 0.01 or p < 0.05). We confirmed that STK40 was a direct target of miR-130a, and STK40 was involved in miR-130a-induced cell functions. Overexpressing miR-130a significantly upregulated NF-κB p65, SOX9, p-c-Jun, p-JNK, and p-p38MAPK proteins and silencing miR-130a downregulated them. In addition, silencing STK40 alleviated the effects of anti-miR-130a on SOX9 expression. Furthermore, silencing SOX9 also decreased levels of p-c-Jun, p-JNK, and p-p38MAPK proteins. MiR-130a regulates human keratinocyte HaCaT viability, migration and apoptosis might be by direct regulation of STK40-mediated NF-κB pathway and by indirect regulation of SOX9-mediated downstream JNK/MAPK signaling pathway.
Assuntos
Apoptose/genética , Movimento Celular/genética , Queratinócitos/metabolismo , MicroRNAs/genética , NF-kappa B/genética , Proteínas Quinases/genética , Fatores de Transcrição SOX9/genética , Regiões 3' não Traduzidas/genética , Adolescente , Adulto , Linhagem Celular , Sobrevivência Celular/genética , Feminino , Regulação da Expressão Gênica , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Queratinócitos/citologia , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Psoríase/genética , Psoríase/patologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais/genética , Adulto Jovem , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Inhibitors of the mechanistic target of rapamycin (mTOR) are currently used to treat advanced metastatic breast cancer. However, whether an aggressive phenotype is sustained through adaptation or resistance to mTOR inhibition remains unknown. Here, complementary studies in human tumors, cancer models and cell lines reveal transcriptional reprogramming that supports metastasis in response to mTOR inhibition. This cancer feature is driven by EVI1 and SOX9. EVI1 functionally cooperates with and positively regulates SOX9, and promotes the transcriptional upregulation of key mTOR pathway components (REHB and RAPTOR) and of lung metastasis mediators (FSCN1 and SPARC). The expression of EVI1 and SOX9 is associated with stem cell-like and metastasis signatures, and their depletion impairs the metastatic potential of breast cancer cells. These results establish the mechanistic link between resistance to mTOR inhibition and cancer metastatic potential, thus enhancing our understanding of mTOR targeting failure.
Assuntos
Neoplasias da Mama/genética , Proteínas de Ligação a DNA/genética , Neoplasias Pulmonares/genética , Proto-Oncogenes/genética , Fatores de Transcrição SOX9/genética , Serina-Treonina Quinases TOR/genética , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Idoso , Neoplasias da Mama/patologia , Proteínas de Transporte/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Células MCF-7 , Proteína do Locus do Complexo MDS1 e EVI1 , Proteínas dos Microfilamentos/genética , Pessoa de Meia-Idade , Metástase Neoplásica , Osteonectina/genética , Proteína Regulatória Associada a mTOR , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
There is little evidence demonstrating the effects of electromagnetic fields (EMFs) generated within the biological entity and the effect of extrinsic fields on cellular programing. Taking the path of the more studied stimuli into attention, mechanical forces, it could be understood that nonchemical factors play a consequential role in transcriptional regulatory networks. Cartilaginous tissue consists of collagen protein that is considered as a piezoelectric substrate and is influenced by electric fields making chondrogenic specific genes an exciting candidate for bioelectromagnetic studies. As electromagnetic properties highly depend on the frequencies applied, this study delves into the ability of two EMFs with the frequency of 25 Hz and 50 Hz in inducing SOX9 and COL2 gene expressions in a three-dimensional (3D) mesenchymal stem cell (MSC)-alginate construct. Cell-alginate beads were divided into six groups and treated for a time period of 21 days. To determine the results, qualitative and quantitative data were both reviewed. On observation of real-time polymerase chain reaction (PCR) data, it was apparent that TGF-ß1 treatment had a greater COL2 and SOX9 gene expression impact on MSCs compared to pulsed electromagnetic field (PEMF) treatments alone. COL2 was shown to have a greater transcriptional tendency to PEMF, whereas under defined electromagnetic parameters applied in this study, no significant difference was detected in SOX9 gene expressions compared to the control group. PEMF co-treatments enhanced the deposition of extracellular matrix molecules, as the matrix-rich beads were positively stained by Alcian blue. This genre of study is the venue for the control and healing of connective tissue defects.
Assuntos
Condrogênese , Regulação da Expressão Gênica , Células-Tronco Mesenquimais/citologia , Alginatos/química , Animais , Células Cultivadas , Campos Eletromagnéticos , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Células-Tronco Mesenquimais/metabolismo , Coelhos , Fatores de Transcrição SOX9/genética , Alicerces Teciduais/químicaRESUMO
Here we report the successful generation and long-term expansion of SOX9-expressing CD271(+)PDGFRα(+)CD73(+) chondrogenic ectomesenchymal cells from the PAX3/SOX10/FOXD3-expressing MIXL1(-)CD271(hi)PDGFRα(lo)CD73(-) neural crest-like progeny of human pluripotent stem cells in a chemically defined medium supplemented with Nodal/Activin/transforming growth factorß (TGFß) inhibitor and fibroblast growth factor (FGF). When "primed" with TGFß, such cells efficiently formed translucent cartilage particles, which were completely mineralized in 12 weeks in immunocompromized mice. The ectomesenchymal cells were expandable without loss of chondrogenic potential for at least 16 passages. They maintained normal karyotype for at least 10 passages and expressed genes representing embryonic progenitors (SOX4/12, LIN28A/B), cranial mesenchyme (ALX1/3/4), and chondroprogenitors (SOX9, COL2A1) of neural crest origin (SOX8/9, NGFR, NES). Ectomesenchyme is a source of many craniofacial bone and cartilage structures. The method we describe for obtaining a large quantity of human ectomesenchymal cells will help to model craniofacial disorders in vitro and potentially provide cells for the repair of craniofacial damage.
Assuntos
Diferenciação Celular , Condrogênese , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição SOX9/metabolismo , Antígenos de Superfície/metabolismo , Biomarcadores , Cartilagem , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células , Autorrenovação Celular/genética , Condrogênese/efeitos dos fármacos , Condrogênese/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imunofenotipagem , Crista Neural/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Fatores de Transcrição SOX9/genética , Transdução de Sinais , Fator de Crescimento Transformador beta/farmacologiaRESUMO
INTRODUCTION: Runx2 is one of the most studied transcription factors expressed in mesenchymal stem cells (MSCs) upon their commitment toward an osteogenic differentiation. During endochondral bone formation in vivo, Sox9 directly interacts with Runx2 and represses its activity; however, the role of Sox9 in direct osteogenesis in vitro has been largely overlooked. METHODS: Bone marrow-derived human MSCs (hMSCs) were cultured in vitro either in the control or osteogenic medium supplemented with dexamethasone (DEX). To further investigate the role of Sox9 in direct osteogenesis in vitro, hMSCs were treated with Sox9 siRNA. RESULTS: We show here that Sox9 is the key early indicator during in vitro osteogenic differentiation of hMSCs. Osteogenic induction leads to a significant decrease of Sox9 gene and protein expression by day 7. Treatment of hMSCs with Sox9 siRNA enhanced mineralization in vitro, suggesting that downregulation of Sox9 is involved in direct osteogenesis. siRNA knockdown of Sox9 did not in itself induce osteogenesis in the absence of DEX, indicating that other factors are still required. CONCLUSION: Screening of not preselected donors of different ages and gender (n=12) has shown that the Runx2/Sox9 ratio on day 7 is correlated to the (45)Ca incorporation on day 28. The impact of Sox9 downregulation in the mineralization of human MSCs in vitro indicates a so far unprecedented role of Sox9 as a major regulator of direct osteogenesis. We propose that the Runx2/Sox9 ratio is a promising, early, in vitro screening method for osteogenicity of human MSCs.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Fatores de Transcrição SOX9/metabolismo , Adulto , Idoso , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Dexametasona/farmacologia , Regulação para Baixo/efeitos dos fármacos , Eletroporação , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Pessoa de Meia-Idade , Osteogênese/efeitos dos fármacos , Osteogênese/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição SOX9/genética , Doadores de Tecidos , Adulto JovemRESUMO
To deliver efficiently osteogenic, chondrogenic or adipogenic induction genes, such as Runx2, SOX9 and C/EBP-α, to adipose tissue-derived stem cells (ADSCs), a photo-mediated nanocomplex internalization gene delivery system was designed using chlorin e6 as a photosensitizer (PS) and polyethyleneimine (PEI) as a gene delivery carrier. In this system, gene delivery efficacy was significantly increased in ADSCs by photo irradiation. The gene transfection efficiency of Runx2, SOX9 and C/EBP-α was increased by 8.6-, 6.7- and 9.3-fold, respectively, by applying 0.7J/cm(2) of irradiation. Osteogenic, chondrogenic and adipogenic differentiation was confirmed by differentiation-related markers and histological analysis. ADSCs transfected with Runx2, SOX9 and C/EBP-α genes via photo irradiation indicated enhanced differentiation in comparison to the non-irradiated cells. These findings demonstrate that photo-mediated internalization is a promising system for efficient gene delivery and differentiation in ADSCs.
Assuntos
Tecido Adiposo/citologia , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Técnicas de Transferência de Genes , Fototerapia/métodos , Adipogenia/genética , Células-Tronco Adultas/efeitos da radiação , Proteínas Estimuladoras de Ligação a CCAAT/genética , Técnicas de Cultura de Células/métodos , Diferenciação Celular/genética , Diferenciação Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Condrogênese/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Proteínas de Fluorescência Verde/genética , Humanos , Nanoestruturas , Osteogênese/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição SOX9/genética , TransfecçãoRESUMO
BACKGROUND: Mesenchymal stem cells (MSC) are multipotent progenitor cells localized in the stromal compartment of the bone marrow (BM). The potential of MSC for mesenchymal differentiation has been well documented in different animal models predominantly on rodents. However, information regarding bovine MSC (bMSC) is limited, and the differentiation potential of bMSC derived from fetal BM remains unknown. In the present study we sought to isolate bMSC from abattoir-derived fetal BM and to characterize the multipotent and differentiation potential under osteogenic, chondrogenic and adipogenic conditions by quantitative and qualitative analyses. RESULTS: Plastic-adherent bMSC isolated from fetal BM maintained a fibroblast-like morphology under monolayer culture conditions. These cells expressed high levels of MSC surface markers (CD73, CD90, and CD105) and low levels of hematopoietic surface markers (CD34 and CD45). Culture of bMSC under osteogenic conditions during a 27-day period induced up-regulation of the osteocalcin (OC) gene expression and alkaline phosphatase (ALPL) activity, and promoted mineralization of the matrix. Increasing supplementation levels of ascorbic acid to culture media enhanced osteogenic differentiation of bMSC; whereas, reduction of FBS supplementation compromised osteogenesis. bMSC increased expression of cartilage-specific genes aggrecan (ACAN), collagen 2A1 (COL2A1) and SRY (sex-determining region Y) box 9 (SOX9) at Day 21 of chondrogenic differentiation. Treatment of bMSC with adipogenic factors increased levels of fatty acid-binding protein 2 (AP2) mRNA and accumulation of lipid vacuoles after 18 days of culture. NANOG mRNA levels in differentiating bMSC were not affected during adipogenic culture; however, osteogenic and chondrogenic conditions induced higher and lower levels, respectively. CONCLUSIONS: Our analyses revealed the potential multilineage differentiation of bMSC isolated from abattoir-derived fetal BM. NANOG mRNA pattern in differentiating bMSC varied according to differentiation culture conditions. The osteogenic differentiation of bMSC was affected by ascorbic acid and FBS concentrations in culture media. The simplicity of isolation and the differentiation potential suggest that bMSC from abattoir-derived fetal BM are appropriate candidate for investigating MSC biology and for eventual applications for regenerative therapy.
Assuntos
Adipogenia/fisiologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/fisiologia , Condrogênese/fisiologia , Células-Tronco Mesenquimais/fisiologia , Osteogênese/fisiologia , Adipogenia/genética , Animais , Células da Medula Óssea/citologia , Bovinos , Diferenciação Celular/genética , Condrogênese/genética , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Feto , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células-Tronco Mesenquimais/citologia , Microscopia de Fluorescência , Fatores de Transcrição de Octâmero/genética , Fatores de Transcrição de Octâmero/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/genética , PPAR gama/genética , PPAR gama/metabolismo , RNA/química , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismoRESUMO
OBJECTIVE: Calcific aortic valve disease (CAVD) is a major public health problem with no effective treatment available other than surgery. We previously showed that mature heart valves calcify in response to retinoic acid (RA) treatment through downregulation of the SRY transcription factor Sox9. In this study, we investigated the effects of excess vitamin A and its metabolite RA on heart valve structure and function in vivo and examined the molecular mechanisms of RA signaling during the calcification process in vitro. METHODS AND RESULTS: Using a combination of approaches, we defined calcific aortic valve disease pathogenesis in mice fed 200 IU/g and 20 IU/g of retinyl palmitate for 12 months at molecular, cellular, and functional levels. We show that mice fed excess vitamin A develop aortic valve stenosis and leaflet calcification associated with increased expression of osteogenic genes and decreased expression of cartilaginous markers. Using a pharmacological approach, we show that RA-mediated Sox9 repression and calcification is regulated by classical RA signaling and requires both RA and retinoid X receptors. CONCLUSIONS: Our studies demonstrate that excess vitamin A dietary intake promotes heart valve calcification in vivo. Therefore suggesting that hypervitaminosis A could serve as a new risk factor of calcific aortic valve disease in the human population.