RESUMEN
Eye size is a key parameter of visual function, but the precise mechanisms of eye size control remain poorly understood. Here, we discovered that the lipogenic transcription factor sterol regulatory element-binding protein 2 (SREBP2) has an unanticipated function in the retinal pigment epithelium (RPE) to promote eye size in postnatal mice. SREBP2 transcriptionally represses low density lipoprotein receptor-related protein 2 (Lrp2), which has been shown to restrict eye overgrowth. Bone morphogenetic protein 2 (BMP2) is the downstream effector of Srebp2 and Lrp2, and Bmp2 is suppressed by SREBP2 transcriptionally but activated by Lrp2. During postnatal development, SREBP2 protein expression in the RPE decreases whereas that of Lrp2 and Bmp2 increases as the eye growth rate reduces. Bmp2 is the key determinant of eye size such that its level in mouse RPE inversely correlates with eye size. Notably, RPE-specific Bmp2 overexpression by adeno-associated virus effectively prevents the phenotypes caused by Lrp2 knock out. Together, our study shows that rapid postnatal eye size increase is governed by an RPE-derived signaling pathway, which consists of both positive and negative regulators of eye growth.
Asunto(s)
Proteína Morfogenética Ósea 2 , Proteína 2 de Unión a Elementos Reguladores de Esteroles , Animales , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Regulación de la Expresión Génica , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Epitelio Pigmentado de la Retina/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismoRESUMEN
Heart failure has become a major life-threatening cause affecting millions globally, characterized by the permanent loss of adult functional cardiomyocytes leading to fibrosis which ultimately deprives the heart of its functional efficacy. Here we investigated the reparative property of embryonic and adult epicardial cells towards cardiomyocyte differentiation under oxidative stress-induced conditions along with the identification of a possible molecular signaling pathway. Isolated epicardial cells from embryonic chick hearts subjected to oxidative stress and hypoxia induction. Initial assessment of successful injury induction reveals hypertrophy of isolated epicardial cells. Detailed marker gene expression analyses and inhibitor studies reveal Bone morphogenic protein (Bmp)2-Smad1/5/8 signaling dependent cardiomyocyte lineage specification via epithelial to mesenchymal transition (EMT) post-injury. EMT is further confirmed by increased proliferation, migration, and differentiation towards cardiomyocyte lineage. We have also established an in-vivo model in adult male rats using Isoproterenol. Successful oxidative stress-mediated injury induction in adult heart was marked by increased activated fibroblasts followed by apoptosis of adult cardiomyocytes. The detailed characterization of adult epicardial cells reveals similar findings to our avian in-vitro data. Both in-vitro and in-vivo results show a significant increase in the expression of cardiomyocyte specific markers indicative of lineage specificity and activation of epicardial cells post oxidative stress mediated injury. Our findings suggest an EMT-induced reactivation of epicardial cells and early cardiomyocyte lineage specification following oxidative stress in a Bmp2- Smad1/5/8 dependent manner. Overall, this regulatory mechanism of cardiomyocyte differentiation induced by oxidative stress may contribute to the field of cardiac repair and regenerative therapeutics.
Asunto(s)
Transición Epitelial-Mesenquimal , Miocitos Cardíacos , Masculino , Ratas , Animales , Miocitos Cardíacos/metabolismo , Transición Epitelial-Mesenquimal/genética , Diferenciación Celular/genética , Transducción de Señal , Células Cultivadas , Proteína Smad1/genética , Proteína Smad1/metabolismoRESUMEN
Vascular calcification is a pathological process commonly associated with atherosclerosis, chronic kidney disease, and diabetes. Paraspeckle protein NONO is a multifunctional RNA/DNA binding protein involved in many nuclear biological processes but its role in vascular calcification remains unclear. Here, we observed that NONO expression was decreased in calcified arteries of mice and patients with CKD. We generated smooth muscle-specific NONO-knockout mice and established three different mouse models of vascular calcification by means of 5/6 nephrectomy, adenine diet to induce chronic kidney failure, or vitamin D injection. The knockout mice were more susceptible to the development of vascular calcification relative to control mice, as verified by an increased calcification severity and calcium deposition. Likewise, aortic rings from knockout mice showed more significant vascular calcification than those from control mice ex vivo. In vitro, NONO deficiency aggravated high phosphate-induced vascular smooth muscle cell osteogenic differentiation and apoptosis, whereas NONO overexpression had a protective effect. Mechanistically, we demonstrated that the regulation of vascular calcification by NONO was mediated by bone morphogenetic protein 2 (BMP2). NONO directly bound to the BMP2 promoter using its C-terminal region, exerting an inhibitory effect on the transcription of BMP2. Thus, our study reveals that NONO is a novel negative regulator of vascular calcification, which inhibits osteogenic differentiation of vascular smooth muscle cell and vascular calcification via negatively regulating BMP2 transcription. Hence, NONO may provide a promising target for the prevention and treatment of vascular calcification.
Asunto(s)
Proteína Morfogenética Ósea 2 , Modelos Animales de Enfermedad , Ratones Noqueados , Músculo Liso Vascular , Miocitos del Músculo Liso , Osteogénesis , Insuficiencia Renal Crónica , Transcripción Genética , Calcificación Vascular , Animales , Humanos , Masculino , Ratones , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/prevención & control , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/metabolismo , Apoptosis/efectos de los fármacos , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 2/genética , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Ratones Endogámicos C57BL , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Miocitos del Músculo Liso/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Regiones Promotoras Genéticas , Insuficiencia Renal Crónica/patología , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/prevención & control , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Calcificación Vascular/patología , Calcificación Vascular/prevención & control , Calcificación Vascular/metabolismo , Calcificación Vascular/genética , Calcificación Vascular/etiologíaRESUMEN
BACKGROUND: Antibiotic-containing carrier systems are one option that offers the advantage of releasing active ingredients over a longer period of time. In vitro sustained drug release from a carrier system consisting of microporous ß-TCP ceramic and alginate has been reported in previous works. Alginate dialdehyde (ADA) gelatin gel showed both better mechanical properties when loaded into a ß-TCP ceramic and higher biodegradability than pure alginate. METHODS: Dual release of daptomycin and BMP-2 was measured on days 1, 2, 3, 6, 9, 14, 21, and 28 by HPLC and ELISA. After release, the microbial efficacy of the daptomycin was verified and the biocompatibility of the composite was tested in cell culture. RESULTS: Daptomycin and the model compound FITC protein A (n = 30) were released from the composite over 28 days. A Daptomycin release above the minimum inhibitory concentration (MIC) by day 9 and a burst release of 71.7 ± 5.9% were observed in the loaded ceramics. Low concentrations of BMP-2 were released from the loaded ceramics over 28 days.
Asunto(s)
Antibacterianos , Proteína Morfogenética Ósea 2 , Fosfatos de Calcio , Cerámica , Daptomicina , Gelatina , Proteína Morfogenética Ósea 2/química , Proteína Morfogenética Ósea 2/metabolismo , Daptomicina/química , Daptomicina/farmacología , Gelatina/química , Cerámica/química , Antibacterianos/química , Antibacterianos/farmacología , Fosfatos de Calcio/química , Animales , Pruebas de Sensibilidad Microbiana , Ratones , Portadores de Fármacos/química , Liberación de FármacosRESUMEN
Our previous studies have shown that lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) is expressed in liver sinusoidal endothelial cells, and oxidized low-density lipoprotein induces liver sinusoidal dysfunction and defenestration through the LOX-1/ROS/NF-kB pathway, revealing that LOX-1 can mediate liver sinusoidal barrier function, involved in the regulation of non-alcoholic fatty liver disease. Here, we investigated whether, in the context of bone metabolic diseases, LOX-1 could affect bone quality and type H blood vessels in diabetic mice. We used db/db mice as model and found that LOX-1 knockdown can ameliorate bone quality and type H blood vessel generation in db/db mice. This further verifies our hypothesis that LOX-1 is involved in the regulation of bone quality and type H blood vessel homeostasis, thus inhibiting osteoporosis progression in db/db mice.
Asunto(s)
Diabetes Mellitus Experimental , Animales , Ratones , Diabetes Mellitus Experimental/metabolismo , Células Endoteliales/metabolismo , Lipoproteínas LDL/metabolismo , FN-kappa B/metabolismo , Receptores Depuradores de Clase E/genética , Receptores Depuradores de Clase E/metabolismoRESUMEN
Beta-tricalcium phosphate (ß-TCP) is considered as one of the most promising biomaterials for bone reconstruction. This study generated a functional molybdenum disulfide (MoS2 )/polydopamine (PDA)/-bone morphogenetic protein 2 (BMP2)-insulin-like growth factor-1 (IGF-1) coating on the ß-TCP scaffold and analyzed the outcomes. The MoS2 /PDA-BMP2-IGF-1@ß-TCP (MPBI@ß-TCP) scaffold was prepared by 3D printing and physical adsorption, followed by characterization to validate its successful construction. The in vitro osteogenic effect of the MPBI@ß-TCP scaffold was evaluated. It was found that MPBI@ß-TCP augmented the adhesion, diffusion and proliferation of mesenchymal stem cells (MSCs). The alkaline phosphatase (ALP) activity, collagen secretion and extracellular matrix (ECM) mineralization along with the expression of Runx2, ALP and OCN were also enhanced in the presence of MPBI@ß-TCP. Additionally, MPBI@ß-TCP stimulated endothelial cells to secrete VEGF and promoted capillary-like tubule formation. We then confirmed the biocompatibility of MPBI@ß-TCP to macrophages and its anti-inflammatory effects. Furthermore, under near-infrared (NIR) laser irradiation, MPBI@ß-TCP produced photothermal effect to not only kill MG-63 osteosarcoma cells, but also enhance bone regeneration in vivo with biosafety. Overall, this work demonstrates that 3D-printed MPBI@ß-TCP with enhanced osteogenic activity under NIR laser irradiation has a vast potential in the field of tissue defects.
Asunto(s)
Neoplasias Óseas , Osteosarcoma , Humanos , Molibdeno , Factor I del Crecimiento Similar a la Insulina/farmacología , Andamios del Tejido , Células Endoteliales , Regeneración Ósea , Osteogénesis , Osteosarcoma/radioterapia , Rayos Infrarrojos , Neoplasias Óseas/radioterapiaRESUMEN
Electrical stimulation (ES) plays an important role in regulating cell osteoblast differentiation. As a noninvasive rehabilitation therapy method, Es has a unique role in postoperative recovery. Bone morphogenetic protein-2 (BMP-2) is the most commonly used bioactive molecule in in situ tissue engineering scaffolds, and it plays an important regulatory role in the whole process of bone injury repair. In this study, the osteogenic regulation of MC-3T3-E1 cells was studied by combining pulsed electrical stimulation (PES) and different concentrations of BMP-2. The results showed that PES and BMP-2 could synergically promote the proliferation of MC-3T3-E1 cells. The qPCR results of osteoblast-related genes showed that PES was synergistic with BMP-2 to promote osteoblast differentiation mainly through the regulation of the Smad/BMP and insulin like growth factor 1 (IGF1) signaling pathways. The expression level of alkaline phosphatase (ALP) and alizarin red staining further demonstrated the synergistic effect of PES and BMP-2 on promoting osteogenic differentiation and mineralization of cells. PES and BMP-2 could also synergically promote cell proliferation, expression of collagen I (COL-I) and ALP, and cell mineralization on the 3D-printed polylactic acid scaffold. These results suggest that the use of PES can enhance the osteogenic effect of in situ bone repair scaffolds containing BMP-2, reduce the dose of BMP-2 alone, and reduce the possible side effects of high-dose BMP-2 in vivo.
RESUMEN
Dealing with bone defects is a significant challenge to global health. Electrospinning in bone tissue engineering has emerged as a solution to this problem. In this study, we designed a PVDF-b-PTFE block copolymer by incorporating TFE, which induced a phase shift in PVDF fromαtoß, thereby enhancing the piezoelectric effect. Utilizing the electrospinning process, we not only converted the material into a film with a significant surface area and high porosity but also intensified the piezoelectric effect. Then we used polydopamine to immobilize BMP-2 onto PVDF-b-PTFE electrospun nanofibrous membranes, achieving a controlled release of BMP-2. The scaffold's characters were examined using SEM and XRD. To assess its osteogenic effectsin vitro, we monitored the proliferation of MC3T3-E1 cells on the fibers, conducted ARS staining, and measured the expression of osteogenic genes.In vivo, bone regeneration effects were analyzed through micro-CT scanning and HE staining. ELISA assays confirmed that the sustained release of BMP-2 can be maintained for at least 28 d. SEM images and CCK-8 results demonstrated enhanced cell viability and improved adhesion in the experimental group. Furthermore, the experimental group exhibited more calcium nodules and higher expression levels of osteogenic genes, including COL-I, OCN, and RUNX2. HE staining and micro-CT scans revealed enhanced bone tissue regeneration in the defective area of the PDB group. Through extensive experimentation, we evaluated the scaffold's effectiveness in augmenting osteoblast proliferation and differentiation. This study emphasized the potential of piezoelectric PVDF-b-PTFE nanofibrous membranes with controlled BMP-2 release as a promising approach for bone tissue engineering, providing a viable solution for addressing bone defects.
Asunto(s)
Proteína Morfogenética Ósea 2 , Regeneración Ósea , Indoles , Nanofibras , Osteogénesis , Polímeros , Ingeniería de Tejidos , Andamios del Tejido , Proteína Morfogenética Ósea 2/farmacología , Proteína Morfogenética Ósea 2/metabolismo , Nanofibras/química , Regeneración Ósea/efectos de los fármacos , Animales , Ratones , Indoles/química , Indoles/farmacología , Polímeros/química , Polímeros/farmacología , Ingeniería de Tejidos/métodos , Osteogénesis/efectos de los fármacos , Andamios del Tejido/química , Proliferación Celular/efectos de los fármacos , Línea Celular , Proteínas Inmovilizadas/farmacología , Proteínas Inmovilizadas/química , Supervivencia Celular/efectos de los fármacosRESUMEN
Plant cells serve as versatile platforms for the production of high-value recombinant proteins. This study explored the efficacy of utilizing an endogenous αAmy3 promoter for the expression of a bioactive pharmaceutical protein, specifically the mature region of human bone morphogenetic protein 2 (hBMP2m). Utilizing a refined CRISPR/Cas9-mediated intron-targeting insertion technique, which incorporates an artificial 3' splicing site upstream of the target gene, we achieved a transformation efficiency of 13.5% in rice calli that carried the rice-codon optimized mature region of hBMP2 cDNA (rhBMP2m) in the αAmy3 intron 1. Both homozygous and heterozygous rhBMP2m knock-in rice suspension cell lines were generated. These lines demonstrated the endogenous αAmy3 promoter regulated rhBMP2m mRNA and rhBMP2m recombinant protein expression, with strongly upregulation in respond to sugar depletion. The homozygous rhBMP2m knock-in cell line yielded an impressive 21.5 µg/mL of rhBMP2m recombinant protein, accounting for 1.03% of the total soluble protein. The high-yield expression was stably maintained across two generations, indicating the genetic stability of rhBMP2m gene knock-in at the αAmy3 intron 1 locus. Additionally, the rice cell-derived rhBMP2m proteins were found to be glycosylated, capable of dimer formation, and bioactive. Our results indicate that the endogenous rice αAmy3 promoter-signal peptide-based expression system is an effective strategy for producing bioactive pharmaceutical proteins. KEY POINTS: ⢠The endogenous αAmy3 promoter-based expression system enhanced the yield of BMP2 ⢠The increased yield of BMP2 accounted for 1.03% of the total rice-soluble proteins ⢠The rice-produced BMP2 showed glycosylation modifications, dimer formation, and bioactivity.
Asunto(s)
Oryza , Humanos , Oryza/genética , Proteína Morfogenética Ósea 2/genética , Intrones , Proteínas Recombinantes/genética , Preparaciones FarmacéuticasRESUMEN
The development of the inflow tract is undoubtedly one of the most complex remodeling events in the formation of the four-chambered heart. It involves the creation of two separate atrial chambers, the formation of an atrial/atrioventricular (AV) septal complex, the incorporation of the caval veins and coronary sinus into the right atrium, and the remodeling events that result in pulmonary venous return draining into the left atrium. In these processes, the atrioventricular mesenchymal complex, consisting of the major atrioventricular (AV) cushions, the mesenchymal cap on the primary atrial septum (pAS), and the dorsal mesenchymal protrusion (DMP), plays a crucial role.
Asunto(s)
Atrios Cardíacos , Animales , Humanos , Seno Coronario/embriología , Seno Coronario/anomalías , Corazón/embriología , Mesodermo/embriología , Venas Pulmonares/anomalíasRESUMEN
MicroRNAs (miRNAs) are related to the regulation of bone metabolism. Delayed fracture healing (DFH) is a common complication after fracture surgery. The study attempted to examine the role of miR-98-5p and bone morphogenetic protein (BMP)-2 with the onset of DFH. A total of 140 patients with femoral neck fracture were recruited, including 80 cases with normal fracture healing (NFH) and 60 cases with DFH. MC3T3-E1 cells were induced cell differentiation for cell function experiments. Real-time quantitative polymerase chain reaction (RT-qPCR) was carried out to test mRNA levels. Cell proliferation and apoptosis were determined via CCK-8 and flow cytometry assay. Luciferase reporter assay was done to verify the targeted regulatory relationship of miR-98-5p with BMP-2. In comparison with NFH cases, DFH patients owned high levels of serum miR-98-5p and low concentration of BMP-2, and the levels of the two indexes are significantly negatively correlated. Both miR-98-5p and BMP-2 had the ability to predict DFH, while their combined diagnostic value is the highest. BMP-2 was demonstrated to be the target gene of miR-98-5p. Overexpression of BMP-2 reversed the role of miR-98-5p in MC3T3-E1 cell proliferation, apoptosis and differentiation. Increased miR-98-5p and decreased BMP-2 serve as potential biomarkers for the diagnosis of DFH. MiR-98-5p overexpression inhibits osteoblast proliferation and differentiation via targeting BMP-2.
Asunto(s)
Apoptosis , Proteína Morfogenética Ósea 2 , Proliferación Celular , Curación de Fractura , MicroARNs , Anciano , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Apoptosis/genética , Secuencia de Bases , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 2/genética , Diferenciación Celular/genética , Línea Celular , Fracturas del Cuello Femoral/metabolismo , Fracturas del Cuello Femoral/genética , Curación de Fractura/genética , MicroARNs/genética , MicroARNs/metabolismoRESUMEN
Bone morphogenetic protein 2 (BMP2) has been reported to regulate adipogenesis, but its role in porcine beige adipocyte formation remains unclear. Our data reveal that BMP2 is significantly induced at the early stages of porcine beige adipocyte differentiation. Additionally, supplementing rhBMP2 during the early stages, but not the late stages of differentiation, significantly enhances porcine SVF adipogenesis, thermogenesis, and proliferation. Furthermore, compared to the empty plasmid-transfected-SVFs, BMP2-overexpressed SVFs had the enhanced lipid accumulation and thermogenesis, while knockdown of BMP2 in SVFs exhibited the opposite effect. The RNA-seq of the above three types of cells revealed the enrichment of the annotation of thermogenesis, brown cell differentiation, etc. In addition, the analysis also highlights the significant enrichment of cell adhesion, the MAPK cascade, and PPARγ signaling. Mechanistically, BMP2 positively regulates the adipogenic and thermogenic capacities of porcine beige adipocytes by activating PPARγ expression through AKT/mTOR and MAPK signaling pathways.
Asunto(s)
Adipogénesis , Proteínas Proto-Oncogénicas c-akt , Porcinos , Animales , Adipogénesis/genética , Proteína Morfogenética Ósea 2/genética , PPAR gamma , Transducción de Señal , Serina-Treonina Quinasas TOR/genéticaRESUMEN
As the global population ages, the number of patients with osteoporosis is rapidly rising. The existing first-line clinical drugs are bone resorption inhibitors that have difficulty restoring the bone mass of elderly patients to the safe range. The range and period of use of existing peptides and monoclonal antibodies are limited, and small-molecule bone formation-promoting drugs are urgently required. We established an I-9 synthesis route with high yield, simple operation, and low cost that was suitable for future large-scale production. I-9 administration promoted bone formation and increased bone mass in mice with low bone mass in an aged C57 mouse model. Our findings revealed a hitherto undescribed pathway involving the BMP2-ERK-ATF4 axis that promotes osteoblast differentiation; I-9 has favorable biosafety in mice. This study systematically investigated the efficacy, safety, and mechanism of I-9 for treating osteoporosis and positions this drug for preclinical research in the future. Thus, this study has promoted the development of small-molecule bone-promoting drugs.
Asunto(s)
Conservadores de la Densidad Ósea , Osteoporosis , Anciano , Ratones , Humanos , Animales , Osteogénesis , Preparaciones Farmacéuticas/metabolismo , Osteoporosis/tratamiento farmacológico , Osteoporosis/metabolismo , Conservadores de la Densidad Ósea/uso terapéutico , Péptidos/metabolismo , Diferenciación Celular , Osteoblastos/metabolismo , Factor de Transcripción Activador 4/metabolismo , Proteína Morfogenética Ósea 2/metabolismoRESUMEN
The pathology of medication-related osteonecrosis of the jaw (MRONJ), often associated with antiresorptive therapy, is still not fully understood. Osteocyte networks are known to play a critical role in maintaining bone homeostasis and repair, but the exact condition of these networks in MRONJ is unknown. On the other hand, the local application of E-coli-derived Recombinant Human Bone Morphogenetic Protein 2/ß-Tricalcium phosphate (E-rhBMP-2/ß-TCP) has been shown to promote bone regeneration and mitigate osteonecrosis in MRONJ-like mouse models, indicating its potential therapeutic application for the treatment of MRONJ. However, the detailed effect of BMP-2 treatment on restoring bone integrity, including its osteocyte network, in an MRONJ condition remains unclear. Therefore, in the present study, by applying a scanning electron microscope (SEM) analysis and a 3D osteocyte network reconstruction workflow on the alveolar bone surrounding the tooth extraction socket of an MRONJ-like mouse model, we examined the effectiveness of BMP-2/ß-TCP therapy on the alleviation of MRONJ-related bone necrosis with a particular focus on the osteocyte network and alveolar bone microstructure (microcrack accumulation). The 3D osteocyte dendritic analysis showed a significant decrease in osteocyte dendritic parameters along with a delay in bone remodeling in the MRONJ group compared to the healthy counterpart. The SEM analysis also revealed a notable increase in the number of microcracks in the alveolar bone surface in the MRONJ group compared to the healthy group. In contrast, all of those parameters were restored in the E-rhBMP-2/ß-TCP-treated group to levels that were almost similar to those in the healthy group. In summary, our study reveals that MRONJ induces osteocyte network degradation and microcrack accumulation, while application of E-rhBMP-2/ß-TCP can restore a compromised osteocyte network and abrogate microcrack accumulation in MRONJ.
Asunto(s)
Proteína Morfogenética Ósea 2 , Fosfatos de Calcio , Modelos Animales de Enfermedad , Osteocitos , Proteínas Recombinantes , Animales , Proteína Morfogenética Ósea 2/farmacología , Proteína Morfogenética Ósea 2/metabolismo , Osteocitos/efectos de los fármacos , Fosfatos de Calcio/farmacología , Ratones , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/administración & dosificación , Osteonecrosis de los Maxilares Asociada a Difosfonatos/etiología , Osteonecrosis de los Maxilares Asociada a Difosfonatos/patología , Humanos , Regeneración Ósea/efectos de los fármacos , Masculino , Extracción Dental/efectos adversos , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Proceso Alveolar/efectos de los fármacos , Proceso Alveolar/patologíaRESUMEN
Recombinant human bone morphogenetic protein 2 (rhBMP-2) is an FDA-approved growth factor for bone regeneration and repair in medical practice. The therapeutic effects of rhBMP-2 may be enhanced through specific binding to extracellular matrix (ECM)-like scaffolds. Here, we report the selection of a novel rhBMP-2-specific DNA aptamer, functionalization of the aptamer in an ECM-like scaffold, and its application in a cellular context. A DNA aptamer BA1 was evolved and shown to have high affinity and specificity to rhBMP-2. A molecular docking model demonstrated that BA1 was probably bound to rhBMP-2 at its heparin-binding domain, as verified with experimental competitive binding assays. The BA1 aptamer was used to functionalize a type I collagen scaffold, and fraction ratios were optimized to mimic the natural ECM. Studies in the myoblast cell model C2C12 showed that the aptamer-enhanced scaffold could specifically augment the osteo-inductive function of rhBMP-2 in vitro. This aptamer-functionalized scaffold may have value in enhancing rhBMP-2-mediated bone regeneration.
Asunto(s)
Aptámeros de Nucleótidos , Proteína Morfogenética Ósea 2 , Humanos , Proteína Morfogenética Ósea 2/farmacología , Proteína Morfogenética Ósea 2/química , Aptámeros de Nucleótidos/farmacología , Andamios del Tejido/química , Simulación del Acoplamiento Molecular , Regeneración Ósea , Factor de Crecimiento Transformador beta/farmacología , Proteínas Recombinantes/químicaRESUMEN
Phosphorylation of the central adaptor protein complex, AP-2 is pivotal for clathrin-mediated endocytosis (CME). Here, we uncover the role of an uncharacterized kinase (BMP-2 inducible kinase-BMP2K) in AP-2 phosphorylation. We demonstrate that BMP2K can phosphorylate AP-2 in vitro and in vivo. Functional impairment of BMP2K impedes AP-2 phosphorylation leading to defects in clathrin-coated pit (CCP) morphology and cargo internalization. BMP2K engages AP-2 via its extended C-terminus and this interaction is important for its CCP localization and function. Notably, endogenous BMP2K levels decline upon functional impairment of AP-2 indicating AP-2 dependent BMP2K stabilization in cells. Further, functional inactivation of BMP2K in zebrafish embryos yields gastrulation phenotypes which mirror AP-2 loss-of-function suggesting physiological relevance of BMP2K in vertebrates. Together, our findings propose involvement of a novel kinase in AP-2 phosphorylation and in the operation of CME.
Asunto(s)
Complejo 2 de Proteína Adaptadora , Clatrina , Complejo 2 de Proteína Adaptadora/metabolismo , Animales , Clatrina/metabolismo , Endocitosis/fisiología , Fosforilación , Pez Cebra/metabolismoRESUMEN
The molecular mechanism for the microgravity-induced decrease in bone formation remains unclear and there is a lack of effective specific preventative therapies. We recently reported that primary cilia of osteoblasts became shorter and even disappeared when the cells were exposed to random positioning machine (RPM)-simulated microgravity and that the microgravity-induced loss of osteogenic potential of osteoblasts could be attenuated when the resorption of primary cilia was prevented by treatment with 0.1 µM cytochalasin D. In the current study, it was further found that the loss of the osteogenic capacity of rat calvarial osteoblasts (ROBs) was associated with the inhibition of the BMP-2/Smad1/5/8 signalling pathway, of which most of the signalling proteins including BMP-2, BMPRII, Smad1/5/8 and p-Smad1/5/8 were found localized to primary cilia. Accompanying the resorption of primary cilia following the cells being exposed to simulated microgravity, the expression levels of these signalling proteins were reduced significantly. Furthermore, the expression of miRNA-129-3p, a microRNA previously reported to control cilium biogenesis, was found to be reduced quickly and changed in a similar tendency with the length of primary cilia. Moreover, overexpression of miRNA-129-3p in ROBs significantly attenuated microgravity-induced inhibition of BMP-2 signalling and loss of osteogenic differentiation and mineralization. These results indicated the important role of miRNA-129-3p in microgravity-induced resorption of primary cilia of osteoblasts and the potential of replenishing the miRNA-129-3p as an effective countermeasure against microgravity-induced loss of primary cilia and impairment of osteoblast function.
Asunto(s)
MicroARNs , Ingravidez , Ratas , Animales , Osteogénesis/genética , Cilios/metabolismo , Ingravidez/efectos adversos , Diferenciación Celular/genética , MicroARNs/metabolismo , Osteoblastos/metabolismoRESUMEN
Betulin derivatives are proposed to serve as an alternative to the drugs already established in oncologic treatment. Drug-induced nephrotoxicity leading to acute kidney injury frequently accompanies cancer treatment, and thus there is a need to research the effects of betulin derivatives on renal cells. The objective of our study was to assess the influence of the betulin derivatives 28-propynylobetulin (EB5) and 29-diethoxyphosphoryl-28-propynylobetulin (ECH147) on the expression of TGFß1, BMP2 and GDF15 in renal proximal tubule epithelial cells (RPTECs) cultured in vitro. The changes in mRNA expression and copy numbers were assessed using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) and the standard curve method, respectively. An enzyme-linked immunosorbent assay (ELISA) was used to evaluate the effect of the betulin derivatives on the protein concentration in the culture media's supernatant. The assessment of the betulin derivatives' influence on gene expression demonstrated that the mRNA level and protein concentration did not always correlate with each other. Each of the tested compounds affected the mRNA expression. The RT-qPCR analyses showed that EB5 and ECH147 induced effects similar to those of betulin or cisplatin and resulted in a decrease in the mRNA copy number of all the analyzed genes. The ELISA demonstrated that EB5 and ECH147 elevated the protein concentration of TGFß1 and GDF15, while the level of BMP2 decreased. The concentration of the derivatives used in the treatment was crucial, but the effects did not always exhibit a simple linear dose-dependent relationship. Betulin and its derivatives, EB5 and ECH147, influenced the gene expression of TGFß1, BMP2 and GDF15 in the renal proximal tubule epithelial cells. The observed effects raise the question of whether treatment with these compounds could promote the development of renal fibrosis.
RESUMEN
This study aimed to treat dental injuries by utilizing one of the most advanced tissue engineering techniques. In this study, an in vitro model was employed to investigate the proliferation and odontogenic differentiation of canine endometrial stem cells (C-EnSCs). Furthermore, the dentin regeneration potential of odontoblast like-cells (OD) derived from C-EnSCs was assessed in rats. The C-EnSCs were isolated by the enzymatic method and identified by flow cytometry. The C-EnSCs were encapsulated in fibrin gel associated with signaling factors to create the proper conditions for cell growth and differentiation. Then, the OD cells were associated with bone morphologic protein-2 (BMP-2) to promote dentin formation in vivo. The animal model used to evaluate the regenerative effect of cells and biomaterials included the preparation of the left maxillary first molar of rats for direct pulp capping operation. Animals were divided into four groups: group 1, a control group without any treatment, group 2, which received fibrin, group 3, which received fibrin with ODs (fibrin/ODs), and group 4, which received fibrin with ODs and BMP-2 (fibrin/ODs/BMP-2). The morphological observations showed the differentiation of C-EnSCs into adipose, bone, neural cells, and ODs. Furthermore, the histomorphometric data of the treated teeth showed how fibrin gel and BMP2 at a concentration of 100 ng/mL provided an optimal microenvironment for regenerating dentin tissue in rats, which was increased significantly with the presence of OD cells within eight weeks. Our study showed that using OD cells derived from C-EnSCs encapsulated in fibrin gel associated with BMP2 can potentially be an appropriate candidate for direct pulp-capping and dentin regeneration.
RESUMEN
BACKGROUND: Calcific aortic valve disease (CAVD) is the second leading cause of adult heart diseases. The purpose of this study is to investigate whether miR-101-3p plays a role in the human aortic valve interstitial cells (HAVICs) calcification and the underlying mechanisms. METHODS: Small RNA deep sequencing and qPCR analysis were used to determine changes in microRNA expression in calcified human aortic valves. RESULTS: The data showed that miR-101-3p levels were increased in the calcified human aortic valves. Using cultured primary HAVICs, we demonstrated that the miR-101-3p mimic promoted calcification and upregulated the osteogenesis pathway, while anti-miR-101-3p inhibited osteogenic differentiation and prevented calcification in HAVICs treated with the osteogenic conditioned medium. Mechanistically, miR-101-3p directly targeted cadherin-11 (CDH11) and Sry-related high-mobility-group box 9 (SOX9), key factors in the regulation of chondrogenesis and osteogenesis. Both CDH11 and SOX9 expressions were downregulated in the calcified human HAVICs. Inhibition of miR-101-3p restored expression of CDH11, SOX9 and ASPN and prevented osteogenesis in HAVICs under the calcific condition. CONCLUSION: miR-101-3p plays an important role in HAVIC calcification through regulation of CDH11/SOX9 expression. The finding is important as it reveals that miR-1013p may be a potential therapeutic target for calcific aortic valve disease.