RESUMO
Pigment epithelium-derived factor (PEDF) is widely recognized as a neuroprotective factor expressed in the retina and has shown therapeutic potential in several retinal diseases. Our study aimed to identify the neuroprotective fragment in PEDF and investigate its protective activity in retinas under ischemia-reperfusion (IR) condition. We synthesized a series of shorter synthetic peptides, 6-mer (Ser93-Gln98) and its d-form variant (6 dS) derived from the 44-mer (Val78-Thr121; a PEDF neurotrophic fragment), to determine their cytoprotective activity in IR injury, which was induced in rat retinas by injection of saline into the anterior chamber to increase the intraocular pressure (IOP) followed by reperfusion. We found the cytoprotective effect of 6-mer on glutamate-treated Neuro-2a cells and tert-butyl hydroperoxide (tBHP)-treated 661W cells were 2.6-fold and 1.5-fold higher than the 44-mer, respectively. The cytoprotective effect was blocked by a chemical inhibitor atglistatin and blocking antibody targeting PEDF receptor (PEDF-R). IR induced several impairments in retina, including cell apoptosis, activation of microglia/macroglia, degeneration of retinal capillaries, reduction in electroretinography (ERG) amplitudes, and retinal atrophy. Such IR injuries were ameliorated by treatment with 6-mer and 6 dS eye drops. Also, the neuroprotective activity of 6-mer and 6 dS in ischemic retinas were dramatically reversed by atglistatin preconditioning. Taken together, our data demonstrate smallest neuroprotective fragment of PEDF has potential to treat retinal degeneration-related diseases.
Assuntos
Proteínas do Olho , Fatores de Crescimento Neural , Traumatismo por Reperfusão , Retina , Retinite , Serpinas , Animais , Ratos , Coelhos , Fatores de Crescimento Neural/administração & dosagem , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/metabolismo , Proteínas do Olho/administração & dosagem , Proteínas do Olho/química , Proteínas do Olho/metabolismo , Serpinas/administração & dosagem , Serpinas/química , Serpinas/metabolismo , Retina/metabolismo , Retina/patologia , Traumatismo por Reperfusão/metabolismo , Citoproteção , Apoptose , Neurônios/metabolismo , Retinite/tratamento farmacológico , Retinite/metabolismo , Administração Tópica , Peptídeos/administração & dosagem , Peptídeos/metabolismoRESUMO
BACKGROUND: Dry eye disease (DED) is a common disease in ophthalmology, affecting millions of people worldwide. Recent studies have shown that inflammation is the core mechanism of DED. IL-20 is a proinflammatory cytokine involved in various inflammatory diseases. Therefore, we aimed to explore the role of this cytokine in the pathogenesis of DED and evaluate the therapeutic potential of the anti-IL-20 monoclonal antibody (mAb) 7E for DED treatment. METHODS: Clinical tear samples from patients with DED and non-DED controls were collected and their IL-20 protein levels were determined. We established three DED animal models to explore the role of IL-20 and the efficacy of IL-20 antibody in DED. Benzalkonium chloride (BAC)-induced over-evaporative DED, extra-orbital lacrimal gland excision (LGE)-induced aqueous tear-deficient DED, and desiccating stress (DS)-induced combined over-evaporative and aqueous tear-deficient DED animal models were established to investigate the role of IL-20. The anti-IL-20 antibody 7E was established to neutralize IL-20 activity. The effects of IL-20 or 7E on human corneal epithelial cells and macrophages under hyperosmotic stress were analyzed. 7E was topically applied to eyes to evaluate the therapeutic effects in the DED animal models. RESULTS: IL-20 was significantly upregulated in the tears of patients with DED and in the tears and corneas of DED animal models. Under hyperosmotic stress, IL-20 expression was induced via NFAT5 activation in corneal epithelial cells. 7E suppressed hyperosmotic stress-induced activation of macrophages. IL-20 induced cell death in corneal epithelial cells and 7E protected cells from hyperosmotic stress-induced cell death. Blocking IL-20 signaling with 7E protected mice from BAC-induced, LGE-induced, and DS-induced DED by reducing DED symptoms and inhibiting inflammatory responses, macrophage infiltration, apoptosis, and Th17 populations in the conjunctiva and draining lymph nodes. CONCLUSIONS: Our results demonstrated the functions of IL-20 in DED and presented a potential therapeutic option for this condition.
Assuntos
Síndromes do Olho Seco , Interleucinas , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Síndromes do Olho Seco/induzido quimicamente , Síndromes do Olho Seco/diagnóstico , Síndromes do Olho Seco/tratamento farmacológico , Humanos , Interleucinas/metabolismo , Camundongos , Lágrimas/metabolismoRESUMO
BACKGROUND: Pigment epithelial-derived factor (PEDF), a 50 kDa secreted glycoprotein, exhibits distinct effects on a range of cell types. PEDF has been shown to inhibit vascular endothelial growth factor (VEGF)-mediated angiogenesis and widely accepted as a promising agent for treatment eye diseases related to neovascularization. A pool of short peptide fragments derived from PEDF reportedly manifests angioinhibitory activity. This study aims to determine the minimal PEDF fragment which can exert the anti-VEGF effect. METHODS: A series of shorter synthetic peptides, derived from the 34-mer (PEDF amino acid positions Asp44-Asn77), were synthesized. An MTT assay was used to evaluate the ability of the 34-mer-derived peptides to inhibit VEGF-induced proliferation of multiple myeloma RPMI8226 cells. Cell apoptosis was monitored by annexin V-FITC staining. Western blot analysis was used to detect phosphorylated kinases, including c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and the expression of apoptosis-associated proteins, including p53, bax and caspase-3. VEGF-mediated angiogenesis of human umbilical vein endothelial cells (HUVECs), rat aortic ring and mouse cornea were used to detect the angioinhibitory activity of the PEDF-derived peptides. RESULTS: The MTT assay showed that the anti-VEGF effect of a 7-mer (Asp64-Ser70) was 1.5-fold greater than the 34-mer. In addition, massive apoptosis (37%) was induced by 7-mer treatment. The 7-mer induced JNK phosphorylation in RPMI8226 cells. Cell apoptosis and apoptosis-associated proteins induced by the 7-mer were blocked by pharmacological inhibition of JNK, but not p38 MAPK. Moreover, the 7-mer prevented VEGF-mediated angiogenesis of endothelial cells (ECs), including tube formation, aortic EC spreading and corneal neovascularization in mice. CONCLUSIONS: This is the first study to show that the PEDF 7-mer peptide manifests anti-VEGF activity, further establishing its potential as an anti-angiogenic agent.
Assuntos
Inibidores da Angiogênese/farmacologia , Proteínas do Olho/farmacologia , Fatores de Crescimento Neural/farmacologia , Peptídeos/farmacologia , Serpinas/farmacologia , Animais , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Proteínas do Olho/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Fatores de Crescimento Neural/metabolismo , Ratos , Serpinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Cell signaling mediated by the αv integrin plays a pivotal role in macrophage activation in various inflammatory processes, but its involvement in the pathogenesis of dry eye disease (DED) remains unclear. In a murine model of DED, we found increased αv integrin expression in ocular surface macrophages. The αv integrins inhibitor c(RGDfK) ameliorated the corneal damage caused by DED, suggesting a pathogenic role for αv integrin. Because tear hyperosmolarity induces ocular inflammation in DED, a hyperosmolar culture of murine bone marrow-derived macrophages (BMDMs) is used to reproduce inflammation in vitro. However, the expression of proinflammatory cytokine mRNA was minimal, even though αv integrin was induced. In searching for components that are involved in αv integrin-mediated inflammation but that are missing from the culture model, we showed that the levels of vitronectin (VTN), a binding ligand of αv integrins, were increased in the tear fluid and conjunctival stroma of DED animals. The addition of VTN prominently enhanced hyperosmolarity-induced inflammation in BMDMs. Mechanistically, we showed that VTN/αv integrins mediated NF-κB activation to induce inflammatory gene expression in the BMDMs. Our findings indicate that interaction the of VTN with αv integrins is a crucial step in the inflammatory process in DED and suggests a novel therapeutic target.
Assuntos
Síndromes do Olho Seco/metabolismo , Inflamação/metabolismo , Integrina alfaV/metabolismo , Macrófagos/metabolismo , Vitronectina/metabolismo , Animais , Linhagem Celular , Citocinas/metabolismo , Olho/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia , Células THP-1 , Lágrimas/metabolismoRESUMO
BACKGROUND: Cardiomyopathy is a common and lethal complication in patients with limb-girdle muscular dystrophy (LGMD), one of the most prevalent forms of muscular dystrophy. The pathogenesis underlying LGMD-related cardiomyopathy remains unclear. NRIP (gene name DCAF6), a Ca2+-dependent calmodulin binding protein, was reduced in dystrophic muscles from LGMD patients. Mice lacking NRIP exhibit a myopathic phenotype resembling that in LGMD patients, making NRIP deficiency a potential culprit leading to cardiomyopathy. This study aimed to determine if NRIP deficiency leads to cardiomyopathy and to explore the underlying molecular mechanisms. METHODS AND RESULTS: NRIP expression was reduced in both human and mouse failing hearts. Muscle-specific NRIP knockout (MCK-Cre::Dcaf6flox/flox) mouse heart and isolated cardiomyocytes exhibited markedly reduced contractility. Transmission electron microscopy revealed abnormal sarcomere structures and mitochondrial morphology in MCK-Cre::Dcaf6flox/flox hearts. Protein co-immunoprecipitation and confocal imaging revealed that NRIP interacts with α-actinin 2 (ACTN2) at the Z-disc. We found that NRIP facilitated ACTN2-mediated F-actin bundling, and that NRIP deficiency resulted in reduced binding between Z-disc proteins ACTN2 and Cap-Z. In addition, NRIP-deficiency led to increased mitochondrial ROS and impaired mitochondrial respiration/ATP production owing to elevated cellular NADH/NAD+ ratios. Treatment with mitochondria-directed antioxidant mitoTEMPO or NAD+ precursor nicotinic acid restored mitochondrial function and cardiac contractility in MCK-Cre::Dcaf6flox/flox mice. CONCLUSIONS: NRIP is essential to maintain sarcomere structure and mitochondrial/contractile function in cardiomyocytes. Our results revealed a novel role for NRIP deficiency in the pathogenesis of LGMD and heart failure. Targeting NRIP, therefore, could be a powerful new approach to treat myocardial dysfunction in LGMD and heart failure patients.
Assuntos
Cardiomiopatias/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteína 1 de Interação com Receptor Nuclear/metabolismo , Sarcômeros/metabolismo , Actinina/metabolismo , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Cardiomiopatias/fisiopatologia , Respiração Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Insuficiência Cardíaca/genética , Homeostase/efeitos dos fármacos , Humanos , Masculino , Camundongos , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/ultraestrutura , Modelos Biológicos , Contração Miocárdica/efeitos dos fármacos , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , NAD/metabolismo , Niacina/farmacologia , Proteína 1 de Interação com Receptor Nuclear/química , Fenótipo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Espécies Reativas de Oxigênio/metabolismo , Sarcômeros/efeitos dos fármacos , Sarcômeros/ultraestruturaRESUMO
Expansion of limbal epithelial stem cells (LSCs) is crucial for the success of limbal transplantation. Previous studies showed that pigment epithelium-derived peptide (PEDF) short peptide 44-mer could effectively expand LSCs and maintain them in a stem-cell state, but the mechanism remained unclear. In the current study, we found that pharmacological inhibition of Sonic Hedgehog (SHh) activity reduced the LSC holoclone number and suppressed LSC proliferation in response to 44-mer. In mice subjected to focal limbal injury, 44-mer facilitated the restoration of the LSC population in damaged limbus, and such effect was impeded by the SHh or ATGL (a PEDF receptor) inhibitor. Furthermore, we showed that 44-mer increased nuclear translocation of Gli1 and Gli3 in LSCs. Knockdown of Gli1 or Gli3 suppressed the ability of 44-mer to induce cyclin D1 expression and LSC proliferation. In addition, ATGL inhibitor suppressed the 44-mer-induced phosphorylation of STAT3 at Tyr705 in LSC. Both inhibitors for ATGL and STAT3 attenuated 44-mer-induced SHh activation and LSC proliferation. In conclusion, our data demonstrate that SHh-Gli pathway driven by ATGL/STAT3 signalling accounts for the 44-mer-mediated LSC proliferation.
Assuntos
Proteínas do Olho/farmacologia , Proteínas Hedgehog/metabolismo , Limbo da Córnea/citologia , Fatores de Crescimento Neural/farmacologia , Peptídeos/farmacologia , Serpinas/farmacologia , Transdução de Sinais , Células-Tronco/citologia , Animais , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Lipase/metabolismo , Camundongos Endogâmicos BALB C , Mitógenos/farmacologia , Coelhos , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Fatores de Transcrição/metabolismoRESUMO
Age-related meibomian gland (MG) atrophy, characterized by decreased meibocyte proliferation, is one of the causes of meibomian gland dysfunction (MGD), which leads to dry eye disease. Currently, there is no available treatment effectively preventing or reversing the decreased cell proliferation and acinar tissue atrophy. In this study, we investigated the therapeutic effects of a pigment epithelium-derived factor (PEDF) peptide in treating this condition. We found abundant expression of PEDF in the nucleus of acinar basal cells, but not in mature meibocytes, and that the expression levels were significantly decreased in the aged mice. We next treated the aged mice (15-month old) with atrophic MGs using a synthetic PEDF-derived peptide 29-mer (residues 93-121). We found that 29-mer effectively stimulated acinar basal cell proliferation and the following mature meibocyte proliferation in the atrophied MGs. In addition, the treatment increased ΔNp63 and Lrig1 expressions in acinar basal cells. Finally, the aged mice receiving the treatment showed MG growth and improved tear film break-up time. In conclusion, the 29-mer treatment is effective in promoting MG acinar basal cell proliferation and enlarging the acinar size of MG, as well as improving MG function in aged mice, suggesting a therapeutic potential of the PEDF-derived short peptide in ameliorating age-related MGD.
Assuntos
Envelhecimento/fisiologia , Proteínas do Olho/uso terapêutico , Glândulas Tarsais/efeitos dos fármacos , Fatores de Crescimento Neural/uso terapêutico , Serpinas/uso terapêutico , Células Acinares/efeitos dos fármacos , Células Acinares/metabolismo , Células Acinares/patologia , Animais , Atrofia/tratamento farmacológico , Atrofia/metabolismo , Atrofia/patologia , Proliferação de Células/efeitos dos fármacos , Túnica Conjuntiva/efeitos dos fármacos , Síndromes do Olho Seco/tratamento farmacológico , Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/patologia , Proteínas do Olho/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Imuno-Histoquímica , Injeções Intraoculares , Glândulas Tarsais/metabolismo , Glândulas Tarsais/patologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Serpinas/metabolismo , Lágrimas/fisiologia , Transativadores/metabolismoRESUMO
Psoriasis is a chronic inflammatory skin disease characterized by excessive growth of keratinocytes and hyperkeratosis in the epidermis. An abnormality of the non-lesional epidermis at an early stage of psoriasis is involved in triggering inflammatory cell infiltration into the dermis. Integrin α5ß1 acts as a receptor for fibronectin and has been found to be overexpressed in non-lesional psoriatic epidermis. To investigate whether α5ß1 integrin has a potential as a drug target for psoriasis treatment, the α5ß1 integrin-binding peptide, C16, was used to obstruct the HaCat keratinocyte cellular responses induced by fibronectin (Fn) in culture and psoriasis-like skin inflammation induced in mice by imiquimod (IMQ). The C16 exhibited antagonistic activity against α5ß1 integrin in HaCat cells, with evidence of suppression of the Fn-mediated proliferative, cytoskeletal, and inflammatory responses. Topical treatment with C16 greatly reduced the IMQ-induced epidermal hyperplasia, infiltration of neutrophils/macrophages, and expression of pro-inflammatory mediators in mouse skin. The C16SP (C16-derived short peptide; DITYVRLKF) also exhibited antagonistic activity, suppressing α5ß1 integrin activity in culture, and reducing IMQ-induced skin inflammation. Taken together, this study provides the first evidence that α5ß1 integrin may be a potential drug target for psoriasis. The synthetic C16 peptide may serve as an agent for psoriasis therapy.
Assuntos
Anti-Inflamatórios/uso terapêutico , Laminina/química , Fragmentos de Peptídeos/uso terapêutico , Psoríase/tratamento farmacológico , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Linhagem Celular , Feminino , Fibronectinas/farmacologia , Humanos , Imiquimode/toxicidade , Integrina alfa5beta1/antagonistas & inibidores , Integrina alfa5beta1/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Psoríase/etiologiaRESUMO
Nuclear receptor interaction protein (NRIP, also known as DCAF6 and IQWD1) is a Ca(2+)-dependent calmodulin-binding protein. In this study, we newly identify NRIP as a Z-disc protein in skeletal muscle. NRIP-knockout mice were generated and found to have reduced muscle strength, susceptibility to fatigue and impaired adaptive exercise performance. The mechanisms of NRIP-regulated muscle contraction depend on NRIP being downstream of Ca(2+) signaling, where it stimulates activation of both 'calcineurin-nuclear factor of activated T-cells, cytoplasmic 1' (CaN-NFATc1; also known as NFATC1) and calmodulin-dependent protein kinase II (CaMKII) through interaction with calmodulin (CaM), resulting in the induction of mitochondrial activity and the expression of genes encoding the slow class of myosin, and in the regulation of Ca(2+) homeostasis through the internal Ca(2+) stores of the sarcoplasmic reticulum. Moreover, NRIP-knockout mice have a delayed regenerative capacity. The amount of NRIP can be enhanced after muscle injury and is responsible for muscle regeneration, which is associated with the increased expression of myogenin, desmin and embryonic myosin heavy chain during myogenesis, as well as for myotube formation. In conclusion, NRIP is a novel Z-disc protein that is important for skeletal muscle strength and regenerative capacity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Calmodulina/metabolismo , Músculo Esquelético/fisiologia , Proteínas Nucleares/metabolismo , Regeneração/fisiologia , Animais , Camundongos , Camundongos Knockout , Contração Muscular/fisiologia , Músculo Esquelético/metabolismo , Transdução de SinaisRESUMO
In response injury, intrinsic repair mechanisms are activated in skeletal muscle to replace the damaged muscle fibers with new muscle fibers. The regeneration process starts with the proliferation of satellite cells to give rise to myoblasts, which subsequently differentiate terminally into myofibers. Here, we investigated the promotion effect of pigment epithelial-derived factor (PEDF) on muscle regeneration. We report that PEDF and a synthetic PEDF-derived short peptide (PSP; residues Ser(93)-Leu(112)) induce satellite cell proliferation in vitro and promote muscle regeneration in vivo. Extensively, soleus muscle necrosis was induced in rats by bupivacaine, and an injectable alginate gel was used to release the PSP in the injured muscle. PSP delivery was found to stimulate satellite cell proliferation in damaged muscle and enhance the growth of regenerating myofibers, with complete regeneration of normal muscle mass by 2 wk. In cell culture, PEDF/PSP stimulated C2C12 myoblast proliferation, together with a rise in cyclin D1 expression. PEDF induced the phosphorylation of ERK1/2, Akt, and STAT3 in C2C12 myoblasts. Blocking the activity of ERK, Akt, or STAT3 with pharmacological inhibitors attenuated the effects of PEDF/PSP on the induction of C2C12 cell proliferation and cyclin D1 expression. Moreover, 5-bromo-2'-deoxyuridine pulse-labeling demonstrated that PEDF/PSP stimulated primary rat satellite cell proliferation in myofibers in vitro. In summary, we report for the first time that PSP is capable of promoting the regeneration of skeletal muscle. The signaling mechanism involves the ERK, AKT, and STAT3 pathways. These results show the potential utility of this PEDF peptide for muscle regeneration.
Assuntos
Proteínas do Olho/farmacologia , Músculo Esquelético/fisiologia , Fatores de Crescimento Neural/farmacologia , Regeneração/fisiologia , Serpinas/farmacologia , Células-Tronco/fisiologia , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Masculino , Camundongos , Músculo Esquelético/efeitos dos fármacos , Mioblastos/efeitos dos fármacos , Mioblastos/fisiologia , Ratos , Ratos Sprague-Dawley , Regeneração/efeitos dos fármacos , Células-Tronco/efeitos dos fármacosRESUMO
Here, we show that dBCAS2 (CG4980, human Breast Carcinoma Amplified Sequence 2 ortholog) is essential for the viability of Drosophila melanogaster. We find that ubiquitous or tissue-specific depletion of dBCAS2 leads to larval lethality, wing deformities, impaired splicing, and apoptosis. More importantly, overexpression of hBCAS2 rescues these defects. Furthermore, the C-terminal coiled-coil domain of hBCAS2 binds directly to CDC5L and recruits hPrp19/PLRG1 to form a core complex for splicing in mammalian cells and can partially restore wing damage induced by knocking down dBCAS2 in flies. In summary, Drosophila and human BCAS2 share a similar function in RNA splicing, which affects cell viability.
Assuntos
Apoptose/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas de Neoplasias/metabolismo , Splicing de RNA/genética , Asas de Animais/anormalidades , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Larva/crescimento & desenvolvimento , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Especificidade de Órgãos , Fenótipo , Regiões Promotoras Genéticas , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão , Asas de Animais/crescimento & desenvolvimentoRESUMO
PURPOSE: Continuous culture of limbal epithelial stem cells (LSCs) slows down proliferation, which inevitably results in differentiation. Transforming growth factor-beta (TGFß)-assisted epithelial-mesenchymal transition (EMT) is often found in the late stage of LSC culture. Thus, EMT is proposed to be part of the mechanism responsible for the loss of LSCs in culture. To explore the regulation mechanism of EMT, we investigated the early stage culture for factor(s) that may potentially prevent EMT. METHODS: LSCs from the corneal limbus region of rabbits were isolated and expanded to confluence in culture (P0), and then serial passage of these LSCs (P1 to P3) was performed. EMT in LSCs was induced with TGFß1, and the corresponding EMT signaling was confirmed with Smad2/3 phosphorylation. The expression of mesenchymal markers, including alpha-smooth muscle actin (α-SMA) and vimentin, was determined with western blot analysis. Proteins extracted from different passaged cells were also subjected to western blot analysis of TGFß signaling components, including TGFß1, TGFß receptor I/II, and Smad2/3 as well as Smad7, the main negative regulator of TGFß signaling. The mitogenic response was measured with the bromodeoxyuridine (BrdU) labeling index and real-time PCR using primers for Ki67. N-(N-[3,5-difluorophenacetyl]-l-alanyl)-S-phenylglycine t-butyl ester (DAPT), a gamma-secretase inhibitor, and Jagged-1 Notch ligand were used to block and activate Notch signaling, respectively, and their efficacy was evaluated by determining the expression of Hes1, a Notch signaling target. RESULTS: Mesenchymal marker induction and growth arrest were found in the TGFß1-treated P1 cells, and the changes were less significant in the TGFß1-treated P0 cells. Western blot analysis confirmed that the expressed levels of TGFß signaling components, including TGFß1, TGFß receptor I/II, and Smad2/3, were relatively stable with passages. In contrast, the expression of Hes1 and Smad7 markedly decreased after the first passage, and with each passage, the levels diminished even further. Hes1 and Smad7 were expressed only in the limbal epithelium and not in the corneal epithelium. DAPT effectively blocked the expression of Hes1. DAPT also dose-dependently suppressed Smad7 expression in P0 cells, which was associated with the susceptibility of P0 cells to TGFß1-induced Smad2/3 phosphorylation, EMT formation, and growth arrest. Reciprocally, Jagged-1 upregulated Smad7 expression in LSCs against TGFß signaling. CONCLUSIONS: These findings indicate that Smad7 plays a crucial role in antagonizing EMT induced by TGFß signaling and support our proposition that Smad7 is a Notch signaling target in LSCs, and may mediate the Notch function in preventing the occurrence of EMT.
Assuntos
Transição Epitelial-Mesenquimal , Limbo da Córnea/citologia , Receptores Notch/metabolismo , Proteína Smad7/metabolismo , Células-Tronco/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Biomarcadores/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteínas de Homeodomínio/metabolismo , Camundongos , Células NIH 3T3 , Coelhos , Ratos , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Limbal epithelial stem cell (LSC) transplantation is a prevalent therapeutic method for patients with LSC deficiency. The maintenance of stem cell characteristics in the process of culture expansion is critical for the success of ocular surface reconstruction. Pigment epithelial-derived factor (PEDF) increased the numbers of holoclone in LSC monolayer culture and preserved the stemness of LSC in suspension culture by evidence of ΔNp63α, Bmi-1, and ABCG2 expression. BrdU pulse-labeling assay also demonstrated that PEDF stimulated LSCs proliferation. In air-lift culture of limbal equivalent, PEDF was capable of increasing the numbers of ΔNp63α-positive cells. The mitogenic effect of PEDF was found to be mediated by the phosphorylations of p38 MAPK and STAT3 in LSCs. Synthetic 44-mer PEDF (residues 78-121) was as effective as the full length PEDF in LSC expansion in suspension culture and limbal equivalent formation, as well as the activation of p38 MAPK and STAT3. In mice subjecting to mechanical removal of cornea epithelium, 44-mer PEDF facilitated corneal wound healing. Microscopically, 44-mer PEDF advanced the early proliferative response in limbus, increased the proliferation of ΔNp63α-positive cells both in limbus and in epithelial healing front, and assisted the repopulation of limbus in the late phase of wound healing. In conclusion, the capability of expanding LSC in cell culture and in animal indicates the potential of PEDF and its fragment (e.g., 44-mer PEDF) in ameliorating limbal stem cell deficiency; and their uses as therapeutics for treating corneal wound.
Assuntos
Epitélio Corneano/patologia , Proteínas do Olho/farmacologia , Limbo da Córnea/citologia , Fatores de Crescimento Neural/farmacologia , Serpinas/farmacologia , Células-Tronco/citologia , Cicatrização/efeitos dos fármacos , Animais , Bromodesoxiuridina/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Epitélio Corneano/efeitos dos fármacos , Imunofluorescência , Humanos , Camundongos , Mitógenos/farmacologia , Células NIH 3T3 , Peptídeos/farmacologia , Coelhos , Fator de Transcrição STAT3/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/enzimologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
PURPOSE: The purpose of this study was to extensively evaluate the efficacy of integrin αvß3 antagonists for the treatment of experimental dry eye (EDE). METHODS: Vitronectin, an αvß3 ligand, was used to induce tumor necrosis factor-α gene expression in human THP-1 macrophages. To induce EDE, C57BL/6 mice were housed in a low-humidity controlled environment chamber and injected subcutaneously with scopolamine for 7 days. Subsequently, αvß3 antagonists, including RGDfD, c(RGDfD), c(RGDiD), c(RGDfK), ATN-161, SB273005, and cilengitide, were administered topically to EDE animals under controlled environment chamber conditions. Corneal epithelial damage in EDE was assessed by fluorescein staining. The density of conjunctival goblet cells and secretion of tears was measured by period acid-Schiff staining and phenol red-impregnated cotton threads, respectively. Inflammation markers, including tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, IL-17A, and metalloproteinase (MMP)-9, in the pooled cornea and conjunctiva tissues were examined by real-time polymerase chain reaction. RESULTS: The inhibitory effects of αvß3 antagonists on the vitronectin-induced tumor necrosis factor-α gene expression and integrin-mediated inflammatory signaling were validated in THP-1 macrophages. αvß3 antagonists ameliorated the impairment of the corneal epithelial barrier with varying therapeutic efficacies, compared with vehicle-treated mice. c(RGDfD) and c(RGDiD) significantly protected against goblet cell loss, tear reduction, and proinflammatory gene expression in EDE. CONCLUSIONS: Topical applications of αvß3 antagonists yield therapeutic benefits in EDE by promoting corneal epithelial defect healing and reducing inflammation. Antagonistic targeting αvß3 may be a novel promising strategy to treat patients with dry eye disease.
Assuntos
Síndromes do Olho Seco , Integrina alfaVbeta3 , Humanos , Animais , Camundongos , Integrina alfaVbeta3/metabolismo , Integrina alfaVbeta3/uso terapêutico , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Vitronectina/metabolismo , Vitronectina/farmacologia , Vitronectina/uso terapêutico , Camundongos Endogâmicos C57BL , Síndromes do Olho Seco/metabolismo , Lágrimas/metabolismo , Túnica Conjuntiva/patologia , Córnea/patologia , Inflamação/metabolismo , Modelos Animais de DoençasRESUMO
Aims: Pigment epithelium-derived factor (PEDF) is known to induce several types of tissue regeneration by activating tissue-specific stem cells. Here, we investigated the therapeutic potential of PEDF 29-mer peptide in the damaged articular cartilage (AC) in rat osteoarthritis (OA). Methods: Mesenchymal stem/stromal cells (MSCs) were isolated from rat bone marrow (BM) and used to evaluate the impact of 29-mer on chondrogenic differentiation of BM-MSCs in culture. Knee OA was induced in rats by a single intra-articular injection of monosodium iodoacetate (MIA) in the right knees (set to day 0). The 29-mer dissolved in 5% hyaluronic acid (HA) was intra-articularly injected into right knees at day 8 and 12 after MIA injection. Subsequently, the therapeutic effect of the 29-mer/HA on OA was evaluated by the Osteoarthritis Research Society International (OARSI) histopathological scoring system and changes in hind paw weight distribution, respectively. The regeneration of chondrocytes in damaged AC was detected by dual-immunostaining of 5-bromo-2'-deoxyuridine (BrdU) and chondrogenic markers. Results: The 29-mer promoted expansion and chondrogenic differentiation of BM-MSCs cultured in different defined media. MIA injection caused chondrocyte death throughout the AC, with cartilage degeneration thereafter. The 29-mer/HA treatment induced extensive chondrocyte regeneration in the damaged AC and suppressed MIA-induced synovitis, accompanied by the recovery of cartilage matrix. Pharmacological inhibitors of PEDF receptor (PEDFR) and signal transducer and activator of transcription 3 (STAT3) signalling substantially blocked the chondrogenic promoting activity of 29-mer on the cultured BM-MSCs and injured AC. Conclusion: The 29-mer/HA formulation effectively induces chondrocyte regeneration and formation of cartilage matrix in the damaged AC.
RESUMO
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron (MN) degeneration, leading to neuromuscular junction (NMJ) dismantling and severe muscle atrophy. The nuclear receptor interaction protein (NRIP) functions as a multifunctional protein. It directly interacts with calmodulin or α-actinin 2, serving as a calcium sensor for muscle contraction and maintaining sarcomere integrity. Additionally, NRIP binds with the acetylcholine receptor (AChR) for NMJ stabilization. Loss of NRIP in muscles results in progressive motor neuron degeneration with abnormal NMJ architecture, resembling ALS phenotypes. Therefore, we hypothesize that NRIP could be a therapeutic factor for ALS. METHODS: We used SOD1 G93A mice, expressing human SOD1 with the ALS-linked G93A mutation, as an ALS model. An adeno-associated virus vector encoding the human NRIP gene (AAV-NRIP) was generated and injected into the muscles of SOD1 G93A mice at 60 days of age, before disease onset. Pathological and behavioral changes were measured to evaluate the therapeutic effects of AAV-NRIP on the disease progression of SOD1 G93A mice. RESULTS: SOD1 G93A mice exhibited lower NRIP expression than wild-type mice in both the spinal cord and skeletal muscle tissues. Forced NRIP expression through AAV-NRIP intramuscular injection was observed in skeletal muscles and retrogradely transduced into the spinal cord. AAV-NRIP gene therapy enhanced movement distance and rearing frequencies in SOD1 G93A mice. Moreover, AAV-NRIP increased myofiber size and slow myosin expression, ameliorated NMJ degeneration and axon terminal denervation at NMJ, and increased the number of α-motor neurons (α-MNs) and compound muscle action potential (CMAP) in SOD1 G93A mice. CONCLUSIONS: AAV-NRIP gene therapy ameliorates muscle atrophy, motor neuron degeneration, and axon terminal denervation at NMJ, leading to increased NMJ transmission and improved motor functions in SOD1 G93A mice. Collectively, AAV-NRIP could be a potential therapeutic drug for ALS.
Assuntos
Esclerose Lateral Amiotrófica , Dependovirus , Modelos Animais de Doenças , Terapia Genética , Camundongos Transgênicos , Neurônios Motores , Atrofia Muscular , Animais , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/terapia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Terapia Genética/métodos , Atrofia Muscular/genética , Atrofia Muscular/terapia , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Dependovirus/genética , Camundongos , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Junção Neuromuscular/metabolismo , Junção Neuromuscular/patologia , Vetores Genéticos/administração & dosagem , Degeneração Neural/genética , Degeneração Neural/terapia , Masculino , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismoRESUMO
Pigment epithelium-derived factor (PEDF) is an intrinsic anti-angiogenic factor and a potential anti-tumor agent. The tumoricidal mechanism of PEDF, however, has not been fully elucidated. Here we report that PEDF induces the apoptosis of TC-1 and SK-Hep-1 tumor cells when they are cocultured with bone marrow-derived macrophages (BMDMs). This macrophage-mediated tumor killing is prevented by blockage of TNF-related apoptosis-inducing ligand (TRAIL) following treatment with the soluble TRAIL receptor. PEDF also increases the amount of membrane-bound TRAIL on cultured mouse BMDMs and on macrophages surrounding subcutaneous tumors. PEDF-induced tumor killing and TRAIL induction are abrogated by peroxisome proliferator-activated receptor γ (PPARγ) antagonists or small interfering RNAs targeting PPARγ. PEDF also induces PPARγ in BMDMs. Furthermore, the activity of the TRAIL promoter in human macrophages is increased by PEDF stimulation. Chromatin immunoprecipitation and DNA pull-down assays confirmed that endogenous PPARγ binds to a functional PPAR-response element (PPRE) in the TRAIL promoter, and mutation of this PPRE abolishes the binding of the PPARγ-RXRα heterodimer. Also, PPARγ-dependent transactivation and PPARγ-RXRα binding to this PPRE are prevented by PPARγ antagonists. Our results provide a novel mechanism for the tumoricidal activity of PEDF, which involves tumor cell killing via PPARγ-mediated TRAIL induction in macrophages.
Assuntos
Apoptose , Proteínas do Olho/metabolismo , Macrófagos/metabolismo , Neoplasias/metabolismo , Fatores de Crescimento Neural/metabolismo , Serpinas/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Linhagem Celular Transformada , Linhagem Celular Tumoral , Técnicas de Cocultura , Proteínas do Olho/imunologia , Humanos , Macrófagos/imunologia , Camundongos , Mutação , Neoplasias/imunologia , Fatores de Crescimento Neural/imunologia , PPAR gama/imunologia , PPAR gama/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Elementos de Resposta/imunologia , Receptor X Retinoide alfa/imunologia , Receptor X Retinoide alfa/metabolismo , Serpinas/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/imunologiaRESUMO
Previously, we found a gene named nuclear receptor interaction protein (NRIP) (or DCAF6 or IQWD1). We demonstrate that NRIP is a novel binding protein for human papillomavirus 16 (HPV-16) E2 protein. HPV-16 E2 and NRIP can directly associate into a complex in vivo and in vitro, and the N-terminal domain of NRIP interacts with the transactivation domain of HPV-16 E2. Only full-length NRIP can stabilize E2 protein and induce HPV gene expression, and NRIP silenced by two designed small interfering RNAs (siRNAs) decreases E2 protein levels and E2-driven gene expression. We found that NRIP can directly bind with calmodulin in the presence of calcium through its IQ domain, resulting in decreased E2 ubiquitination and increased E2 protein stability. Complex formation between NRIP and calcium/calmodulin activates the phosphatase calcineurin to dephosphorylate E2 and increase E2 protein stability. We present evidences for E2 phosphorylation in vivo and show that NRIP acts as a scaffold to recruit E2 and calcium/calmodulin to prevent polyubiquitination and degradation of E2, enhancing E2 stability and E2-driven gene expression.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Calcineurina/metabolismo , Proteínas de Ligação a Calmodulina/genética , Linhagem Celular , Células HEK293 , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Humanos , FosforilaçãoRESUMO
Purpose: To evaluate the efficacy of a pigment epithelium-derived factor (PEDF)-derived short peptide 29-mer, on the treatment and prevention of experimental dry eye (EDE). Methods: C57BL/6 mice were housed in a low humidity controlled environment chamber for 14 days to induce EDE. The 29-mer was administered topically to their eyes, for treatment or dosing, from the point of housing in the controlled environment chamber. The efficacy of the 29-mer on EDE was evaluated in terms of corneal epithelial integrity, tear secretion, and the density of conjunctival goblet cells. PEDF and inflammatory factors, including tumor necrosis factor-α, IL-1ß, IL-6, monocyte chemotactic protein (MCP)-1, matrix metalloproteinase-9, and macrophage infiltration, were examined by real-time polymerase chain reaction, Western blotting, and immunostaining. The involvement of the PEDF receptor/PNPLA2 on the 29-mer effects was evaluated by a specific inhibitor, atglistatin. Rabbit corneal epithelial cells were exposed to hyperosmotic medium to induce inflammatory responses. Results: The levels of PEDF protein increased in the corneal epithelium of EDE, compared with the nonstressed mice. The 29-mer showed a therapeutic effect on EDE and prevented the development of EDE, accompanied by amelioration of the inflammatory factors. The 29-mer effects of inflammatory relief were dramatically reversed by atglistatin. The 29-mer also suppressed the expression of matrix metalloproteinase-9 and proinflammatory cytokines in rabbit corneal epithelial cells induced by hyperosmolarity. Conclusions: Through this animal study, we provide a proof of concept of the anti-inflammatory domain of PEDF having potential to treat dry eye disease. Translational Relevance: This study shows the 29-mer has novel potential as an ophthalmic drop treatment for dry eye disease.
Assuntos
Síndromes do Olho Seco , Metaloproteinase 9 da Matriz , Animais , Anti-Inflamatórios/uso terapêutico , Citocinas/metabolismo , Citocinas/uso terapêutico , Modelos Animais de Doenças , Síndromes do Olho Seco/tratamento farmacológico , Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/patologia , Proteínas do Olho , Inflamação/tratamento farmacológico , Interleucina-6/uso terapêutico , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quimioatraentes de Monócitos/uso terapêutico , Fatores de Crescimento Neural , Compostos de Fenilureia , Coelhos , Serpinas , Fator de Necrose Tumoral alfa/uso terapêuticoRESUMO
BACKGROUND: Breast carcinoma-amplified sequence 2 (BCAS2) regulates ß-catenin gene splicing. The conditional knockout of BCAS2 expression in the forebrain (BCAS2 cKO) of mice confers impaired learning and memory along with decreased ß-catenin expression. Because ß-catenin reportedly regulates adult neurogenesis, we wondered whether BCAS2 could regulate adult neurogenesis via ß-catenin. METHODS: BCAS2-regulating neurogenesis was investigated by characterizing BCAS2 cKO mice. Also, lentivirus-shBCAS2 was intracranially injected into the hippocampus of wild-type mice to knock down BCAS2 expression. We evaluated the rescue effects of BCAS2 cKO by intracranial injection of adeno-associated virus encoding BCAS2 (AAV-DJ8-BCAS2) and AAV-ß-catenin gene therapy. RESULTS: To show that BCAS2-regulating adult neurogenesis via ß-catenin, first, BCAS2 cKO mice showed low SRY-box 2-positive (Sox2+) neural stem cell proliferation and doublecortin-positive (DCX+) immature neurons. Second, stereotaxic intracranial injection of lentivirus-shBCAS2 knocked down BCAS2 in the hippocampus of wild-type mice, and we confirmed the BCAS2 regulation of adult neurogenesis via ß-catenin. Third, AAV-DJ8-BCAS2 gene therapy in BCAS2 cKO mice reversed the low proliferation of Sox2+ neural stem cells and the decreased number of DCX+ immature neurons with increased ß-catenin expression. Moreover, AAV-ß-catenin gene therapy restored neuron stem cell proliferation and immature neuron differentiation, which further supports BCAS2-regulating adult neurogenesis via ß-catenin. In addition, cells targeted by AAV-DJ8 injection into the hippocampus included Sox2 and DCX immature neurons, interneurons, and astrocytes. BCAS2 may regulate adult neurogenesis by targeting Sox2+ and DCX+ immature neurons for autocrine effects and interneurons or astrocytes for paracrine effects. CONCLUSIONS: BCAS2 can regulate adult neurogenesis in mice via ß-catenin.