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BACKGROUND: Currently, no perfect treatment for neovascularization and lymphangiogenesis exist, and each treatment method has its complications and side effects. This study aimed to investigate the anti-angiogenic and anti-inflammatory effects of cannabidiol and its mechanism of action. METHOD: An in vivo corneal neovascularization (CNV) model was established using the suture method to investigate the inhibitory effects of CBD on suture-induced corneal inflammation, pathological blood vessel formation, and lymphangiogenesis. Additionally, the impact of CBD on immune cells was studied. In vitro methodologies, including cell sorting and co-culture, were employed to elucidate its mechanism of action. RESULTS: Compared with the CNV group, CBD can inhibit CNV, lymphangiogenesis, and inflammation induced via the suture method. In addition, CBD specifically induced CD45+CD11b+Gr-1+ cell upregulation, which significantly inhibited the proliferation of CD4+ T lymphocytes in vitro and exhibited a CD31+ phenotype, proving that they were myeloid-derived suppressor cells (MDSCs). We administered anti-Gr-1 to mice to eliminate MDSCs in vivo and found that anti-Gr-1 partially reversed the anti-inflammatory and angiogenic effects of CBD. Furthermore, we found that compared with MDSCs in the normal group, CBD-induced MDSCs overexpress peroxisome proliferator-activated receptor-gamma (PPAR-γ). Administering PPAR-γ inhibitor in mice almost reversed the induction of MDSCs by CBD, demonstrating the role of PPAR-γ in the function of CBD. CONCLUSION: This study indicates that CBD may induce MDSCs upregulation by activating the nuclear receptor PPAR-γ, exerting anti-inflammatory, antiangiogenic, and lymphangiogenic effects, and revealing potential therapeutic targets for corneal neovascularization and lymphangiogenesis.
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Anti-Inflamatórios , Canabidiol , Neovascularização da Córnea , Linfangiogênese , Camundongos Endogâmicos C57BL , Células Supressoras Mieloides , Animais , Canabidiol/farmacologia , Canabidiol/uso terapêutico , Neovascularização da Córnea/tratamento farmacológico , Neovascularização da Córnea/patologia , Células Supressoras Mieloides/efeitos dos fármacos , Células Supressoras Mieloides/imunologia , Camundongos , Linfangiogênese/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Modelos Animais de Doenças , Suturas , PPAR gama/metabolismo , Humanos , Inflamação/tratamento farmacológico , Masculino , Córnea/patologia , Córnea/efeitos dos fármacos , Células CultivadasRESUMO
BACKGROUND: Cataracts are the main cause of reversible blindness worldwide. The ageing of the lens caused by ultraviolet B (UVB) radiation is mostly related to oxidative stress (OS). Little is known about whether OS induced by UVB enhances the sensitivity of lens epithelial cells to ferroptotic stress, which may be a new mechanism leading to age-related cataracts (ARCs). METHODS: Ferroptosis was detected by transmission electron microscopy (TEM), iron assay, lipid peroxidation (MDA) assay, real-time PCR, western blotting, and immunofluorescence. Genetic engineering technology was used to investigate the regulatory relationship among Sirtuin 6 (SIRT6), nuclear factor erythroid 2-related factor 2 (Nrf2), nuclear receptor coactivator 4 (NCOA4), glutathione peroxidase 4 (GPX4) and ferritin heavy chain (FTH1). Knockdown and overexpression of SIRT6 locally in vivo in rats were performed to probe the regulatory mechanism of SIRT6 in ferroptosis in ARCs. FINDINGS: Here, we observed that UVB can drastically induce ferroptosis in lens epithelial cells in vivo and in vitro. Surprisingly, inhibition of ferroptosis was the direct reason that melatonin rescued B-3, SRA01/04 and HEK-293 T cells survival; the pan-caspase inhibitor Z-Vad-FMK did not significantly reverse the death of UVB-irradiated cells compared with that in the UVB+DMSO group. SIRT6 was an upstream regulator of phosphorylated Nrf2 (p-Nrf2) and NCOA4 in B-3, SRA01/04 and HEK-293 T cells. Melatonin inhibited ferroptosis through the SIRT6/p-Nrf2/GPX4 and SIRT6/COA4/FTH1 pathways to neutralize lipid peroxidation toxicity, which protected cells against ferroptotic stress in vitro and delayed cataract formation caused by UVB exposure in rats. INTERPRETATION: Our findings reveal a novel causal role of melatonin in the pathogenesis of ARCs, which raises the possibility of selectively targeting the activation of SIRT6 and ferroptotic resistance as a latent antioxidative therapeutic strategy for ARCs.
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Catarata , Ferroptose , Melatonina , Sirtuínas , Animais , Humanos , Ratos , Catarata/prevenção & controle , Catarata/metabolismo , Ferritinas , Células HEK293 , Melatonina/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Coativadores de Receptor Nuclear/metabolismo , Oxirredutases/metabolismo , Sirtuínas/metabolismoRESUMO
Retinoblastoma (RB) is the most common intraocular tumor in childhood. Long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NTAT1) has been reported to be related to RB progression. This study aims to study the molecular mechanism of NEAT1 in regulating cell cycle, proliferation, apoptosis, migration, and invasion in RB. The expression levels of NEAT1 and miR-3619-5p were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression of LIM and SH3 domain protein 1 (LASP1) was measured by western blot. The proliferation of RB cells was analyzed by cell counting kit-8 (CCK-8) and cell colony formation assays. Cell migration and invasion were evaluated by transwell assay. Cell cycle and apoptosis were assessed by flow cytometry analysis. The association between miR-3619-5p and NEAT1 or LASP1 was predicted by starBase 3.0 database and identified by dual-luciferase reporter assay. The effects of NEAT1 knockdown on the tumor growth in vivo were detected by in vivo tumor formation assay. NEAT1 expression was dramatically up-regulated, and miR-3619-5p expression was obviously downregulated in RB tissues and cells compared with control groups. The protein level of LASP1 was obviously increased in RB tissues or cells relative to paracancerous normal tissues or cells, respectively. Functionally, NEAT1 silencing inhibited RB cell migration, invasion, and proliferation, whereas induced cell apoptosis and cell cycle arrest in RB; this phenomenon was partially abolished by miR-3619-5p inhibitor. Mechanistically, NEAT1 acted as a sponge of miR-3619-5p, and miR-3619-5p was associated with LASP1. In addition, NEAT1 knockdown decreased the volume and weight of RB tumor in vivo. Together, NEAT1 silencing repressed cell migration, invasion, and proliferation, whereas induced cell apoptosis and cycle arrest by sponging miR-3619-5p to inhibit LASP1 expression in RB cells. This study may provide a theoretical basis for RB therapy.
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PURPOSE: The present study investigated a pathogenic mutation and its mechanism on membranous cataract in a congenital membranous cataract family. METHODS: An autosomal dominant four-generation Chinese congenital membranous cataract family was recruited and whole-exome sequencing was performed to screen for sequence variants. Candidate variants were validated using polymerase chain reaction and Sanger sequencing. Wild-type and mutant low-density lipoprotein receptor-related protein 5-like (LRP5L) plasmids were constructed and transfected into human lens epithelial cells (HLE B-3) and human anterior lens capsules. The cell lysates, nuclear and cytoplasmic proteins, and basement membrane components of HLE B-3 cells were harvested. LRP5L and laminin γ1 were knocked down in HLE B-3 cells using specific small-interfering RNA. The protein expression levels of LRP5L, laminin γ1, and c-MAF were detected using immunoblotting and immunofluorescence. RESULTS: We identified a novel suspected pathogenic mutation in LRP5L (c.107C > G, p.P36R) in the congenital membranous cataract family. This mutation was absent in 300 normal controls and 300 age-related cataract patients. Bioinformatics analysis with PolyPhen-2 and SIFT suggested that LRP5L-P36R was pathogenic. LRP5L upregulated laminin γ1 expression in the cytoplasmic proteins of HLE B-3 cells and human anterior lens capsules, and LRP5L-P36R inhibited the effects of LRP5L. LRP5L upregulated c-MAF expression in the nucleus and cytoplasm of HLE B-3 cells, and LRP5L-P36R inhibited c-MAF expression via inhibition of laminin γ1. CONCLUSION: Our study identified a novel gene, LRP5L, associated with congenital membranous cataract, and its mutant LRP5L-P36R contributed to membranous cataract development via inhibition of laminin γ1 and c-MAF.
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Catarata , Laminina , Povo Asiático , Catarata/genética , Humanos , Laminina/genética , Mutação , Mutação de Sentido Incorreto , Linhagem , Proteínas Proto-Oncogênicas c-maf/genéticaRESUMO
RATIONALE: Autologous peripheral nerve injury caused by crush syndrome due to alcohol intoxication is relatively rare, and to our knowledge, the compression of 3 upper limb nerves at the same time has not been reported previously. If a compressive peripheral nerve injury is not treated in a timely manner, it is difficult to recover neurological function, and the prognosis is poor. PATIENT CONCERNS: Here, we present a case of a 50-year-old man with ipsilateral radial nerve, median nerve, and ulnar nerve injuries caused by autogenous compression after drunkenness. DIAGNOSIS: Electromyography and nerve conduction studies suggested peripheral nerve injury in the left upper limb. The diagnosis was injury to the radial nerve, median nerve, and ulnar nerve in the left upper arm. INTERVENTIONS: Exploratory neurolysis surgery of the radial nerve, median nerve, and ulnar nerve was performed in the left upper arm. Postoperative oral neurotrophic drugs were administered, and functional exercise was performed. OUTCOMES: After timely diagnosis and treatment, the strength of the left upper arm muscle recovered, and the prognosis of neurological function was satisfactory during 3 years of follow-up sessions. LESSONS: In the treatment of such patients, a comprehensive understanding of their medical history and a strict physical examination should be performed. Combined with neuroelectrophysiological and imaging examination, the diagnosis can be confirmed. After timely diagnosis and treatment, the prognosis is mostly excellent.
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Intoxicação Alcoólica/complicações , Síndrome de Esmagamento/etiologia , Nervo Mediano/lesões , Nervo Radial/lesões , Nervo Ulnar/lesões , Intoxicação Alcoólica/patologia , Síndrome de Esmagamento/patologia , Síndrome de Esmagamento/terapia , Humanos , Masculino , Nervo Mediano/patologia , Pessoa de Meia-Idade , Nervo Radial/patologia , Nervo Ulnar/patologiaRESUMO
Senescence is a leading cause of age-related cataract (ARC). The current study indicated that the senescence-associated protein, p53, total laminin (LM), LMα4, and transforming growth factor-beta1 (TGF-ß1) in the cataractous anterior lens capsules (ALCs) increase with the grades of ARC. In cataractous ALCs, patient age, total LM, LMα4, TGF-ß1, were all positively correlated with p53. In lens epithelial cell (HLE B-3) senescence models, matrix metalloproteinase-9 (MMP-9) alleviated senescence by decreasing the expression of total LM and LMα4; TGF-ß1 induced senescence by increasing the expression of total LM and LMα4. Furthermore, MMP-9 silencing increased p-p38 and LMα4 expression; anti-LMα4 globular domain antibody alleviated senescence by decreasing the expression of p-p38 and LMα4; pharmacological inhibition of p38 MAPK signaling alleviated senescence by decreasing the expression of LMα4. Finally, in cataractous ALCs, positive correlations were found between LMα4 and total LM, as well as between LMα4 and TGF-ß1. Taken together, our results implied that the elevated LMα4, which was possibly caused by the decreased MMP-9, increased TGF-ß1 and activated p38 MAPK signaling during senescence, leading to the development of ARC. LMα4 and its regulatory factors show potential as targets for drug development for prevention and treatment of ARC.
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Catarata/metabolismo , Senescência Celular/fisiologia , Células Epiteliais/fisiologia , Laminina/metabolismo , Cápsula do Cristalino/metabolismo , Envelhecimento , Anticorpos , Catarata/patologia , Linhagem Celular , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Laminina/genética , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Proteína Supressora de Tumor p53 , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Corneal neovascularization (angiogenesis and lymphangiogenesis) compromises corneal transparency and transplant survival, however, the molecular mechanisms of corneal host epithelial and stromal cells in neovascularization have not yet been fully elucidated. Furthermore, the contribution and mechanism of corneal host endothelial cells involved in neovascularization are largely unexplored. METHODS: Liquid chromatography-mass spectrometry, immunoblotting, and ELISA were used to screen and identify potential neovascularization-related factors in human full-thickness vascularized corneal tissues. Lipopolysaccharide was used to induce inflammation in three kinds of corneal host cells in vitro, including corneal epithelial, stromal, and endothelial cells. Fungus was used to establish an animal model of corneal neovascularization in vivo. Tube formation and spheroid sprouting assays were used to evaluate the contribution of three kinds of corneal host cells to the degree of neovascularization under various stimuli. Matrix metalloproteinase (MMP)-2, alpha-crystallin A chain (CRYAA), galectin-8, Bcl-2, neuropilin-2, MMP-9 plasmids, and recombinant human fibronectin were used to identify the key proteins of corneal host cells involved in corneal inflammatory neovascularization. FINDINGS: All three kinds of corneal host cells influenced corneal neovascularization to varying degrees. MMP-9 in human corneal epithelial cells, MMP-2, and CRYAA in human corneal stromal cells, and MMP-2 and galectin-8 in human corneal endothelial cells are potential key proteins that participate in corneal inflammatory neovascularization. INTERPRETATION: Our data indicated that both the effects of key proteins and corneal host cells involved should be considered for the treatment of corneal inflammatory neovascularization.
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Córnea/citologia , Neovascularização da Córnea/etiologia , Neovascularização da Córnea/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores , Linhagem Celular , Cromatografia Líquida , Córnea/metabolismo , Neovascularização da Córnea/metabolismo , Meios de Cultivo Condicionados , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/metabolismo , Feminino , Expressão Gênica , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Proteoma , Proteômica , Ratos , Células Estromais/metabolismo , Adulto JovemRESUMO
BACKGROUND: Cervical tuberculosis accounts for only 4.2%-12% of the total incidence of spinal tuberculosis cases. Although antituberculosis drugs have been the mainstay treatment of cervical tuberculosis, they have been ineffective against the symptoms of existing spinal deformities and spinal cord compression, which often require surgical intervention. The conventional surgical methods have been anterior debridement and titanium mesh, cage bone graft fusion and internal fixation. However, all have certain deficiencies regarding the stability of fixation. CASE DESCRIPTION: We have presented the case of a 41-year-old Chinese man who had been experiencing neck pain and stiffness for 1 month. The symptoms had been accompanied by low-grade fever and repeated night sweats. The purified protein derivative test result was positive and the antituberculosis test result was negative. Imaging examination showed destruction of the C5 and C6 vertebral bodies and C5 andC6 intervertebral discs, with an intensive abscess at the C5-C6 vertebral level. After 3-dimensional printing-assisted anterior debridement and artificial vertebral body replacement, his preoperative symptoms of neck pain and stiffness had been alleviated. Also, his symptoms of numbness in both upper limbs had disappeared completely. At the last follow-up examination, he had recovered well and the tuberculosis focus had been completely cured. CONCLUSION: To the best of our knowledge, we have reported the first clinical application of 3-dimensional printing-assisted cervical anterior bilateral pedicle screw fixation of an artificial vertebral body. We accomplished ultrashort segment fixation, with excellent clinical outcomes obtained, which were maintained at the recent 2-year follow-up examination.
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Parafusos Pediculares , Procedimentos de Cirurgia Plástica/instrumentação , Procedimentos de Cirurgia Plástica/métodos , Impressão Tridimensional , Tuberculose da Coluna Vertebral/cirurgia , Adulto , Vértebras Cervicais/cirurgia , Humanos , Masculino , Fusão Vertebral/métodos , Vértebras Torácicas/cirurgia , Resultado do TratamentoRESUMO
Fuchs endothelial dystrophy (FED) and late cornea allograft failure of cornea transplantation are associated with human corneal endothelial cells (HCEC) senescence. Kojic acid has various functions, however, its anti-senescence effect has never been identified. In this study, we investigated the anti-senescence effect of kojic acid on HCEC. Cell viability, migration ability and senescence were evaluated by MTT assay, migration assay, and senescence-associated beta-galactosidase (SA-ß-Gal) staining, respectively. Senescence-related protein expression was analyzed by western blotting and immunofluorescence assay. Angiogenesis of human umbilical vein endothelial cells (HUVEC) was examined by tube formation assay and spheroid sprouting assay. The results showed that kojic acid could inhibit HCEC senescence, characterized by enhancing migration, decreasing the levels of SA-ß-Gal staining, galectin 8, laminin α1, laminin α2, laminin γ1 and p21, and increasing that of p-NF-κB of senescent HCEC. The p-NF-κB inhibitor could reverse the anti-senescent effect of kojic acid, and p21 siRNA showed similar anti-senescence effect with kojic acid. In addition, kojic acid could alleviate HUVEC tube formation induced by senescent HCEC, which could be reversed by p-NF-κB inhibitor. The p21 siRNA could alleviate HUVEC spheroid sprouting induced by senescent HCEC. These results indicated that kojic acid might inhibit HCEC senescence and following resulted angiogenesis via NF-κB and p21 signaling pathways, possibly through downregulation of galectin 8 and laminins. Therefore, kojic acid is a promising drug for HCEC senescence-related diseases such as FED and late cornea allograft failure.
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Antioxidantes/farmacologia , Senescência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Endotélio Corneano/patologia , NF-kappa B/metabolismo , Pironas/farmacologia , Transdução de Sinais , Western Blotting , Linhagem Celular , Movimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Endotélio Corneano/metabolismo , Imunofluorescência , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , TransfecçãoRESUMO
INTRODUCTION: Here we intended to investigate the changes in corneal endothelial cells and foveal thickness after phacoemulsification surgery on the eyes of diabetic and non-diabetic cataract patients. MATERIAL AND METHODS: A total of 120 cataract patients who were scheduled for phacoemulsification surgery and intraocular lens implantation were recruited and divided into 2 categories according to the diagnosis of diabetes mellitus. Changes in integrity, endothelial cell density (ECD), coefficient of variation (CV), percentage of hexagonal cells (PHC), central corneal thickness (CCT), and central foveal thickness (CFT) were all recorded at preoperative day 1 and postoperative day 3, 1 week, 1 month, 3 months and 6 months. RESULTS: None of the recorded variables showed any difference between the nondiabetic and diabetic groups before surgery (p > 0.05). During the postoperative 6 months, ECD and PHC decreased and CV increased in both groups (all ptime < 0.05), whereas CCT and CFT fluctuated in both groups significantly (both ptime < 0.05), with their individual peaks at postoperative 1 week in the diabetic group. The groups differed significantly in ECD, PHC, and CV at each time point postoperatively (all pgroup < 0.05). Furthermore, the diabetic group had improved CFT during the postoperative 1 month and higher CCT during the 6 months postoperatively than the nondiabetic group (all pgroup < 0.05). The time and group interactions were significant for ECD, CV, PHC, CCT and CFT (all pgroup × time < 0.05). CONCLUSIONS: The diabetic group had more changes in corneal endothelial cells and foveal thickness than the nondiabetic group postoperatively.
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Cataracts are the most common eye disease to cause blindness in patients. The abnormal deposition of laminins (LMs) in the lens capsule and the disruption of capsular epithelium contribute to cataract development, although the mechanism by which this occurs is currently unclear. The present study aimed to reproduce HLE B3 basement membranes (BMs) using HLE B3 cells and to analyze the similarities of LM expression between HLE B3 BMs and human anterior lens capsule (ALC). Immunohistochemistry (IHC), ELISA, western blot analysis and immunoprecipitation (IP)western blot analysis were used to detect total LMs, LM trimers and 11 LM subunits in HLE B3 cells, HLE B3 BMs and human ALCs. In IHC staining, HLE B3 cells and human ALCs were positive for LMs. In LM ELISA, all samples analyzed were positive for LMs. Western blot analysis detected all LM subunits except for LMγ3 in HLE B3 cell lysate, 4 subunits (LMα4, LMα2, LMα1 and LMγ1) in HLE B3 cell culture supernatant, 5 subunits (LMα4, LMα2, LMα1, LMß3 and LMγ1) in HLE B3 BMs, and 3 subunits (LMα4, LMγ2 and LMγ1) in human ALCs. The results of IPwestern blot analysis revealed that the LM411 trimer was detected in HLE B3 cell culture supernatant. These results indicated that HLE B3 BMs were similar to human ALCs in terms of LM expression. Therefore, HLE B3 BMs could be used as an in vitro ALC model to determine the role of LMs in ALC in the pathogenesis of cataracts and to select potential anticataract drugs.
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Células Epiteliais/metabolismo , Laminina/metabolismo , Cristalino/metabolismo , Adulto , Idoso , Membrana Basal/metabolismo , Biomarcadores , Linhagem Celular , Feminino , Humanos , Imuno-Histoquímica , Laminina/química , Masculino , Pessoa de Meia-Idade , Multimerização ProteicaRESUMO
Aberrantly expressed microRNAs (miRNAs/miRs) and their role in cancer development have recently gained more attention. However, the potential role of miRNAs in hepatocellular carcinoma (HCC) remains largely unknown. In this study, we demonstrated that miR-377 was markedly downregulated in HCC cell lines and primary human HCC tissues. The decreased expression of miR-377 contributes to the upregulation of Bcl-xL expression by targeting its 3'-untranslated region (3'-UTR). Functionally, knockdown of miR-377 noticeably increased HCC cell growth and colony formation and inhibited apoptosis. In contrast, overexpression of miR-377 suppressed cell proliferation and increased apoptosis. This study provides new insights for the use of miR-377 as a potential molecular target in HCC therapy.
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Apoptose/genética , Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , MicroRNAs/genética , Interferência de RNA , Proteína bcl-X/genética , Regiões 3' não Traduzidas , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , HumanosRESUMO
Arsenic trioxide (ATO) has been used to treat patients with acute promyelocytic leukemia. Recently, studies have shown that ATO can induce apoptosis in leukemic cells and blood vessel endothelial cells in a time- and dose-dependent manner through the inhibition of vascular endothelial growth factor A (VEGFA) production. VEGFA is a key factor in angiogenesis initiation. Targeted inhibition of VEGF or VEGFA expression can suppress angiogenesis; however, little is known about the mechanism by which ATO inhibits VEGFA expression. In this study, we investigated the role of miRNA-126 in the mechanism of action of ATO in human umbilical vein endothelial cells (HUVECs). ATO significantly decreased the viability and proliferation of HUVECs and decreased their migration at 48 h. Cell proliferation was inhibited by 50% (IC50) when 5.0 µmol/L ATO was used. ATO treatment induced miR-126 upregulation and HUVEC apoptosis. Transfection with a miR-126 mimic significantly downregulated VEGFA mRNA levels, and transfection with a miR-126 inhibitor significantly upregulated VEGFA mRNA levels. Finally, we showed that ATO treatment upregulated Ets-2 and miR-126 expression in HUVECs. These results demonstrate that ATO inhibits the growth of HUVECs and induces apoptosis by downregulating VEGFA. One mechanism by which this occurs is Ets-2 upregulation, which results in an increase in miR-126 levels and downregulation of VEGFA expression.
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Arsenicais/farmacologia , Regulação para Baixo/efeitos dos fármacos , MicroRNAs/biossíntese , Óxidos/farmacologia , Proteína Proto-Oncogênica c-ets-2/biossíntese , Regulação para Cima/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/biossíntese , Apoptose/efeitos dos fármacos , Trióxido de Arsênio , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , HumanosRESUMO
PURPOSE: To construct a scaffold using silk fibroin (SF) and chitosan (CS) that could replace the corneal stroma with the biological characteristics of the scaffold materials still intact. METHODS: To develop an organotypic corneal stroma, SF and CS were chosen to synthesise the tissue-engineered bioscaffold. We cultured primary rabbit corneal epithelial cells and corneal stromal cells in vitro. Keratocytes were used to assess cytotoxicity on SF-CS (SFCS) blends, which was determined by a Cell Counting Kit-8 assay. The corneal lamellar scaffolds were developed with sequential culture techniques to form cell-scaffold constructs. Implantation was tested in 15 New Zealand White rabbits. The corneal substitutes were analysed by light and electron microscopy. RESULTS: The reconstructed lamellar cornea was comparable to native tissue, with high levels of K3/12 expression in the corneal epithelial cells and vimentin in the stromal cells; moreover, the morphology and the position of the cells could be distinguished by histological methods. There was no sign of any immune reaction in or around the transplanted discs 12 weeks after implantation. CONCLUSION: A SFCS scaffold might be a suitable blend for corneal tissue engineering.
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Quitosana/química , Córnea/citologia , Substância Própria/citologia , Células Epiteliais/citologia , Fibroínas/química , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , CoelhosRESUMO
An Arg-Gly-Asp (RGD) motif in the fourth FAS1 domain of the human BIGH3 (transforming growth factor-ß1-inducible gene-h3) protein has been reported to play an important role in mediating tumor angiogenesis. The aim of this study was to investigate the inhibitory effect of a modified C-terminal fragment BIGH3 protein with an RGDRGD motif on corneal neovascularization in vitro and in vivo. Recombinant C-terminal fragment BIGH3 protein with wild-type sequence and modified C-terminal fragment BIGH3 protein containing an RGDRGD motif were successfully expressed and purified. We demonstrated that both proteins significantly inhibited vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cell (HUVEC) adhesion, migration, and tube formation and induced cell apoptosis but failed to inhibit HUVEC proliferation. We determined that the mechanism underlying this activity was an interaction between BIGH3 and αvß3 integrin, which blocked the phosphorylation of PI3K/Akt and ERK signaling pathways. The inhibitory effects of wild-type and modified C-terminal fragment BIGH3 proteins on angiogenesis were confirmed by a rabbit corneal neovascularization assay. More importantly, we provided evidence that the modified C-terminal fragment BIGH3 protein with an RGDRGD motif inhibited angiogenic activity far more effectively than did wild-type C-terminal fragment BIGH3. Collectively, our data show that a C-terminal fragment BIGH3 protein containing an RGDRGD motif might be promising as an effective drug in treating corneal neovascularization.
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Inibidores da Angiogênese/farmacologia , Neovascularização da Córnea/prevenção & controle , Proteínas da Matriz Extracelular/farmacologia , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Motivos de Aminoácidos , Inibidores da Angiogênese/química , Inibidores da Angiogênese/genética , Animais , Apoptose/efeitos dos fármacos , Vasos Sanguíneos/efeitos dos fármacos , Western Blotting , Caspases/metabolismo , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neovascularização da Córnea/metabolismo , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Citometria de Fluxo , Células Endoteliais da Veia Umbilical Humana , Integrina alfaVbeta3/metabolismo , Mutagênese Sítio-Dirigida , Oligopeptídeos/química , Oligopeptídeos/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Plasmídeos , Dobramento de Proteína , Proteínas Proto-Oncogênicas c-akt/metabolismo , Coelhos , Fator de Crescimento Transformador beta/química , Fator de Crescimento Transformador beta/genética , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
PURPOSE: Endostatin plays an important role in inhibiting corneal neovascularization (CNV). The aim of this study was to evaluate the antiangiogenic activities of lipid-mediated subconjunctival injection of the modified RGDRGD (arginine- glycin- aspartic- arginine- glycin- aspartic- endostatin gene in a rabbit model of neovascularization in vivo. METHODS: A modified human endostatin gene containing an RGDRGD motif was obtained by rapid site-directed mutagenesis. Forty New Zealand white rabbits underwent alkaline burn and developed CNV, which were randomly divided into four groups: an experimental control group, a PCI empty vector group, a PCI-endostatin group, and a PCI-RGDRGD-endostatin group. The vector, endostatin, and RGDRGD-endostatin groups received injections into the superior bulbar conjunctiva after the burn. An injection of 5 µg was given twice at 1-week intervals. Four eyes of two rabbits received neither treatment nor alkaline burn and served as absolute normal controls. The areas of CNV were monitored after 7 and 14 days. Corneas were examined by histology, and VEGF (vascular endothelial growth factor) and CD31 (platelet endothelial cell adhesion molecule-1) expression was detected by immunohistochemistry after 7 and 14 days. Retina, liver, and kidney were examined by histology, and CD38 expression in the inflammatory cells was detected by immunohistochemistry at 90 days. RESULTS: Subconjunctival injection of both native endostatin and modified RGDRGD-endostatin genes resulted in a significant suppression of CNV in vivo, with modified RGDRGD-endostatin being more effective than native endostatin. The mean concentration of VEGF in the PCI-RGDRGD-endostatin group significantly decreased compared to the means in the other groups. Upon histological examination, the endostatin-treated and RGDRGD-endostatin-treated eyes showed significantly less neovascular area and fewer vessels than the control and vector-injected groups. Retinal, hepatic, and renal tissue sections were normal, and there was no inflammatory cell infiltration observed. CONCLUSIONS: Native and modified endostatin can significantly inhibit CNV by suppressing the expression of VEGF. However, modified endostatin with the RGDRGD motif is far more effective than the endostatin gene in antiangiogenic activity.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Córnea/efeitos dos fármacos , Neovascularização da Córnea/tratamento farmacológico , Endostatinas/administração & dosagem , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Inibidores da Angiogênese/química , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/uso terapêutico , Animais , Sequência de Bases , Córnea/metabolismo , Córnea/patologia , Neovascularização da Córnea/genética , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Neovascularização da Córnea/prevenção & controle , Modelos Animais de Doenças , Endostatinas/química , Endostatinas/genética , Endostatinas/uso terapêutico , Feminino , Vetores Genéticos/uso terapêutico , Humanos , Imuno-Histoquímica , Injeções Intraoculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
PURPOSE: To investigate the inhibitory effects of anti-mouse interleukin-17 (IL-17) monoclonal antibody (mAb) in high-responder corneal allograft rejection. METHODS: C57BL/6 or BALB/c mice corneal grafts were grafted onto BALB/c hosts. The neutralizing mouse IL-17 antibody and isotype control were injected intraperitoneally immediately after transplantation for experimental treatment. At appropriate times after treatment, recipient grafts were assessed clinically and histologically, and recipient corneal graft- infiltrating cells were detected by immunohistochemistry and quantified by real-time PCR. The cytokine spleen levels of T helper type 1 (Th1), Th2, and Th17 were analyzed by enzyme-linked immunosorbent assay. Flow cytometric analysis was used to evaluate the frequencies of IL-17-producing Th17 cells. RESULTS: Neutralization of IL-17 with anti-IL-17 mAb obviously prolonged allograft survival compared to the group that received isotype control. Neovascularizations and inflammatory immune cells in corneal stroma decreased in the allogeneic recipients treated with anti-IL-17 mAb. The mRNA (mRNA) level of graft-infiltrating cells, including neutrophiles, cluster of differentiation 4 (CD4) T cells, and CD8 T cells, decreased dramatically in the IL-17 neutralization group. At days 14 and 42, splenocytes from recipients treated with anti-IL-17 mAb produced significantly less of the pro-inflammatory cytokines interferon-gamma (IFN-γ), IL-12p40, and IL-17 compared to those from control Ig-treated recipients at day 14. However, Th2 cytokine IL-4 and IL-5 production increased, and IL-13 levels were not significantly different among the three groups. IL-6 production was elevated in recipients treated with anti-IL-17 mAb. Anti-IL-17 mAb reduced the percentage of Th17 in CD4+ T cells, but there was no statistical significance between anti-IL-17 mAb and the control group. CONCLUSIONS: Neutralization of mouse IL-17 bioactivity with anti-IL-17 mAb improves allogeneic corneal graft survival and inhibits corneal allograft rejection to a certain extent by inhibiting production of graft-infiltrating inflammatory cells and decreasing the secretion of pro-inflammatory cytokines.
Assuntos
Anticorpos Monoclonais/farmacologia , Transplante de Córnea , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/efeitos dos fármacos , Interleucina-17/antagonistas & inibidores , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Sobrevivência de Enxerto/imunologia , Interferon gama/biossíntese , Interferon gama/imunologia , Subunidade p40 da Interleucina-12/biossíntese , Subunidade p40 da Interleucina-12/imunologia , Interleucina-13/biossíntese , Interleucina-13/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-4/biossíntese , Interleucina-4/imunologia , Interleucina-5/biossíntese , Interleucina-5/imunologia , Interleucina-6/biossíntese , Interleucina-6/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Baço/citologia , Baço/imunologia , Linfócitos T/citologia , Transplante HomólogoRESUMO
OBJECTIVE: To construct the eukaryotic express vector of wild type and R555W mutated transforming growth factor beta induced (BIGH3) gene, and to determine the effects of overexpression of R555W mutated BIGH3 in human corneal epithelial (HCE) cells. METHODS: The full coding domain sequence of BIGH3 gene was cloned from human corneal tissue from operative spackman with RT-PCR. Mutagenesis of R555W-BIGH3 was performed in rapid site-directed mutagenesis technique in the expression vector pIRES2-EGFP. The two types of BIGH3 gene were transient transfected to HCE cells. Immunoblotting was used to detect the expression of BIGH3. The cell apoptosis rates were observed by flow cytometry. The morphological changes of the cells were examined by electron microscopy. The viability of caspase-3 was examined. RESULTS: Wild type and mutated BIGH3 gene were successfully amplified by PCR. After HCE cells transfected with two types eukaryotic expression plasmid, the BIGH3 were detected in the HCE cells. The cell apoptosis were observed by flow cytometry and electron microscopy. The viability of caspase-3 was increased in the R555W mutated type. CONCLUSIONS: The R555W mutated BIGH3 is successfully obtained by rapid site-directed mutagenesis technique. Overexpression of R555W mutated BIGH3 induces apoptosis in HCE cells through activation of caspase-3.