RESUMO
Spastic paraplegia type 11 (SPG11) is a common autosomal recessive form of hereditary spastic paraplegia (HSP) characterized by the degeneration of cortical motor neuron axons, leading to muscle spasticity and weakness. Impaired lipid trafficking is an emerging pathology in neurodegenerative diseases including SPG11, though its role in axonal degeneration of human SPG11 neurons remains unknown. Here, we established a pluripotent stem cell-based SPG11 model by knocking down the SPG11 gene in human embryonic stem cells (hESCs). These stem cells were then differentiated into cortical projection neurons (PNs), the cell types affected in HSP patients, to examine axonal defects and cholesterol distributions. Our data revealed that SPG11 deficiency led to reduced axonal outgrowth, impaired axonal transport, and accumulated swellings, recapitulating disease-specific phenotypes. In SPG11-knockdown neurons, cholesterol was accumulated in lysosome and reduced in plasma membrane, revealing impairments in cholesterol trafficking. Strikingly, the liver-X-receptor (LXR) agonists restored cholesterol homeostasis, leading to the rescue of subsequent axonal defects in SPG11-deficient cortical PNs. To further determine the implication of impaired cholesterol homeostasis in SPG11, we examined the cholesterol distribution in cortical PNs generated from SPG11 disease-mutation knock-in hESCs, and observed a similar cholesterol trafficking impairment. Moreover, LXR agonists rescued the aberrant cholesterol distribution and mitigated the degeneration of SPG11 disease-mutated neurons. Taken together, our data demonstrate impaired cholesterol trafficking underlying axonal degeneration of SPG11 human neurons, and highlight the therapeutic potential of LXR agonists for SPG11 through restoring cholesterol homeostasis.
Assuntos
Paraplegia Espástica Hereditária , Humanos , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/metabolismo , Proteínas/metabolismo , Neurônios/metabolismo , Mutação , Colesterol/metabolismo , Fígado/patologiaRESUMO
Hereditary spastic paraplegias are characterized by lower limb spasticity resulting from degeneration of long corticospinal axons. SPG11 is one of the most common autosomal recessive hereditary spastic paraplegias, and the SPG11 protein spatacsin forms a complex with the SPG15 protein spastizin and heterotetrameric AP5 adaptor protein complex, which includes the SPG48 protein AP5Z1. Using the integration-free episomal method, we established SPG11 patient-specific induced pluripotent stem cells (iPSCs) from patient fibroblasts. We differentiated SPG11 iPSCs, as well as SPG48 iPSCs previously established, into cortical projection neurons and examined protective effects by targeting mitochondrial dynamics using P110, a peptide that selectively inhibits mitochondrial fission GTPase Drp1. P110 treatment mitigates mitochondrial fragmentation, improves mitochondrial motility, and restores mitochondrial health and ATP levels in SPG11 and SPG48 neurons. Neurofilament aggregations are increased in SPG11 and SPG48 axons, and these are also suppressed by P110. Similarly, P110 mitigates neurofilament disruption in both SPG11 and SPG48 knockdown cortical projection neurons, confirming the contribution of hereditary spastic paraplegia gene deficiency to subsequent neurofilament and mitochondrial defects. Strikingly, neurofilament aggregations in SPG11 and SPG48 deficient neurons double stain with ubiquitin and autophagy related proteins, resembling the pathological hallmark observed in SPG11 autopsy brain sections. To confirm the cause-effect relationship between the SPG11 mutations and disease phenotypes, we knocked-in SPG11 disease mutations to human embryonic stem cells (hESCs) and differentiated these stem cells into cortical projection neurons. Reduced ATP levels and accumulated neurofilament aggregations along axons are observed, and both are mitigated by P110. Furthermore, rescue experiment with expression of wild-type SPG11 in cortical projection neurons derived from both SPG11 patient iPSCs and SPG11 disease mutation knock-in hESCs leads to rescue of mitochondrial dysfunction and neurofilament aggregations in these SPG11 neurons. Finally, in SPG11 and SPG48 long-term cultures, increased release of phosphoNF-H, a biomarker for nerve degeneration, is significantly reduced by inhibiting mitochondrial fission pharmacologically using P110 and genetically using Drp1 shRNA. Taken together, our results demonstrate that impaired mitochondrial dynamics underlie both cytoskeletal disorganization and axonal degeneration in SPG11 and SPG48 neurons, highlighting the importance of targeting these pathologies therapeutically.
Assuntos
Paraplegia Espástica Hereditária , Humanos , Paraplegia Espástica Hereditária/patologia , Dinâmica Mitocondrial , Neurônios/metabolismo , Mutação , Trifosfato de Adenosina/metabolismo , Proteínas/genéticaRESUMO
Mechanisms by which long corticospinal axons degenerate in hereditary spastic paraplegia (HSP) are largely unknown. Here, we have generated induced pluripotent stem cells (iPSCs) from patients with two autosomal recessive forms of HSP, SPG15 and SPG48, which are caused by mutations in the ZFYVE26 and AP5Z1 genes encoding proteins in the same complex, the spastizin and AP5Z1 proteins, respectively. In patient iPSC-derived telencephalic glutamatergic and midbrain dopaminergic neurons, neurite number, length and branching are significantly reduced, recapitulating disease-specific phenotypes. We analyzed mitochondrial morphology and noted a significant reduction in both mitochondrial length and their densities within axons of these HSP neurons. Mitochondrial membrane potential was also decreased, confirming functional mitochondrial defects. Notably, mdivi-1, an inhibitor of the mitochondrial fission GTPase DRP1, rescues mitochondrial morphology defects and suppresses the impairment in neurite outgrowth and late-onset apoptosis in HSP neurons. Furthermore, knockdown of these HSP genes causes similar axonal defects, also mitigated by treatment with mdivi-1. Finally, neurite outgrowth defects in SPG15 and SPG48 cortical neurons can be rescued by knocking down DRP1 directly. Thus, abnormal mitochondrial morphology caused by an imbalance of mitochondrial fission and fusion underlies specific axonal defects and serves as a potential therapeutic target for SPG15 and SPG48.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas de Transporte/genética , GTP Fosfo-Hidrolases/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Mitocondriais/genética , Paraplegia Espástica Hereditária/genética , Axônios/efeitos dos fármacos , Axônios/patologia , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Dinaminas , Humanos , Células-Tronco Pluripotentes Induzidas , Potencial da Membrana Mitocondrial/genética , Mesencéfalo/metabolismo , Mesencéfalo/patologia , Mitocôndrias/genética , Mitocôndrias/patologia , Dinâmica Mitocondrial/genética , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Mutação , Crescimento Neuronal/efeitos dos fármacos , Crescimento Neuronal/genética , Quinazolinonas/farmacologia , Paraplegia Espástica Hereditária/tratamento farmacológico , Paraplegia Espástica Hereditária/fisiopatologiaRESUMO
Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, is characterized by the deterioration of alpha motor neurons in the brainstem and spinal cord. Currently, there is no cure for SMA, which calls for an urgent need to explore affordable and effective therapies and to maximize patients' independence and quality of life. Adeno-associated virus (AAV) vector, one of the most promising and well-investigated vehicles for delivering transgenes, is a compelling candidate for gene therapy. Some of the hallmarks of AAVs are their nonpathogenicity, inability to incur an immune response, potential to achieve robust transgene expression, and varied tropism for several tissues of the body. Recently, these features were harnessed in a clinical trial conducted by AveXis in SMA patients, where AAV9 was employed as a vehicle for one-time administration of the SMN gene, the causative gene in SMA. The trial demonstrated remarkable improvements in motor milestones and rates of survival in the patients. This review focuses on the advent of SMA gene therapy and summarizes different preclinical studies that were conducted leading up to the AAV9-SMA trial in SMA patients.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Atrofia Muscular Espinal/terapia , Animais , Dependovirus/metabolismo , Terapia Genética/efeitos adversos , Humanos , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
Cell culture has become an indispensable tool to help uncover fundamental biophysical and biomolecular mechanisms by which cells assemble into tissues and organs, how these tissues function, and how that function becomes disrupted in disease. Cell culture is now widely used in biomedical research, tissue engineering, regenerative medicine, and industrial practices. Although flat, two-dimensional (2D) cell culture has predominated, recent research has shifted toward culture using three-dimensional (3D) structures, and more realistic biochemical and biomechanical microenvironments. Nevertheless, in 3D cell culture, many challenges remain, including the tissue-tissue interface, the mechanical microenvironment, and the spatiotemporal distributions of oxygen, nutrients, and metabolic wastes. Here, we review 2D and 3D cell culture methods, discuss advantages and limitations of these techniques in modeling physiologically and pathologically relevant processes, and suggest directions for future research.
Assuntos
Técnicas de Cultura de Células/métodos , Animais , Pesquisa Biomédica/métodos , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Matriz Extracelular/fisiologia , Humanos , Engenharia Tecidual/métodosRESUMO
Telocyte (TC) as a special stromal cell exists in mammary gland and might play an important role in the balance of epithelium-stroma of mammary gland. Considering that different types of breast interstitial cells influence the development and progression of breast cancer, TCs may have its distinct role in this process. We here studied the roles of TCs in the self-assembly of reconstituted breast cancer tissue. We co-cultured primary isolated TCs and other breast stromal cells with breast cancer EMT-6 cells in collagen/Matrigel scaffolds to reconstitute breast cancer tissue in vitro. Using histology methods, we investigated the immunohistochemical characteristics and potential functions of TCs in reconstituted breast cancer tissue. TCs in primary mammary gland stromal cells with long and thin overlapping cytoplasmic processes, expressed c-kit/CD117, CD34 and vimentin in reconstitute breast cancer tissue. The transmission electron microscopy showed that the telocyte-like cells closely communicated with breast cancer cells as well as other stromal cells, and might serve as a bridge that directly linked the adjacent cells through membrane-to-membrane contact. Compared with cancer tissue sheets of EMT-6 alone, PCNA proliferation index analysis and TUNEL assay showed that TCs and other breast stromal cells facilitated the formation of typical nest structure, promoted the proliferation of breast cancer cells, and inhibited their apoptosis. In conclusion, we successfully reconstituted breast cancer tissue in vitro, and it seems to be attractive that TCs had potential functions in self-assembly of EMT-6/stromal cells reconstituted breast cancer tissue.
Assuntos
Comunicação Celular , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Cocultura , Colágeno , Combinação de Medicamentos , Feminino , Expressão Gênica , Laminina , Glândulas Mamárias Animais/fisiologia , Neoplasias Mamárias Experimentais/genética , Camundongos , Camundongos Endogâmicos BALB C , Cultura Primária de Células , Proteoglicanas , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais , Células Estromais/patologia , Células Estromais/fisiologia , Técnicas de Cultura de Tecidos , Vimentina/genética , Vimentina/metabolismoRESUMO
BACKGROUND: Biallelic mutations in CYP27A1 and CYP7B1, two critical genes regulating cholesterol and bile acid metabolism, cause cerebrotendinous xanthomatosis (CTX) and hereditary spastic paraplegia type 5 (SPG5), respectively. These rare diseases are characterized by progressive degeneration of corticospinal motor neuron axons, yet the underlying pathogenic mechanisms and strategies to mitigate axonal degeneration remain elusive. METHODS: To generate induced pluripotent stem cell (iPSC)-based models for CTX and SPG5, we reprogrammed patient skin fibroblasts into iPSCs by transducing fibroblast cells with episomal vectors containing pluripotency factors. These patient-specific iPSCs, as well as control iPSCs, were differentiated into cortical projection neurons (PNs) and examined for biochemical alterations and disease-related phenotypes. RESULTS: CTX and SPG5 patient iPSC-derived cortical PNs recapitulated several disease-specific biochemical changes and axonal defects of both diseases. Notably, the bile acid chenodeoxycholic acid (CDCA) effectively mitigated the biochemical alterations and rescued axonal degeneration in patient iPSC-derived neurons. To further examine underlying disease mechanisms, we developed CYP7B1 knockout human embryonic stem cell (hESC) lines using CRISPR-cas9-mediated gene editing and, following differentiation, examined hESC-derived cortical PNs. Knockout of CYP7B1 resulted in similar axonal vesiculation and degeneration in human cortical PN axons, confirming a cause-effect relationship between gene deficiency and axonal degeneration. Interestingly, CYP7B1 deficiency led to impaired neurofilament expression and organization as well as axonal degeneration, which could be rescued with CDCA, establishing a new disease mechanism and therapeutic target to mitigate axonal degeneration. CONCLUSIONS: Our data demonstrate disease-specific lipid disturbances and axonopathy mechanisms in human pluripotent stem cell-based neuronal models of CTX and SPG5 and identify CDCA, an established treatment of CTX, as a potential pharmacotherapy for SPG5. We propose this novel treatment strategy to rescue axonal degeneration in SPG5, a currently incurable condition.
Assuntos
Células-Tronco Pluripotentes Induzidas , Paraplegia Espástica Hereditária , Xantomatose Cerebrotendinosa , Humanos , Ácido Quenodesoxicólico/farmacologia , Ácido Quenodesoxicólico/uso terapêutico , Ácido Quenodesoxicólico/metabolismo , Xantomatose Cerebrotendinosa/genética , Neurônios/metabolismo , Neurônios/patologia , Paraplegia Espástica Hereditária/metabolismo , Ácidos e Sais Biliares , Paraplegia/metabolismoRESUMO
Axonal degeneration underlies many debilitating diseases including hereditary spastic paraplegias (HSPs). HSPs are a large heterogeneous group of neurodegenerative diseases characterized by axonopathy involving the long corticospinal tract. How axons of these cortical projection neurons specifically degenerate in HSPs remains largely unclear partially due to the lack of human models to monitor the dynamic process of axonal degeneration. With the development of induced pluripotent stem cell (iPSC) technology, patient-specific iPSCs are successfully generated from HSP patients, providing a unique paradigm to study the axonal degeneration in patient-derived neurons in live cultures. In this chapter, we will summarize the procedures to examine axonal defects in iPSC models of HSPs and discuss the challenges and future applications in order to rescue axonal degeneration in HSPs.
Assuntos
Células-Tronco Pluripotentes Induzidas , Paraplegia Espástica Hereditária , Axônios , Humanos , Neurônios , Paraplegia Espástica Hereditária/genéticaRESUMO
Charcot-Marie-Tooth (CMT) disease is one of the most common genetically inherited neurological disorders and CMT type 2A (CMT 2A) is caused by dominant mutations in the mitofusin-2 (MFN2) gene. MFN2 is located in the outer mitochondrial membrane and is a mediator of mitochondrial fusion, with an essential role in maintaining normal neuronal functions. Although loss of MFN2 induces axonal neuropathy, the detailed mechanism by which MFN2 deficiency results in axonal degeneration of human spinal motor neurons remains largely unknown. In this study, we generated MFN2-knockdown human embryonic stem cell (hESC) lines using lentivirus expressing MFN2 short hairpin RNA (shRNA). Using these hESC lines, we found that MFN2 loss did not affect spinal motor neuron differentiation from hESCs but resulted in mitochondrial fragmentation and dysfunction as determined by live-cell imaging. Notably, MFN2-knockodwn spinal motor neurons exhibited CMT2A disease-related phenotypes, including extensive perikaryal inclusions of phosphorylated neurofilament heavy chain (pNfH), frequent axonal swellings, and increased pNfH levels in long-term cultures. Importantly, MFN2 deficit impaired anterograde and retrograde mitochondrial transport within axons, and reduced the mRNA and protein levels of kinesin and dynein, indicating the interfered motor protein expression induced by MFN2 deficiency. Our results reveal that MFN2 knockdown induced axonal degeneration of spinal motor neurons and defects in mitochondrial morphology and function. The impaired mitochondrial transport in MFN2-knockdown spinal motor neurons is mediated, at least partially, by the altered motor proteins, providing potential therapeutic targets for rescuing axonal degeneration of spinal motor neurons in CMT2A disease.
RESUMO
Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with "MultiKymograph", "Bioformat importer", and "Macros" plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Mitocôndrias/metabolismo , Neurônios/metabolismo , Paraplegia Espástica Hereditária/metabolismo , Animais , Axônios/metabolismo , Transporte Biológico , Células Cultivadas , Humanos , Dinâmica Mitocondrial , Neurônios/citologia , Prosencéfalo/citologiaRESUMO
Hereditary spastic paraplegias (HSPs) are caused by a length-dependent axonopathy of long corticospinal neurons, but how axons of these cortical projection neurons (PNs) degenerate remains elusive. We generated isogenic human pluripotent stem cell (hPSC) lines for two ATL1 missense mutations associated with SPG3A, the most common early-onset autosomal dominant HSP. In hPSC-derived cortical PNs, ATL1 mutations resulted in reduced axonal outgrowth, impaired axonal transport, and accumulated axonal swellings, recapitulating disease-specific phenotypes. Importantly, ATL1 mutations dysregulated proteolipid gene expression, reduced lipid droplet size in astrocytes, and unexpectedly disrupted cholesterol transfer from glia to neurons, leading to cholesterol deficiency in SPG3A cortical PNs. Applying cholesterol or conditioned medium from control astrocytes, a major source of cholesterol in the brain, rescued aberrant axonal transport and swellings in SPG3A cortical PNs. Furthermore, treatment with the NR1H2 agonist GW3965 corrected lipid droplet defects in SPG3A astrocytes and promoted cholesterol efflux from astrocytes, leading to restoration of cholesterol levels and rescue of axonal degeneration in SPG3A cortical PNs. These results reveal a non-cell autonomous mechanism underlying axonal degeneration of cortical PNs mediated by impaired cholesterol homeostasis in glia.
Assuntos
Astrócitos/metabolismo , Córtex Cerebral/metabolismo , Colesterol/metabolismo , Metabolismo dos Lipídeos/fisiologia , Neurônios/metabolismo , Tratos Piramidais/metabolismo , Paraplegia Espástica Hereditária/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Axônios/efeitos dos fármacos , Axônios/metabolismo , Axônios/patologia , Benzoatos/farmacologia , Benzilaminas/farmacologia , Córtex Cerebral/citologia , Proteínas de Ligação ao GTP/genética , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Receptores X do Fígado/agonistas , Proteínas de Membrana/genética , Neurônios/efeitos dos fármacos , Neurônios/patologia , Células-Tronco Pluripotentes , Tratos Piramidais/citologia , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/patologiaRESUMO
Impaired axonal development and degeneration underlie debilitating neurodegenerative diseases including hereditary spastic paraplegia, a large group of inherited diseases. Hereditary spastic paraplegia is caused by retrograde degeneration of the long corticospinal tract axons, leading to progressive spasticity and weakness of leg and hip muscles. There are over 70 subtypes with various underlying pathophysiological processes, such as defective vesicular trafficking, lipid metabolism, organelle shaping, axonal transport, and mitochondrial dysfunction. Although hereditary spastic paraplegia consists of various subtypes with different pathological characteristics, defects in mitochondrial morphology and function emerge as one of the common cellular themes in hereditary spastic paraplegia. Mitochondrial morphology and function are remodeled by mitochondrial dynamics regulated by several key fission and fusion mediators. However, the role of mitochondrial dynamics in axonal defects of hereditary spastic paraplegia remains largely unknown. Recently, studies reported perturbed mitochondrial morphology in hereditary spastic paraplegia neurons. Moreover, downregulation of mitochondrial fission regulator dynamin-related protein 1, both pharmacologically and genetically, could rescue axonal outgrowth defects in hereditary spastic paraplegia neurons, providing a potential therapeutic target for treating these hereditary spastic paraplegia. This mini-review will describe the regulation of mitochondrial fission/fusion, the link between mitochondrial dynamics and axonal defects, and the recent progress on the role of mitochondrial dynamics in axonal defects of hereditary spastic paraplegia.
RESUMO
Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, is caused by reduced levels of functional survival motor neuron (SMN) protein. To identify therapeutic agents for SMA, we established a versatile SMN2-GFP reporter line by targeting the human SMN2 gene. We then screened a compound library and identified Z-FA-FMK as a potent candidate. Z-FA-FMK, a cysteine protease inhibitor, increased functional SMN through inhibiting the protease-mediated degradation of both full-length and exon 7-deleted forms of SMN. Further studies reveal that CAPN1, CAPN7, CTSB, and CTSL mediate the degradation of SMN proteins, providing novel targets for SMA. Notably, Z-FA-FMK mitigated mitochondriopathy and neuropathy in SMA patient-derived motor neurons and showed protective effects in SMA animal model after intracerebroventricular injection. E64d, another cysteine protease inhibitor which can pass through the blood-brain barrier, showed even more potent therapeutic effects after subcutaneous delivery to SMA mice. Taken together, we have successfully established a human SMN2 reporter for future drug discovery and identified the potential therapeutic value of cysteine protease inhibitors in treating SMA via stabilizing SMN proteins.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Genes Reporter/genética , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/genética , Estabilidade Proteica/efeitos dos fármacos , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Células HEK293 , Humanos , Cetonas/farmacologia , Leucina/análogos & derivados , Leucina/farmacologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Substâncias Protetoras/farmacologia , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo , Transfecção , Resultado do TratamentoRESUMO
Physico-chemical characteristics of the CoCrMo degradation products have played an important role in cytotoxicity and clinical complications on the orthopedic patients who have metal implants. Previous studies have limited reflection on the physicochemical characteristics of the degradation products generated in vivo, which are very different from individual metal particles and/or ions obtained from different commercial sources. In this study, we aimed to understand the differences in toxicity induced by the degradation products in as-synthesized form as well as those obtained after post-processing. The degradation products were generated using a hip-simulator by maintaining physiological conditions closer to in vivo and separated into two batches, one with processing by washing and drying called processed degradation products (PDP) and another batch as 'as-synthesized' degradation product (DP). We studied the dose-dependent toxicity response by neural cells derived from induced pluripotent stem cells. The results of the study show that as-synthesized DPs are more toxic to neural cells even at lower concentrations studied with evident low TC50 (1-5 µg/ml) concentrations compared to PDP (25 µg/ml). Flow cytometric analysis showed a significant (p<.01) increase in uptake of the particles after 24 h and corresponding ROS production in DP-treated cells. RT-PCR analysis of oxidative specific gene expression showed, elevated mRNA levels of NADPH oxidase-1, nuclear transcription factor, superoxide dismutase-2 and glutaredoxin-2 in DP-treated cells after 6 h. The results of the study provided a clear evidence of the differential response of neural cells on the degradation products as a function of concentrations and their chemical nature.
Assuntos
Prótese de Quadril , Neurônios/efeitos dos fármacos , Vitálio/química , Vitálio/toxicidade , Apoptose/efeitos dos fármacos , Diferenciação Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Teste de Materiais , Pessoa de Meia-Idade , Neurônios/metabolismo , Neurônios/patologia , Oxirredução , Coroa de Proteína/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Propriedades de SuperfícieRESUMO
Although iron oxide nanoparticles (IRONs) were applied in clinical magnetic resonance imaging in vivo and magnetic tissue engineering in vitro widely, the underlying effects of IRONs on the development of cardiomyocytes especially the intercellular junctions, intercalated discs (IDs), remain an unknown issue. Given the critical role of three-dimensional (3D) engineered cardiac tissues (ECTs) in evaluation of nanoparticles toxicology, it remained necessary to understand the effects of IRONs on IDs assembly of cardiomyocytes in 3D environment. In this study, we first reconstituted collagen/Matrigel based ECTs in vitro and prepared IRONs with 2,3-dimercaptosuccinic acid (DMSA-IRONs). We found that the internalization of DMSA-IRONs by cardiac cells in dose-dependent manner was not associated with the cell distribution in 3D environment by determination of Prussian blue staining and transmission electronic microscopy. Significantly, through determination of western blotting and immunofluorescence of connexin 43, N-cadherin, desmoplakin, and plakoglobin, DMSA-IRONs enhanced the assembly of gap junctions, decreased mechanical junctions (adherens junctions and desmosomes) of cardiac cells but not in dose-dependent manner in ECTs at seventh day. In addition, DMSA-IRONs increased the vesicles secretion of cardiac cells in ECTs apparently compared to control groups. Overall, we conclude that the internalization of DMSA-IRONs by cardiac cells in dose-dependent manner enhanced the assembly of electrochemical junctions and decreased the mechanical related microstructures. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 121-130, 2018.
Assuntos
Nanopartículas de Magnetita/química , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Succímero/química , Engenharia Tecidual , Alicerces Teciduais/química , Animais , Camundongos , Miocárdio/citologia , Miócitos Cardíacos/citologiaRESUMO
Carbon nanotube (CNT)-based hydrogels have been shown to support cardiomyocyte growth and function. However, their role in cellular integrity among cardiomyocytes has not been studied in detail and the mechanisms underlying this process remain unclear. Here, single walled CNTs incorporated into gelatin with methacrylate anhydride (CNT/GelMA) hydrogels were utilized to construct cardiac tissues, which enhanced cardiomyocyte adhesion and maturation. Furthermore, through the use of immunohistochemical staining, transmission electron microscopy and intracellular calcium transient measurement, the incorporation of CNTs into the scaffolds was observed to markedly enhance the assembly and formation in the cardiac constructs. Importantly, we further explored the underlying mechanism behind these effects through the use of immunohistochemical staining and western blotting. The ß1-integrin-mediated FAK and RhoA signaling pathways were found to be responsible for CNT-induced upregulation of electrical and mechanical junction proteins respectively. Together, our study provides new insights into the facilitative effects of CNTs on ID formation, which has important significance for improving the quality of engineered cardiac tissue and applying them to cardiac regenerative therapies. STATEMENT OF SIGNIFICANCE: Currently, the bottleneck to engineering cardiac tissues (ECTs) for cardiac regeneration is the lack of efficient cellular integrity among adjacent cells, especially the insufficient remodeling of intercalated discs (IDs) in ECTs. Recently, carbon nanotube (CNT) hydrogels provide an advantageous supporting microenvironment and therefore benefit greatly the functional performance of ECTs. Although their beneficial effect in modulating ECT performance is evident, the influence of CNTs on structural integrity of ECTs has not been studied in detail, and the mechanisms underlying the process remain to be determined. Here, we utilized carbon nanotube incorporated into gelatin with methacrylate anhydride (CNT/GelMA) hydrogels to construct cardiac tissues, determined the influence of CNTs on intercalated discs (IDs) assembly and formation and explored the underlying mechanisms.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Coração/fisiologia , Hidrogéis/química , Integrina beta1/metabolismo , Nanocompostos/química , Nanotubos de Carbono/química , Engenharia Tecidual/métodos , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Cálcio/metabolismo , Morte Celular , Conexina 43/metabolismo , Imunofluorescência , Junções Comunicantes/metabolismo , Gelatina/química , Metacrilatos/química , Miocárdio/ultraestrutura , Nanotubos de Carbono/ultraestrutura , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Stem cell-based therapy remains one of the promising approaches for cardiac repair and regeneration. However, its applications are restricted by the limited efficacy of cardiac differentiation. To address this issue, we examined whether carbon nanotubes (CNTs) would provide an instructive extracellular microenvironment to facilitate cardiogenesis in brown adipose-derived stem cells (BASCs) and to elucidate the underlying signaling pathways. In this study, we systematically investigated a series of cellular responses of BASCs due to the incorporation of CNTs into collagen (CNT-Col) substrates that promoted cell adhesion, spreading, and growth. Moreover, we found that CNT-Col substrates remarkably improved the efficiency of BASCs cardiogenesis by using fluorescence staining and quantitative real-time reverse transcription-polymerase chain reaction. Critically, CNTs in the substrates accelerated the maturation of BASCs-derived cardiomyocytes. Furthermore, the underlying mechanism for promotion of BASCs cardiac differentiation by CNTs was determined by immunostaining, quantitative real-time reverse transcription-polymerase chain reaction, and Western blotting assay. It is notable that ß1-integrin-dependent TGF-ß1 signaling pathway modulates the facilitative effect of CNTs in cardiac differentiation of BASCs. Therefore, it is an efficient approach to regulate cardiac differentiation of BASCs by the incorporation of CNTs into the native matrix. Importantly, our findings can not only facilitate the mechanistic understanding of molecular events initiating cardiac differentiation in stem cells, but also offer a potentially safer source for cardiac regenerative medicine.
RESUMO
Carbon nanotubes (CNTs) offer a new paradigm for constructing functional cardiac patches and repairing myocardial infarction (MI). However, little is known about how CNTs enhance the mechanical integrity and electrophysiological function of cardiac myocytes. To address this issue, we investigated the regularity and precise mechanism of the influence of CNTs on the assembly of intercalated disc (IDs). Here, single walled CNTs incorporated into collagen substrates were utilized as growth supports for neonatal cardiomyocytes, which enhanced cardiomyocyte adhesion and maturation. Furthermore, through the use of immunohistochemical staining, western blotting, transmission electron microscopy, and intracellular calcium transient measurement, we discovered that the addition of CNTs remarkably increased ID-related protein expression and enhanced ID assembly and functionality. On that basis, we further explored the underlying mechanism for how CNTs enhanced ID assembly through the use of immunohistochemical staining and western blotting. We found that the ß1-integrin-mediated signaling pathway mediated CNT-induced upregulation of electrical and mechanical junction proteins. Notably, CNTs remarkably accelerated gap junction formation via activation of the ß1-integrin-mediated FAK/ERK/GATA4 pathway. These findings provide valuable insight into the mechanistic effects that CNTs have on neonatal cardiomyocyte performance and will have a significant impact on the future of nanomedical research.
Assuntos
Integrina beta1/metabolismo , Miócitos Cardíacos/citologia , Nanotubos de Carbono/química , Transdução de Sinais , Animais , Animais Recém-Nascidos , Cálcio/química , Adesão Celular , Sobrevivência Celular , Colágeno/química , Conexina 43/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Junções Comunicantes , Imuno-Histoquímica , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Miócitos Cardíacos/efeitos dos fármacos , Nanomedicina/métodos , Ratos , Ratos Sprague-Dawley , Estresse Mecânico , Alicerces Teciduais/químicaRESUMO
Interstitial Cajal-like cells are a distinct type of interstitial cell with a wide distribution in mammalian organs and tissues, and have been given the name "telocytes". Recent studies have demonstrated the potential roles of telocytes in heart development, renewal, and repair. However, further research on the functions of telocytes is limited by the complicated in vivo environment. This study was designed to construct engineered heart tissue (EHT) as a three-dimensional model in vitro to better understand the role of telocytes in the architectural organization of the myocardium. EHTs were constructed by seeding neonatal cardiomyocytes in collagen/Matrigel scaffolds followed by culture under persistent static stretch. Telocytes in EHTs were identified by histology, toluidine blue staining, immunofluorescence, and transmission electron microscopy. The results from histology and toluidine blue staining demonstrated widespread putative telocytes with compact toluidine blue-stained nuclei, which were located around cardiomyocytes. Prolongations from the cell bodies showed a characteristic dichotomous branching pattern and formed networks in EHTs. Immunofluorescence revealed positive staining of telocytes for CD34 and vimentin with typical moniliform prolongations. A series of electron microscopy images further showed that typical telocytes embraced the cardiomyocytes with their long prolongations and exhibited a marked appearance of nursing cardiomyocytes during the construction of EHTs. This finding highlights the great importance of telocytes in the architectural organization of EHTs. It also suggests that EHT is an appropriate physical and pathological model system in vitro to study the roles of telocytes during heart development and regeneration.
Assuntos
Miocárdio/citologia , Engenharia Tecidual , Animais , Células Cultivadas , Imunofluorescência , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Ratos , Ratos Wistar , Coloração e RotulagemRESUMO
Recently, carbon nanotubes together with other types of conductive materials have been used to enhance the viability and function of cardiomyocytes in vitro. Here we demonstrated a paradigm to construct ECTs for cardiac repair using conductive nanomaterials. Single walled carbon nanotubes (SWNTs) were incorporated into gelatin hydrogel scaffolds to construct three-dimensional ECTs. We found that SWNTs could provide cellular microenvironment in vitro favorable for cardiac contraction and the expression of electrochemical associated proteins. Upon implantation into the infarct hearts in rats, ECTs structurally integrated with the host myocardium, with different types of cells observed to mutually invade into implants and host tissues. The functional measurements showed that SWNTs were essential to improve the performance of ECTs in inhibiting pathological deterioration of myocardium. This work suggested that conductive nanomaterials hold therapeutic potential in engineering cardiac tissues to repair myocardial infarction.