RESUMO
Airway epithelium, the first defense barrier of the respiratory system, facilitates mucociliary clearance against inflammatory stimuli, such as pathogens and particulates inhaled into the airway and lung. Inhaled particulate matter 2.5 (PM2.5) can penetrate the alveolar region of the lung, and it can develop and exacerbate respiratory diseases. Although the pathophysiological effects of PM2.5 in the respiratory system are well known, its impact on mucociliary clearance of airway epithelium has yet to be clearly defined. In this study, we used two different 3D in vitro airway models, namely the EpiAirway-full-thickness (FT) model and a normal human bronchial epithelial cell (NHBE)-based air-liquid interface (ALI) system, to investigate the effect of diesel exhaust particles (DEPs) belonging to PM2.5 on mucociliary clearance. RNA-sequencing (RNA-Seq) analyses of EpiAirway-FT exposed to DEPs indicated that DEP-induced differentially expressed genes (DEGs) are related to ciliary and microtubule function and inflammatory-related pathways. The exposure to DEPs significantly decreased the number of ciliated cells and shortened ciliary length. It reduced the expression of cilium-related genes such as acetylated α-tubulin, ARL13B, DNAH5, and DNAL1 in the NHBEs cultured in the ALI system. Furthermore, DEPs significantly increased the expression of MUC5AC, whereas they decreased the expression of epithelial junction proteins, namely, ZO1, Occludin, and E-cadherin. Impairment of mucociliary clearance by DEPs significantly improved the release of epithelial-derived inflammatory and fibrotic mediators such as IL-1ß, IL-6, IL-8, GM-CSF, MMP-1, VEGF, and S100A9. Taken together, it can be speculated that DEPs can cause ciliary dysfunction, hyperplasia of goblet cells, and the disruption of the epithelial barrier, resulting in the hyperproduction of lung injury mediators. Our data strongly suggest that PM2.5 exposure is directly associated with ciliary and epithelial barrier dysfunction and may exacerbate lung injury.
Assuntos
Lesão Pulmonar , Emissões de Veículos , Humanos , Emissões de Veículos/toxicidade , Lesão Pulmonar/metabolismo , Mucosa Respiratória , Material Particulado/metabolismo , Células Epiteliais , EpitélioRESUMO
The rapid aging of the population worldwide presents a significant social and economic challenge, particularly due to osteoporotic fractures, primarily resulting from an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. While conventional therapies offer benefits, they also present limitations and a range of adverse effects. This study explores the protective impact of Neorhodomela munita ethanol extract (EN) on osteoporosis by modulating critical pathways in osteoclastogenesis and apoptosis. Raw264.7 cells and Saos-2 cells were used for in vitro osteoclast and osteoblast models, respectively. By utilizing various in vitro methods to detect osteoclast differentiation/activation and osteoblast death, it was demonstrated that the EN's potential to inhibit RANKL induced osteoclast formation and activation by targeting the MAPKs-NFATc1/c-Fos pathway and reducing H2O2-induced cell death through the downregulation of apoptotic signals. This study highlights the potential benefits of EN for osteoporosis and suggests that EN is a promising natural alternative to traditional treatments.
Assuntos
Apoptose , Osteoblastos , Osteoclastos , Ligante RANK , Rodófitas , Animais , Humanos , Camundongos , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Etanol/química , Peróxido de Hidrogênio/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Ligante RANK/metabolismo , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Rodófitas/químicaRESUMO
Vascular calcification (VC) and osteoporosis are age-related diseases and significant risk factors for the mortality of elderly. VC and osteoporosis may share common risk factors such as renin-angiotensin system (RAS)-related hypertension. In fact, inhibitors of RAS pathway, such as angiotensin type 1 receptor blockers (ARBs), improved both vascular calcification and hip fracture in elderly. However, a sex-dependent discrepancy in the responsiveness to ARB treatment in hip fracture was observed, possibly due to the estrogen deficiency in older women, suggesting that blocking the angiotensin signaling pathway may not be effective to suppress bone resorption, especially if an individual has underlying osteoclast activating conditions such as estrogen deficiency. Therefore, it has its own significance to find alternative modality for inhibiting both vascular calcification and osteoporosis by directly targeting osteoclast activation to circumvent the shortcoming of ARBs in preventing bone resorption in estrogen deficient individuals. In the present study, a natural compound library was screened to find chemical agents that are effective in preventing both calcium deposition in vascular smooth muscle cells (vSMCs) and activation of osteoclast using experimental methods such as Alizarin red staining and Tartrate-resistant acid phosphatase staining. According to our data, citreoviridin (CIT) has both an anti-VC effect and anti-osteoclastic effect in vSMCs and in Raw 264.7 cells, respectively, suggesting its potential as an effective therapeutic agent for both VC and osteoporosis.
Assuntos
Aurovertinas , Reabsorção Óssea , Osteoporose , Calcificação Vascular , Humanos , Antagonistas de Receptores de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Reabsorção Óssea/metabolismo , Cálcio/metabolismo , Estrogênios/farmacologia , Músculo Liso Vascular , Miócitos de Músculo Liso , Osteoporose/metabolismo , Calcificação Vascular/metabolismo , Animais , Camundongos , Células RAW 264.7 , Aurovertinas/farmacologiaRESUMO
Recently published clinical trials involving the use of adipose-derived stem cells (ADSCs) indicated that approximately one-third of the studies were conducted on musculoskeletal disorders (MSD). MSD refers to a wide range of degenerative conditions of joints, bones, and muscles, and these conditions are the most common causes of chronic disability worldwide, being a major burden to the society. Conventional treatment modalities for MSD are not sufficient to correct the underlying structural abnormalities. Hence, ADSC-based cell therapies are being tested as a form of alternative, yet more effective, therapies in the management of MSDs. Therefore, in this review, MSDs subjected to the ADSC-based therapy were further categorized as arthritis, craniomaxillofacial defects, tendon/ligament related disorders, and spine disorders, and their brief characterization as well as the corresponding conventional therapeutic approaches with possible mechanisms with which ADSCs produce regenerative effects in disease-specific microenvironments were discussed to provide an overview of under which circumstances and on what bases the ADSC-based cell therapy was implemented. Providing an overview of the current status of ADSC-based cell therapy on MSDs can help to develop better and optimized strategies of ADSC-based therapeutics for MSDs as well as help to find novel clinical applications of ADSCs in the near future.
Assuntos
Tecido Adiposo/citologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células-Tronco Mesenquimais/citologia , Doenças Musculoesqueléticas/terapia , Medicina Regenerativa/métodos , Transplante de Células-Tronco/métodos , Animais , Diferenciação Celular , Ensaios Clínicos como Assunto/métodos , Ensaios Clínicos como Assunto/estatística & dados numéricos , Humanos , Doenças Musculoesqueléticas/patologia , Doenças Musculoesqueléticas/fisiopatologiaRESUMO
The acute demise of stem cells following transplantation significantly compromises the efficacy of stem cell-based cell therapeutics for infarcted hearts. As the stem cells transplanted into the damaged heart are readily exposed to the hostile environment, it can be assumed that the acute death of the transplanted stem cells is also inflicted by the same environmental cues that caused massive death of the host cardiac cells. Pyroptosis, a highly inflammatory form of programmed cell death, has been added to the list of important cell death mechanisms in the damaged heart. However, unlike the well-established cell death mechanisms such as necrosis or apoptosis, the exact role and significance of pyroptosis in the acute death of transplanted stem cells have not been explored in depth. In the present study, we found that M1 macrophages mediate the pyroptosis in the ischemia/reperfusion (I/R) injured hearts and identified miRNA-762 as an important regulator of interleukin 1ß production and subsequent pyroptosis. Delivery of exogenous miRNA-762 prior to transplantation significantly increased the post-transplant survival of stem cells and also significantly ameliorated cardiac fibrosis and heart functions following I/R injury. Our data strongly suggest that suppressing pyroptosis can be an effective adjuvant strategy to enhance the efficacy of stem cell-based therapeutics for diseased hearts.
Assuntos
MicroRNAs , Traumatismo por Reperfusão Miocárdica , Piroptose , Transplante de Células-Tronco , Células-Tronco , Animais , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , MicroRNAs/genética , MicroRNAs/farmacologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/terapia , Piroptose/efeitos dos fármacos , Piroptose/genética , Células RAW 264.7 , Ratos , Ratos Sprague-Dawley , Células-Tronco/metabolismo , Células-Tronco/patologiaRESUMO
Cancer immunotherapy is a clinically validated therapeutic modality for cancer and has been rapidly advancing in recent years. Adoptive transfer of immune cells such as T cells and natural killer (NK) cells has emerged as a viable method of controlling the immune system against cancer. Recent evidence indicates that even immune-cell-released vesicles such as NK-cell-derived exosomes also exert anticancer effect. Nevertheless, the underlying mechanisms remain elusive. In the present study, the anticancer potential of isolated extracellular vesicles (EVs) from expanded and activated NK-cell-enriched lymphocytes (NKLs) prepared by house-developed protocol was evaluated both in vitro and in vivo. Moreover, isolated EVs were characterized by using two-dimensional electrophoresis (2-DE)-based proteome and network analysis, and functional study using identified factors was performed. Our data indicated that the EVs from expanded and active NKLs had anticancer properties, and a number of molecules, such as Fas ligand, TRAIL, NKG2D, ß-actin, and fibrinogen, were identified as effector candidates based on the proteome analysis and functional study. The results of the present study suggest the possibility of NK-cell-derived EVs as a viable immunotherapeutic strategy for cancer.
Assuntos
Vesículas Extracelulares/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Neoplasias/imunologia , Proteoma , Actinas/química , Animais , Anticorpos Neutralizantes/química , Linhagem Celular Tumoral , Sobrevivência Celular , Meios de Cultura , Eletroforese em Gel Bidimensional , Exossomos/metabolismo , Proteína Ligante Fas , Fibrinogênio/química , Citometria de Fluxo , Células Hep G2 , Humanos , Imunoterapia , Linfócitos/metabolismo , Células MCF-7 , Camundongos , Camundongos Nus , Transplante de Neoplasias , Proteômica , Coloração pela PrataRESUMO
Human adipose-derived stem cells (hASCs) can be isolated from fat tissue and have attracted interest for their potential therapeutic applications in metabolic disease. hASCs can be induced to undergo adipogenic differentiation in vitro by exposure to chemical agents or inductive growth factors. We investigated the effects and mechanism of differentiating hASC-derived white adipocytes into functional beige and brown adipocytes with isoliquiritigenin (ILG) treatment. Here, we showed that hASC-derived white adipocytes could promote brown adipogenesis by expressing both uncoupling protein 1 (UCP1) and PR/SET Domain 16 (PRDM16) following low-dose ILG treatments. ILG treatment of white adipocytes enhanced the expression of brown fat-specific markers, while the expression levels of c-Jun N-terminal kinase (JNK) signaling pathway proteins were downregulated. Furthermore, we showed that the inhibition of JNK phosphorylation contributed to white adipocyte differentiation into beige adipocytes, which was validated by the use of SP600125. We identified distinct regulatory effects of ILG dose responses and suggested that low-dose ILG induced the beige adipocyte potential of hASCs via JNK inhibition.
Assuntos
Adipócitos Marrons/citologia , Adipogenia , Chalconas/farmacologia , Inibidores Enzimáticos/farmacologia , MAP Quinase Quinase 4/antagonistas & inibidores , Células-Tronco Mesenquimais/citologia , Adipócitos Marrons/efeitos dos fármacos , Adipócitos Marrons/enzimologia , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologiaRESUMO
Pathological cardiac hypertrophy is characterized by an abnormal increase in cardiac muscle mass in the left ventricle, resulting in cardiac dysfunction. Although various therapeutic approaches are being continuously developed for heart failure, several studies have suggested natural compounds as novel potential strategies. Considering relevant compounds, we investigated a new role for Pterosin B for which the potential life-affecting biological and therapeutic effects on cardiomyocyte hypertrophy are not fully known. Thus, we investigated whether Pterosin B can regulate cardiomyocyte hypertrophy induced by angiotensin II (Ang II) using H9c2 cells. The antihypertrophic effect of Pterosin B was evaluated, and the results showed that it reduced hypertrophy-related gene expression, cell size, and protein synthesis. In addition, upon Ang II stimulation, Pterosin B attenuated the activation and expression of major receptors, Ang II type 1 receptor and a receptor for advanced glycation end products, by inhibiting the phosphorylation of PKC-ERK-NF-κB pathway signaling molecules. In addition, Pterosin B showed the ability to reduce excessive intracellular reactive oxygen species, critical mediators for cardiac hypertrophy upon Ang II exposure, by regulating the expression levels of NAD(P)H oxidase 2/4. Our results demonstrate the protective role of Pterosin B in cardiomyocyte hypertrophy, suggesting it is a potential therapeutic candidate.
Assuntos
Angiotensina II/química , Hipertrofia/tratamento farmacológico , Indanos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Citosol/metabolismo , Proteína HMGB1/metabolismo , Coração/efeitos dos fármacos , NF-kappa B/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Transdução de SinaisRESUMO
Bone diseases may not be imminently life-threatening or a leading cause of death such as heart diseases or cancers. However, as aging population grows in almost every part of the world, they surely impose significant socioeconomic burden on the society, not to mention the patients and their families. Osteoporosis is the most common type of bone disease, which frequently develops in seniors, especially in postmenopausal women. Although currently several anti-osteoclastic drugs designed to suppress excessive osteoclast activation, a major cause of osteoporosis, are commercially available, accompanying adverse effects ranging from mild to severe have been reported as well. Natural products have become increasingly popular because of their effectiveness with fewer side effects. Isoliquiritigenin (ILG), a natural flavonoid from licorice, has been reported to suppress osteoclast differentiation and activation. In the present study, newly synthesized ILG derivatives were screened for their anti-osteoporotic activity as more potent substitute candidates to ILG. Out of the 12 ILG derivatives tested, two compounds demonstrated significantly improved bone loss in vitro by inhibiting both osteoclastogenesis and osteoclast activity. The results of the present study indicate that these compounds may serve as a potential drug for osteoporosis and warrant further studies to evaluate their in vivo efficacy.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Chalconas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ligante RANK/metabolismo , Animais , Camundongos , NF-kappa B/metabolismo , Osteoclastos/patologia , Células RAW 264.7 , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Adoptive transfer of immune cells such as T cells and natural killer (NK) cells has emerged as a targeted method of controlling the immune system against cancer. Despite their significant therapeutic potential, efficient methods to generate adequate numbers of NK cells are lacking and ex vivo-expansion and activation of NK cells is currently under intensive investigation. The primary purpose of this study was to develop an effective method for expansion and activation of the effector cells with high proportion of NK cells and increasing cytotoxicity against liver cancer in a short time period. METHODS: Expanded NK cell-enriched lymphocytes (NKL) designated as "MYJ1633" were prepared by using autologous human plasma, cytokines (IL-2, IL-12 and IL-18) and agonistic antibodies (CD16, CD56 and NKp46) without an NK cell-sorting step. The characteristics of NKL were compared to those of freshly isolated PBMCs. In addition, the cytotoxic effect of the NKL on liver cancer cell was examined in vitro and in vivo. RESULTS: The total cell number after ex vivo-expansion increased about 140-fold compared to that of freshly isolated PBMC within 2 weeks. Approximately 78% of the expanded and activated NKL using the house-developed protocol was NK cell and NKT cells even without a NK cell-sorting step. In addition, the expanded and activated NKL demonstrated potent cytotoxicity against liver cancer in vitro and in vivo. CONCLUSION: The house-developed method can be a new and effective strategy to prepare clinically applicable NKL for autologous NK cell-based anti-tumor immunotherapy.
Assuntos
Transferência Adotiva/métodos , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Neoplasias Hepáticas/terapia , Animais , Antígeno CD56/metabolismo , Sobrevivência Celular , Citocinas/metabolismo , Proteínas Ligadas por GPI/metabolismo , Células Hep G2 , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Nus , Modelos Animais , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Receptores de IgG/metabolismo , Carga TumoralRESUMO
During the past decade, microRNAs have continuously been suggested as a promising therapeutic tool due to their beneficial effects, such as their multi-targets and multi-functions in pathologic conditions. As a pathologic phenotype is generally regulated by multiple signaling pathways, in this study we identified a microRNA regulating multiple target genes within cardiac hypertrophic signaling pathways. microRNA-133a is known to play a crucial role in cardiac hypertrophy. However, the role of microRNA-133a, which may regulate several signaling pathways in norepinephrine-induced cardiac hypertrophy via multi-targeting, has not been investigated. In the current study, we showed that microRNA-133a can protect cardiomyocyte hypertrophy against norepinephrine stimulation in neonatal rat ventricular cardiomyocytes via new targets, PKCδ and Gq, all of which are related to downstream signaling pathways of the α1-adrenergic receptor. Taken together, these results suggest the advantages of the therapeutic use of microRNAs as an effective potential drug regulating multiple signaling pathways under pathologic conditions.
Assuntos
Cardiomegalia/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Proteína Quinase C-delta/metabolismo , Transdução de Sinais , Animais , Cardiomegalia/patologia , Miócitos Cardíacos/patologia , Ratos , Receptor A1 de Adenosina/metabolismoRESUMO
Adipose-derived stromal vascular fractions (SVFs) are a heterogeneous collection of cells, and their regenerative modality has been applied in various animal experiments and clinical trials. Despite the attractive advantages of SVFs in clinical interventions, the recent status of clinical studies involving the application of SVFs in many diseases has not been fully evaluated. Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into a variety of cell types despite their low numbers in heart tissue. Here, we sought to determine if SVF implantation into impaired heart tissue affected endogenous MSCs in the heart. Therefore, we investigated the expression levels of proteins associated with oxidation, inflammation, and apoptosis in MSCs co-cultured with adipose-derived adherent stromal cells (ADASs) from 6 donors' SVFs under oxidative stress conditions for their roles in many physiological processes in the heart. Interestingly, p53 pathway proteins and mitogen-activated protein kinase (MAPK) signalling pathway components were up-regulated by H2 O2 but exhibited a downward trend in MSCs co-cultured with ADASs. These data suggest that ADASs may inhibit oxidative stress-induced apoptosis in MSCs via the p53 and MAPK pathways. Our findings also suggest that the positive effects of SVF implantation into damaged heart tissue may be attributed to the various responses of MSCs. This finding may provide new insights for the clinical application of adipose-derived SVF transplantation in cardiac diseases. SIGNIFICANCE OF THE STUDY: We investigated the expression levels of proteins associated with oxidation, inflammation, and apoptosis in MSCs co-cultured with isolated ADASs from 6 donors' SVFs under oxidative stress conditions. Our results imply that isolated ADASs from SVFs may inhibit oxidative stress-induced cell cycle arrest and/or apoptosis in MSCs via a p53-dependent pathway. Furthermore, we identified an anti-apoptotic mechanism involving oxidative stress-induced apoptosis by adipose-derived ADASs in MSCs for the first time. Our findings suggest that the positive effects of SVF implantation into damaged heart tissue may be attributed to the various actions of MSCs.
Assuntos
Adipócitos/metabolismo , Apoptose , Células Estromais/metabolismo , Adipócitos/efeitos dos fármacos , Adipócitos/patologia , Adulto , Apoptose/efeitos dos fármacos , Técnicas de Cocultura , Feminino , Humanos , Peróxido de Hidrogênio/farmacologia , Masculino , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Células Estromais/patologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Osteoarthritis (OA) can be defined as degradation of articular cartilage of the joint, and is the most common degenerative disease. To regenerate the damaged cartilage, different experimental approaches including stem cell therapy have been tried. One of the major limitations of stem cell therapy is the poor post-transplantation survival of the stem cells. Anoikis, where insufficient matrix support and adhesion to extracellular matrix causes apoptotic cell death, is one of the main causes of the low post-transplantation survival rate of stem cells. Therefore, enhancing the initial interaction of the transplanted stem cells with chondrocytes could improve the therapeutic efficacy of stem cell therapy for OA. Previously, protein kinase C activator phorbol 12-myristate 13-acetate (PMA)-induced increase of mesenchymal stem cell adhesion via activation of focal adhesion kinase (FAK) has been reported. In the present study, we examine the effect PMA on the adipose-derived stem cells (ADSCs) adhesion and spreading to culture substrates, and further on the initial interaction between ADSC and chondrocytes. RESULTS: PMA treatment increased the initial adhesion of ADSC to culture substrate and cellular spreading with increased expression of adhesion molecules, such as FAK, vinculin, talin, and paxillin, at both RNA and protein level. Priming of ADSC with PMA increased the number of ADSCs attached to confluent layer of cultured chondrocytes compared to that of untreated ADSCs at early time point (4 h after seeding). CONCLUSION: Taken together, the results of this study suggest that priming ADSCs with PMA can increase the initial interaction with chondrocytes, and this proof of concept can be used to develop a non-invasive therapeutic approach for treating OA. It may also accelerate the regeneration process so that it can relieve the accompanied pain faster in OA patients. Further in vivo studies examining the therapeutic effect of PMA pretreatment of ADSCs for articular cartilage damage are required.
Assuntos
Cartilagem Articular/citologia , Condrócitos/citologia , Proteína Quinase C/farmacologia , Células-Tronco/efeitos dos fármacos , Western Blotting , Adesão Celular , Comunicação Celular , Técnicas de Cultura de Células , Diferenciação Celular , Sobrevivência Celular , Condrócitos/efeitos dos fármacos , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND/AIMS: Stromal vascular fraction (SVF) cells are a mixed cell population, and their regenerative capacity has been validated in various therapeutic models. The purpose of this study was to investigate the regenerative mechanisms utilized by implanted SVF cells. Using an in vitro co-culture system, we sought to determine whether SVF implantation into impaired tissue affects endogenous mesenchymal stem cell (MSC) differentiation; MSCs can differentiate into a variety of cell types, and they have a strong regenerative capacity despite their low numbers in impaired tissue. METHODS: Adipose-derived SVF cells obtained from four donors were co-cultured with bone marrow-derived MSCs, and the differential expression of osteogenic markers and osteogenic differentiation inducers over time was analyzed in mono-cultured MSCs and MSCs co-cultured with SVF cells. RESULTS: The co-cultivation of MSCs with SVF cells significantly and mutually induced the expression of osteogenic-specific markers via paracrine and/or autocrine regulation but did not induce adipocyte, chondrocyte or myoblast marker expression. More surprisingly, subsequent osteogenesis and/or comparable effects were rapidly induced within 48 h. CONCLUSION: To the best of our knowledge, this is the first study in which osteogenesis and/or comparable effects were rapidly induced in bone marrow-derived MSCs and adipose-derived SVF cells through co-cultivation. Our findings suggest that the positive effects of SVF implantation into impaired bone may be attributed to the rapid induction of MSC osteogenesis, and the transplantation of co-cultured and preconditioned SVF cells and/or MSCs may be more effective than the transplantation of untreated cells for the treatment of bone defects.
Assuntos
Células-Tronco Mesenquimais/citologia , Células Estromais/citologia , Tecido Adiposo/citologia , Adulto , Células da Medula Óssea/citologia , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese , Osteopontina/genética , Osteopontina/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Células Estromais/metabolismoRESUMO
Connexin 43 (Cx43), a ubiquitous connexin expressed in the heart and skin, is associated with a variety of hereditary conditions. Therefore, the characterization of Cx43-interacting proteins and their dynamics is important to understand not only the molecular mechanisms underlying pathological malfunction of gap junction-mediated intercellular communication but also to identify novel and unanticipated biological functions of Cx43. In the present study, we observed potential targets of Cx43 to determine new molecular functions in cardio-protection. MALDI-TOF mass spectrometry analysis of Cx43 co-immunoprecipitated proteins showed that Cx43 interacts with several proteins related to metabolism. In GeneMANIA network analysis, SGSM3, which has not been previously associated with Cx43, was highly correlated with Cx43 in heart functions, and high levels of SGSM3 appeared to induce the turnover of Cx43 through lysosomal degradation in myocardial infarcted rat hearts. Moreover, we confirmed that lysosomal degradation of Cx43 is dependent upon the interaction between SGSM3 and Cx43 in H9c2 cardiomyocytes. The functional importance of the interaction between SGSM3 and Cx43 was confirmed by results showing that Cx43 expression was enhanced by SGSM3 siRNA knockdown in H9c2 cells. In summary, the results of this study elucidate the molecular mechanisms in which Cx43 with SGSM3 is degraded in myocardial infarcted rat hearts, which may contribute to the establishment of new therapeutic targets to modulate cardiac function in physiological and pathological conditions.
Assuntos
Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Comunicação Celular , Linhagem Celular , Conexina 43/genética , Vasos Coronários/patologia , Vasos Coronários/cirurgia , Junções Comunicantes/patologia , Junções Comunicantes/ultraestrutura , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ligadura , Lisossomos/metabolismo , Masculino , Proteínas Monoméricas de Ligação ao GTP/antagonistas & inibidores , Proteínas Monoméricas de Ligação ao GTP/genética , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Miocárdio/patologia , Miócitos Cardíacos/patologia , Miócitos Cardíacos/ultraestrutura , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteólise , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
In the endothelial recovery process, bone marrow-derived MSCs are a potential source of cells for both research and therapy, and their capacities to self-renew and to differentiate into all the cell types in the human body make them a promising therapeutic agent for remodeling cellular differentiation and a valuable resource for the treatment of many diseases. Based on the results provided in a miRNA database, we selected miRNAs with unique targets in cell fate-related signaling pathways. The tested miRNAs targeting GSK-3ß (miR-26a), platelet-derived growth factor receptor, and CD133 (miR-26a and miR-29b) induced MSC differentiation into functional ECs, whereas miRNAs targeting VEGF receptor (miR-15, miR-144, miR-145, and miR-329) inhibited MSC differentiation into ECs through VEGF stimulation. In addition, the expression levels of these miRNAs were correlated with in vivo physiological endothelial recovery processes. These findings indicate that the miRNA expression profile is distinct for cells in different stages of differentiation from MSCs to ECs and that specific miRNAs can function as regulators of endothelialization.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular/genética , Linhagem da Célula , Células Endoteliais/citologia , Células-Tronco Mesenquimais/citologia , MicroRNAs/genética , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Ratos Sprague-Dawley , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Stromal vascular fractions (SVFs) are a heterogeneous collection of cells within adipose tissue that are being studied for various clinical indications. In this study, we aimed to determine whether SVF transplantation into impaired tissues has differential effects on inflammatory and angiogenetic properties with regard to gender. As reactive oxygen species have been implicated in cardiovascular disease development, we investigated differences in gene and protein expression related to inflammation and angiogenesis in HUVECs co-cultured with adipose-derived SVFs from male (M group) and female (F group) individuals under oxidative stress conditions. The expression of several inflammatory (interleukin (IL)-33) and angiogenetic (platelet-derived growth factor (PDGF)) factors differed dramatically between male and female donors. Anti-inflammatory and pro-angiogenetic responses were observed in HUVECs co-cultured with SVFs under oxidative stress conditions, and these characteristics may exhibit partially differential effects according to gender. Using network analysis, we showed that co-culturing HUVECs with SVFs ameliorated pyroptosis/apoptosis via an increase in oxidative stress. Activation of caspase-1 and IL-1B was significantly altered in HUVECs co-cultured with SVFs from female donors. These findings regarding gender-dimorphic regulation of adipose-derived SVFs provide valuable information that can be used for evidence-based gender-specific clinical treatment of SVF transplantation for understanding of cardiovascular disease, allowing for the development of additional treatment.
Assuntos
Inflamação/genética , Neovascularização Patológica/genética , Obesidade/genética , Estresse Oxidativo/genética , Células Estromais/citologia , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Apoptose/genética , Diferenciação Celular , Linhagem da Célula/genética , Técnicas de Cocultura , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação/patologia , Interleucina-33/genética , Masculino , Neovascularização Patológica/patologia , Obesidade/patologia , Fator de Crescimento Derivado de Plaquetas , Espécies Reativas de Oxigênio/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Caracteres Sexuais , Células Estromais/metabolismoRESUMO
BACKGROUND: Pathologic vascular smooth muscle cell (VSMC) proliferation and migration after vascular injury promotes the development of occlusive vascular disease. Therefore, an effective chemical agent to suppress aberrant proliferation and migration of VSMCs can be a potential therapeutic modality for occlusive vascular disease such as atherosclerosis and restenosis. To find an anti-proliferative chemical agent for VSMCs, we screened an in-house small molecule library, and the selected small molecule was further validated for its anti-proliferative effect on VSMCs using multiple approaches, such as cell proliferation assays, wound healing assays, transwell migration assays, and ex vivo aortic ring assay. RESULTS: Among 43 initially screened small molecule inhibitors of kinases that have no known anti-proliferative effect on VSMCs, a spleen tyrosine kinase (Syk) inhibitor (BAY61-3606) showed significant anti-proliferative effect on VSMCs. Further experiments indicated that BAY61 attenuated the VSMC proliferation in both concentration- and time-dependent manner, and it also significantly suppressed the migration of VSMCs as assessed by both wound healing assays and transwell assays. Additionally, BAY61 suppressed the sprouting of VSMCs from endothelium-removed aortic rings. CONCLUSION: The present study identified a Syk kinase inhibitor as a potent VSMC proliferation and migration inhibitor and warrants further studies to elucidate its underlying molecular mechanisms, such as its primary target, and to validate its in vivo efficacy as a therapeutic agent for restenosis and atherosclerosis.
Assuntos
Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Niacinamida/análogos & derivados , Pirimidinas/farmacologia , Quinase Syk/antagonistas & inibidores , Animais , Aorta Torácica/efeitos dos fármacos , Western Blotting , Ensaios de Migração Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Músculo Liso Vascular/citologia , Niacinamida/farmacologia , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Fatores de Tempo , Cicatrização/efeitos dos fármacosRESUMO
The restoration of damaged articular cartilage is a long-pursued goal in regenerative medicine. Chondrocyte-specific differentiation of mesenchymal stem cells (MSCs) may be an effective means of repairing damaged cartilage. We identified small molecule 6 with sulfonamide as an agent that promotes specific chondrogenic differentiation of human adipose-derived MSCs (hASCs). Unlike other chondrogenic differentiation media composed of various defined components, simply adding compound 6 into culture medium was sufficient to induce chondrogenesis in this study. In an animal osteoarthritis model, both the small molecule 6 and the 6-treated hASCs exhibited enhanced recovery of injured articular cartilage. This work provides new insight into MSC differentiation induced by small molecules and potential new therapeutic approaches for articular cartilage injury.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Sulfonamidas/farmacologia , Cartilagem Articular/citologia , Diferenciação Celular , Condrócitos/citologia , Meios de Cultura , Ensaio de Imunoadsorção Enzimática , Humanos , Células-Tronco Mesenquimais/citologia , RegeneraçãoRESUMO
Dynamin-related protein-1 (Drp1) plays a critical role in mitochondrial fission which allows cell proliferation and Mdivi-1, a specific small molecule Drp1 inhibitor, is revealed to attenuate proliferation. However, few molecular mechanisms-related to Drp1 under stimulus for restenosis or atherosclerosis have been investigated in vascular smooth muscle cells (vSMCs). Therefore, we hypothesized that Drp1 inhibition can prevent vascular restenosis and investigated its regulatory mechanism. Angiotensin II (Ang II) or hydrogen peroxide (H2 O2 )-induced proliferation and migration in SMCs were attenuated by down-regulation of Drp1 Ser 616 phosphorylation, which was demonstrated by in vitro assays for migration and proliferation. Excessive amounts of ROS production and changes in mitochondrial membrane potential were prevented by Drp1 inhibition under Ang II and H2 O2 . Under the Ang II stimulation, activated Drp1 interacted with PKCδ and then activated MEK1/2-ERK1/2 signaling cascade and MMP2, but not MMP9. Furthermore, in ex vivo aortic ring assay, inhibition of the Drp1 had significant anti-proliferative and -migration effects for vSMCs. A formation of vascular neointima in response to a rat carotid artery balloon injury was prevented by Drp1 inhibition, which shows a beneficial effect of Drp1 regulation in the pathologic vascular condition. Drp1-mediated SMC proliferation and migration can be prevented by mitochondrial division inhibitor (Mdivi-1) in in vitro, ex vivo and in vivo, and these results suggest the possibility that Drp1 can be a new therapeutic target for restenosis or atherosclerosis.