RESUMO
BACKGROUND: Corneal neovascularization (CoNV) threatens vision by disrupting corneal avascularity, however, current treatments, including pharmacotherapy and surgery, are hindered by limitations in efficacy and adverse effects. Minocycline, known for its anti-inflammatory properties, could suppress CoNV but faces challenges in effective delivery due to the cornea's unique structure. Therefore, in this study a novel drug delivery system using minocycline-loaded nano-hydroxyapatite/poly (lactic-co-glycolic acid) (nHAP/PLGA) nanoparticles was developed to improve treatment outcomes for CoNV. RESULTS: Ultra-small nHAP was synthesized using high gravity technology, then encapsulated in PLGA by a double emulsion method to form nHAP/PLGA microspheres, attenuating the acidic by-products of PLGA degradation. The MINO@PLGA nanocomplex, featuring sustained release and permeation properties, demonstrated an efficient delivery system for minocycline that significantly inhibited the CoNV area in an alkali-burn model without exhibiting apparent cytotoxicity. On day 14, the in vivo microscope examination and ex vivo CD31 staining corroborated the inhibition of neovascularization, with the significantly smaller CoNV area (29.40% ± 6.55%) in the MINO@PLGA Tid group (three times daily) than that of the control group (86.81% ± 15.71%), the MINO group (72.42% ± 30.15%), and the PLGA group (86.87% ± 14.94%) (p < 0.05). Fluorescein sodium staining show MINO@PLGA treatments, administered once daily (Qd) and three times daily (Tid) demonstrated rapid corneal epithelial healing while the Alkali injury group and the DEX group showed longer healing times (p < 0.05). Additionally, compared to the control group, treatments with dexamethasone, MINO, and MINO@PLGA were associated with an increased expression of TGF-ß as evidenced by immunofluorescence, while the levels of pro-inflammatory cytokines IL-1ß and TNF-α demonstrated a significant decrease following alkali burn. Safety evaluations, including assessments of renal and hepatic biomarkers, along with H&E staining of major organs, revealed no significant cytotoxicity of the MINO@PLGA nanocomplex in vivo. CONCLUSIONS: The novel MINO@PLGA nanocomplex, comprising minocycline-loaded nHAP/PLGA microspheres, has shown a substantial capacity for preventing CoNV. This study confirms the complex's ability to downregulate inflammatory pathways, significantly reducing CoNV with minimal cytotoxicity and high biosafety in vivo. Given these findings, MINO@PLGA stands as a highly promising candidate for ocular conditions characterized by CoNV.
Assuntos
Neovascularização da Córnea , Minociclina , Humanos , Minociclina/farmacologia , Neovascularização da Córnea/tratamento farmacológico , Neovascularização da Córnea/prevenção & controle , Microesferas , Angiogênese , ÁlcalisRESUMO
Inflammation plays an important role in pathological angiogenesis. Receptor-interacting protein 1 (RIP1) is highly expressed in inflammatory cells and is known to play an important role in the regulation of apoptosis, necroptosis, and inflammation; however, a comprehensive description of its role in angiogenesis remains elusive. Here, we show that RIP1 is abundantly expressed in infiltrating macrophages during angiogenesis, and genetic or pharmacological inhibition of RIP1 kinase activity using kinase-inactive RIP1K45A/K45A mice or necrostatin-1 attenuates angiogenesis in laser-induced choroidal neovascularization, Matrigel plug angiogenesis, and alkali injury-induced corneal neovascularization in mice. The inhibitory effect on angiogenesis is mediated by caspase activation through a kinase-independent function of RIP1 and RIP3. Mechanistically, infiltrating macrophages are the key target of RIP1 kinase inhibition to attenuate pathological angiogenesis. Inhibition of RIP1 kinase activity is associated with caspase activation in infiltrating macrophages and decreased expression of proangiogenic M2-like markers but not M1-like markers. Similarly, in vitro, catalytic inhibition of RIP1 down-regulates the expression of M2-like markers in interleukin-4-activated bone marrow-derived macrophages, and this effect is blocked by simultaneous caspase inhibition. Collectively, these results demonstrate a nonnecrotic function of RIP1 kinase activity and suggest that RIP1-mediated modulation of macrophage activation may be a therapeutic target of pathological angiogenesis.
Assuntos
Proteínas Ativadoras de GTPase/fisiologia , Macrófagos/fisiologia , Neovascularização Patológica/enzimologia , Animais , Biomarcadores , Caspases/metabolismo , Células Cultivadas , Colágeno , Lesões da Córnea/induzido quimicamente , Lesões da Córnea/etiologia , Neovascularização da Córnea/enzimologia , Neovascularização da Córnea/etiologia , Neovascularização da Córnea/patologia , Neovascularização da Córnea/prevenção & controle , Combinação de Medicamentos , Ativação Enzimática , Fator 2 de Crescimento de Fibroblastos/farmacologia , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Células Endoteliais da Veia Umbilical Humana , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Marcação In Situ das Extremidades Cortadas , Indóis/farmacologia , Indóis/uso terapêutico , Laminina , Lasers/efeitos adversos , Macrófagos/classificação , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Neovascularização Patológica/patologia , Oligopeptídeos/farmacologia , Proteoglicanas , RNA Mensageiro/biossíntese , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Receptores de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/uso terapêuticoRESUMO
PURPOSE: The inhibitory effect of conbercept on corneal neovascularization (CNV) after penetrating keratoplasty (PKP) and its effect on postoperative wound healing and corneal strength recovery was investigated. METHODS: New Zealand white rabbits were randomly divided into 3 groups, two experimental arms A and B and one control arm C. Topical conbercept and subconjunctival injection were carried out respectively after PKP. Slit lamp microscope was used to observe the growth of CNV. The expression of vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and vimentin (Vim) were determined via real-time quantitative polymerase chain reaction (RT-qPCR). The placenta growth factor and vimentin, determination of corneal biomechanical machine strength changed. To measure the maximal strength of the corneal, uniaxial tensile test was carried out on the electroforce 3220-AT biomechanics machine. RESULTS: Two weeks after PKP, CNV appeared, inflammatory cell infiltration and new blood vessel formation were observed in the corneal stroma and superficial stroma layer. Compared with the control arm, the expression levels of VEGF and PlGF in the experimental arms were significantly decreased after using conbercept (P < 0.05), and the expression levels reached the maximum at the 4th week and then decreased gradually. The expression level of Vimentin and corneal intensity increased gradually over time. CONCLUSION: Conbercept effectively inhibited the formation of CNV after PKP in rabbits, and did not affect postoperative wound healing, nor did it affect postoperative corneal strength recovery.
Assuntos
Neovascularização da Córnea , Ceratoplastia Penetrante , Animais , Córnea/metabolismo , Neovascularização da Córnea/tratamento farmacológico , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/prevenção & controle , Feminino , Fator de Crescimento Placentário/metabolismo , Coelhos , Proteínas Recombinantes de Fusão , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Vimentina/metabolismo , CicatrizaçãoRESUMO
Limbal stem cells (LSC) maintain the transparency of the corneal epithelium. Chemical burns lead the loss of LSC inducing an up-regulation of pro-inflammatory and pro-angiogenic factors, triggering corneal neovascularization and blindness. Adipose tissue-derived mesenchymal stem cells (AT-MSC) have shown promise in animal models to treat LSC deficiency (LSCD), but there are not studies showing their efficacy when primed with different media before transplantation. We cultured AT-MSC with standard medium and media used to culture LSC for clinical application. We demonstrated that different media changed the AT-MSC paracrine secretion showing different paracrine effector functions in an in vivo model of chemical burn and in response to a novel in vitro model of corneal inflammation by alkali induction. Treatment of LSCD with AT-MSC changed the angiogenic and inflammatory cytokine profile of mice corneas. AT-MSC cultured with the medium that improved their cytokine secretion, enhanced the anti-angiogenic and anti-inflammatory profile of the treated corneas. Those corneas also presented better outcome in terms of corneal transparency, neovascularization and histologic reconstruction. Priming human AT-MSC with LSC specific medium can potentiate their ability to improve corneal wound healing, decrease neovascularization and inflammation modulating paracrine effector functions in an in vivo optimized rat model of LSCD.
Assuntos
Córnea/citologia , Doenças da Córnea/prevenção & controle , Neovascularização da Córnea/prevenção & controle , Inflamação/prevenção & controle , Células-Tronco Mesenquimais/citologia , Regeneração , Cicatrização , Animais , Diferenciação Celular , Células Cultivadas , Córnea/metabolismo , Doenças da Córnea/patologia , Neovascularização da Córnea/patologia , Humanos , Inflamação/patologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , RatosRESUMO
The cornea actively maintains its own avascular status to preserve its ultimate optical function. This corneal avascular state is also defined as "corneal angiogenic privilege", which results from a critical and sensitive balance between anti-angiogenic and pro-angiogenic mechanisms. In our review, we aim to explore the complex equilibrium among multiple mediators which prevents neovascularization in the resting cornea, as well as to unveil the evolutive process which leads to corneal angiogenesis in response to different injuries.
Assuntos
Córnea/fisiopatologia , Neovascularização da Córnea/prevenção & controle , Inibidores da Angiogênese/uso terapêutico , Animais , Neovascularização da Córnea/fisiopatologia , Humanos , Neovascularização Patológica/fisiopatologiaRESUMO
Hydrocinnamoyl-L-valylpyrrolidine (AS-1), a synthetic low-molecule mimetic of myeloid differentiation primary response gene 88 (MyD88), inhibits inflammation by disrupting the interaction between the interleukin-1 receptor (IL-1R) and MyD88. Here, we describe the effects of AS-1 on injury-induced increases in inflammation and neovascularization in mouse corneas. Mice were administered a subconjunctival injection of 8 µL AS-1 diluent before or after corneal alkali burn, followed by evaluation of corneal resurfacing and corneal neovascularization (CNV) by slit-lamp biomicroscopy and clinical assessment. Corneal inflammation was assessed by whole-mount CD45+ immunofluorescence staining, and corneal hemangiogenesis and lymphangiogenesis following injury were evaluated by immunostaining for the vascular markers isolectin B4 (IB4) and the lymphatic vascularized marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), respectively. Additionally, corneal tissues were collected to determine the expression of 35 cytokines, and we detected activation of IL-1RI, MyD88, and mitogen-activated protein kinase (MAPK). The results showed that alkali conditions increased the number of CD45+ cells and expression of vascular endothelial growth factor (VEGF)-A, VEGF-C, and LYVE1 in corneas, with these levels decreased in the AS-1-treated group. Moreover, AS-1 effectively prevented alkali-induced cytokine production, blocked interactions between IL-1RI and MyD88, and inhibited MAPK activation post-alkali burn. These results indicated that AS-1 prevented alkali-induced corneal hemangiogenesis and lymphangiogenesis by blocking IL-1RI-MyD88 interaction, as well as extracellular signal-regulated kinase phosphorylation, and could be efficacious for the prevention and treatment of corneal alkali burn.
Assuntos
Queimaduras Químicas/prevenção & controle , Neovascularização da Córnea/prevenção & controle , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Queimaduras Oculares/induzido quimicamente , Pirrolidinas/uso terapêutico , Valina/análogos & derivados , Inibidores da Angiogênese , Animais , Biomarcadores/metabolismo , Western Blotting , Queimaduras Químicas/enzimologia , Queimaduras Químicas/patologia , Neovascularização da Córnea/enzimologia , Neovascularização da Córnea/patologia , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Queimaduras Oculares/enzimologia , Queimaduras Oculares/patologia , Proteínas do Olho/metabolismo , Humanos , Imunoprecipitação , Linfangiogênese/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Reação em Cadeia da Polimerase em Tempo Real , Hidróxido de Sódio , Valina/uso terapêuticoRESUMO
PURPOSE: Corneal avascularity is critical for corneal transparency; therefore, a tailored process has been presumed to minimize corneal neovascularization (NV). In most cell types, the transcription of vascular endothelial growth factor (VEGF) is up-regulated, and the stability of VEGF mRNA is sustained by human antigen R (HuR) during hypoxia; however, whether such response applies to corneal epithelial cells is unclear. METHODS: Human corneal epithelial cells (HCECs) and MCF-7 cells that serves as the control were incubated under 0.5% oxygen, and the levels of VEGF and HuR were examined time-dependently. The alteration of HuR was also examined in vivo using the closed-eye contact lens-induced corneal neovascularization rabbit model and immunohistochemistry. Additionally, the expression of HuR was modulated by transfection of plasmids encoding HuR or siRNA targeting HuR to validate the role of HuR in VEGF expression. RESULTS: We found that, unlike in control cells, the level of VEGF was not up-regulated, and the HuR expression was declined in HCECs following hypoxia. The HuR immunostaining intensities were decreased in corneal epithelial cells of rabbits wearing contact lenses. In addition, HuR overexpression restored the ability of HCECs to up-regulate VEGF under hypoxia; however, knockdown of HuR suppressed hypoxia-induced VEGF in control cells but did not further decrease VEGF in HCECs. These findings suggest that HCECs may modulate HuR to suppress hypoxia-mediated up-regulation of VEGF. CONCLUSION: Our study revealed a distinct regulation of VEGF via HuR in HCECs following hypoxia, which likely contributes to minimizing corneal NV and/or maintenance of corneal avascularity.
Assuntos
Córnea/metabolismo , Neovascularização da Córnea/prevenção & controle , Proteína Semelhante a ELAV 1/metabolismo , Epitélio Corneano/citologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Hipóxia Celular , Células Cultivadas , Córnea/irrigação sanguínea , Córnea/patologia , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Proteína Semelhante a ELAV 1/genética , Ensaio de Imunoadsorção Enzimática , Humanos , RNA Mensageiro/metabolismo , Coelhos , Transfecção , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Peroxisome proliferator-activated receptor alpha (PPARα) and gamma (PPARγ) agonists have anti-inflammatory and anti-neovascularization effects, but few reports have tested the combination of PPARα and PPARγ agonists. In this study, we investigated the therapeutic effects of ophthalmic solutions of agonists of PPARα, PPARγ, and the combination in a rat corneal alkali burn model. After alkali injury, an ophthalmic solution of 0.05% fenofibrate (PPARα group), 0.1% pioglitazone (PPARγ group), 0.05% fenofibrate + 0.1% pioglitazone (PPARα+γ group), or vehicle (vehicle group) was topically instilled onto the rat's cornea twice a day. After instillation, upregulation was seen of PPAR mRNA corresponding to each agonist group. Administration of agonists for PPARα, PPARγ, and PPARα+γ suppressed inflammatory cells, neovascularization, and fibrotic changes. In addition, the PPARγ agonist upregulated M2 macrophages, which contributed to wound healing, whereas the PPARα agonist suppressed immature blood vessels in the early phase. Administration of PPARα+γ agonists showed therapeutic effects in corneal wound healing, combining the characteristics of both PPARα and PPARγ agonists. The results indicate that the combination of PPARα and γ agonists may be a new therapeutic strategy.
Assuntos
Queimaduras Químicas/tratamento farmacológico , Lesões da Córnea/tratamento farmacológico , Queimaduras Oculares/tratamento farmacológico , PPAR alfa/agonistas , PPAR gama/agonistas , Animais , Queimaduras Químicas/metabolismo , Queimaduras Químicas/patologia , Lesões da Córnea/metabolismo , Lesões da Córnea/patologia , Neovascularização da Córnea/prevenção & controle , Citocinas/genética , Modelos Animais de Doenças , Quimioterapia Combinada , Queimaduras Oculares/metabolismo , Queimaduras Oculares/patologia , Fenofibrato/administração & dosagem , Fibrose , Ceratite/prevenção & controle , Masculino , Soluções Oftálmicas , PPAR alfa/genética , PPAR alfa/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Pioglitazona/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
Chemical burns are a major cause of corneal haze and blindness. Corticosteroids are commonly used after corneal burns to attenuate the severity of the inflammation-related fibrosis. While research efforts have been aimed toward application of novel therapeutics. In the current study, a novel drug delivery system based nanostructured lipid carriers (NLCs) were designed to treat corneal alkaline burn injury. Rapamycin, a potent inhibitor of mammalian target of rapamycin pathway, was loaded in NLCs (rapa-NLCs), and the NLCs were characterized. Cell viability assay, cellular uptake of NLCs, and in vitro evaluation of the fibrotic/angiogenic genes suppression by rapa-NLCs were carried out on human isolated corneal fibroblasts. Immunohistochemistry (IHC) assays were also performed after treatment of murine model of corneal alkaline burn with rapa-NLCs. According to the results, rapamycin was efficiently loaded in NLCs. NLCs could enhance coumarin-6 fibroblast uptake by 1.5 times. Rapa-NLCs efficiently downregulated platelet-derived growth factor and transforming growth factor beta genes in vitro. Furthermore, proliferation of fibroblasts, a major cause of corneal haze after injury, reduced. IHC staining of treated cornea with alpha-smooth muscle actin and CD34 + antibodies showed efficient prevention of myofibroblasts differentiation and angiogenesis, respectively. In conclusion, ocular delivery of rapamycin using NLCs after corneal injury may be considered as a promising antifibrotic/angiogenic treatment approach to preserve patient eyesight.
Assuntos
Queimaduras Químicas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Córnea/efeitos dos fármacos , Lesões da Córnea/tratamento farmacológico , Opacidade da Córnea/tratamento farmacológico , Portadores de Fármacos , Queimaduras Oculares/tratamento farmacológico , Fibroblastos/efeitos dos fármacos , Lipídeos/química , Nanopartículas , Sirolimo/administração & dosagem , Administração Oftálmica , Animais , Queimaduras Químicas/etiologia , Queimaduras Químicas/metabolismo , Queimaduras Químicas/patologia , Células Cultivadas , Córnea/metabolismo , Córnea/patologia , Lesões da Córnea/induzido quimicamente , Lesões da Córnea/metabolismo , Lesões da Córnea/patologia , Neovascularização da Córnea/induzido quimicamente , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Neovascularização da Córnea/prevenção & controle , Opacidade da Córnea/induzido quimicamente , Opacidade da Córnea/metabolismo , Opacidade da Córnea/patologia , Modelos Animais de Doenças , Composição de Medicamentos , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/metabolismo , Queimaduras Oculares/patologia , Fibroblastos/metabolismo , Fibrose , Humanos , Masculino , Camundongos Endogâmicos BALB C , Nanomedicina , Sirolimo/química , Hidróxido de Sódio , Cicatrização/efeitos dos fármacosRESUMO
Neutrophil-mediated inflammation plays a critical role in corneal damage following injury or infection. Previous studies demonstrated that membrane-bound FasL (mFasL) induces neutrophil chemokine production. However, the extracellular domain of mFasL is normally cleaved by matrix metalloproteinases to release a soluble form of FasL (sFasL) and sFasL antagonizes mFasL-mediated chemokine production. Therefore, we hypothesized that sFasL could be used to prevent neutrophil-mediated corneal inflammation associated with injury and bacterial keratitis. To test this hypothesis, GFP-only, sFasL-GFP, or mFasL-GFP were expressed in the corneal stroma of C57BL/6 mice, using intra-stromal injections of plasmid DNA or adenoviral vectors (AV) and the role of mFasL and sFasL in corneal inflammation was examined in models of corneal injury and LPS-induced keratitis. Our work addresses an important area of disagreement in the field of FasL, with regard to the mechanism by which sFasL regulates ocular inflammation. Herein, we demonstrate that an intrastromal injection of GFP-only, sFasL-GFP, or mFasL-GFP plasmid DNA resulted in GFP expression throughout the corneal stroma for up to two weeks with little to no evidence of inflammation in the GFP-only and sFasL-GFP groups and mild corneal inflammation in the mFasL-GFP group. Similarly, following epithelial debridement, corneas expressing GFP-only or sFasL-GFP showed no significant signs of corneal inflammation, with clear corneas at 15 days post debridement. By contrast, epithelial debridement of corneas expressing mFasL-GFP triggered persistent corneal inflammation and the development of central corneal opacities that was blocked by sFasL. Similar to the mFasL-GFP plasmid DNA, intrastromal injection of mFasL-GFP AV triggered mild corneal inflammation, but it was transient and resolved by day 10 with corneas remaining clear out to 30 days post injection. Nevertheless, intrastromal expression of mFasL-GFP AV exacerbated LPS-induced keratitis, corneal opacity, and neovascularization, while sFasL-GFP AV expression prevented LPS-induced keratitis, resulting in a clear cornea. Histological analysis of corneas with LPS-induced keratitis revealed a robust infiltration of macrophages and neutrophils and sFasL expression specifically blocked the neutrophil influx. Overall, our data demonstrate that stromal expression of mFasL is inflammatory, while sFasL is non-inflammatory, and opposes the effects of mFasL in mouse models of epithelial debridement and LPS-induced keratitis. These data demonstrate that a delicate balance between sFasL and mFasL regulates ocular inflammation. This study further identifies sFasL as a potent inhibitor of neutrophil-mediated corneal damage, and supports the potential use of sFasL in the treatment of neutrophil-mediated keratitis. These results strongly support the hypothesis that, in the immune privileged environment of the eye, the isoform of FasL regulates immune privilege and determines the extent of inflammation: mFasL promotes inflammation and sFasL blocks inflammation.
Assuntos
Desbridamento , Modelos Animais de Doenças , Epitélio Corneano/cirurgia , Proteína Ligante Fas/fisiologia , Ceratite/prevenção & controle , Adenoviridae/genética , Animais , Western Blotting , Neovascularização da Córnea/etiologia , Neovascularização da Córnea/prevenção & controle , Substância Própria/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Regulação da Expressão Gênica/fisiologia , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Ceratite/etiologia , Ceratite/patologia , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/genéticaRESUMO
Corneal neovascularization (CNV) leads to the loss of corneal transparency and vision impairment, and can ultimately cause blindness. Topical corticosteroids are the first line treatment for suppressing CNV, but poor ocular bioavailability and rapid clearance of eye drops makes frequent administration necessary. Patient compliance with frequent eye drop application regimens is poor. We developed biodegradable nanoparticles (NP) loaded with dexamethasone sodium phosphate (DSP) using zinc ion bridging, DSP-Zn-NP, with dense coatings of poly(ethylene glycol) (PEG). DSP-Zn-NP were safe and capable of providing sustained delivery of DSP to the front of the eye following subconjunctival (SCT) administration in rats. We reported that a single SCT administration of DSP-Zn-NP prevented suture-induced CNV in rats for two weeks. In contrast, the eyes receiving SCT administration of either saline or DSP solution developed extensive CNV in less than 1 week. SCT administration of DSP-Zn-NP could be an effective strategy in preventing and treating CNV.
Assuntos
Neovascularização da Córnea/prevenção & controle , Preparações de Ação Retardada/química , Dexametasona/análogos & derivados , Glucocorticoides/administração & dosagem , Zinco/química , Animais , Dexametasona/administração & dosagem , Dexametasona/uso terapêutico , Glucocorticoides/uso terapêutico , Nanopartículas/química , Polietilenoglicóis/química , RatosRESUMO
MicroRNA-204 (miR-204) is highly expressed in cornea, here we explored the role and mechanism of miR-204 in corneal neovascularization (CNV). Mouse CNV was induced by intrastromal placement of suture in BALB/c mice with the subconjunctival injection of miR-204 agomir or negative control. Human primary limbal epithelial cells (LECs) and immortalized microvascular endothelial cells (HMECs) were used to evaluate the expression changes and anti-angiogenic effects of miR-204 under biomechanical stress (BS). The expression and localization of miR-204, vascular endothelial growth factor (VEGF) and their receptors were detected by quantitative real-time PCR, in situ hybridization, immunohistochemistry and Western blot. The results showed that miR-204 expression was mainly localized in epithelium and down-expressed in vascularized cornea. Subconjunctival injection of miR-204 agomir inhibited CNV and reduced the expression of VEGF and VEGF receptor 2. Similarly, miR-204 overexpression attenuated the increased expression of VEGF by biomechanical stress in LECs, and suppressed the proliferation, migration, and tube formation of HMECs. These novel findings indicate that epithelium-derived miR-204 inhibits suture-induced CNV through regulating VEGF and VEGF receptor 2.
Assuntos
Neovascularização da Córnea/prevenção & controle , Modelos Animais de Doenças , Epitélio Corneano/metabolismo , MicroRNAs/fisiologia , Animais , Vasos Sanguíneos/efeitos dos fármacos , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Células Endoteliais/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
To evaluate the effectiveness of epigallocatechin gallate (EGCG) in inhibiting corneal neovascularization in rat alkaline burn model. Corneal neovascularization model was induced by sodium hydroxide alkaline burn injury in SD rats. Rats were randomly divided into two groups and were given intraperitoneal injection with EGCG or PBS per day for up to 14 days respectively. Corneal inflammation and neovascularization area were assessed on days 3, 7, and 14 after cauterization with digital photographs. Vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) mRNA levels were measured by reverse transcription-polymerase chain reaction (qRT-PCR). The nuclear transfactor-Κb (NF-κB) subunit P65 protein was assayed by immunohistochemistry. The differences of corneal inflammation scores between two groups were significant. The area of CNV between two groups had no significant difference on day 3 but have significant difference on days 7 and 14.The PDEF mRNA expression in EGCG group was significantly higher and the expression of VEGF mRNA was lower than those in PBS group. The results of immunohistochemistry showed from day 7, expression of NF-κB P65protein was suppressed considerably in EGCG group. This study demonstrates that EGCG inhibits corneal neovascularization in a rat model induced by alkali burn.
Assuntos
Catequina/análogos & derivados , Neovascularização da Córnea/prevenção & controle , Animais , Queimaduras Químicas/fisiopatologia , Catequina/farmacologia , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Neovascularização da Córnea/fisiopatologia , Proteínas do Olho/biossíntese , Inflamação/induzido quimicamente , Inflamação/patologia , Inflamação/prevenção & controle , Masculino , Proteínas de Neoplasias/metabolismo , Fatores de Crescimento Neural/biossíntese , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ratos , Serpinas/biossíntese , Hidróxido de Sódio , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
CONTEXT: Abnormal angiogenesis and evasion of apoptosis are hallmarks of cancer. Accordingly, anti-angiogenic and pro-apoptotic therapies are effective strategies for cancer treatment. Medicinal plants, namely, Eugenia jambolana Lam. (Myrtaceae), Musa paradisiaca L. (Musaceae), and Coccinia indica Wight & Arn. (Cucurbitaceae), have not been greatly investigated for their anticancer potential. OBJECTIVE: We investigated the anti-angiogenic and pro-apoptotic efficacy of ethyl acetate (EA) and n-butanol (NB) extracts of E. jambolana (seeds), EA extracts of M. paradisiaca (roots) and C. indica (leaves) with respect to mammary neoplasia. MATERIALS AND METHODS: Effect of extracts (2-200 µg/mL) on cytotoxicity and MCF-7, MDA-MB-231 and endothelial cell (EC) proliferation and in vitro angiogenesis were evaluated by MTT, 3[H]thymidine uptake and EC tube formation assays, respectively. In vivo tumour proliferation, VEGF secretion and angiogenesis were assessed using the Ehrlich ascites tumour (EAT) model followed by rat corneal micro-pocket and chicken chorioallantoic membrane (CAM) assays. Apoptosis induction was assessed by morphological and cell cycle analysis. RESULTS: EA extracts of E. jambolana and M. paradisiaca exhibited the highest cytotoxicity (IC50 25 and 60 µg/mL), inhibited cell proliferation (up to 81%), and tube formation (83% and 76%). In vivo treatment reduced body weight (50%); cell number (16.5- and 14.7-fold), secreted VEGF (â¼90%), neoangiogenesis in rat cornea (2.5- and 1.5-fold) and CAM (3- and 1.6-fold) besides EAT cells accumulation in sub-G1 phase (20% and 18.38%), respectively. DISCUSSION AND CONCLUSION: Considering the potent anti-angiogenic and pro-apoptotic properties, lead molecules from EA extracts of E. jambolana and M. paradisiaca can be developed into anticancer drugs.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Carcinoma de Ehrlich/prevenção & controle , Membrana Corioalantoide/irrigação sanguínea , Cucurbitaceae/química , Musa/química , Neovascularização Patológica , Neovascularização Fisiológica/efeitos dos fármacos , Extratos Vegetais/farmacologia , Syzygium/química , Fator A de Crescimento do Endotélio Vascular/farmacologia , 1-Butanol/química , Acetatos/química , Inibidores da Angiogênese/isolamento & purificação , Animais , Antineoplásicos Fitogênicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Carcinoma de Ehrlich/sangue , Carcinoma de Ehrlich/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Neovascularização da Córnea/patologia , Neovascularização da Córnea/fisiopatologia , Neovascularização da Córnea/prevenção & controle , Relação Dose-Resposta a Droga , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Células MCF-7 , Camundongos , Fitoterapia , Extratos Vegetais/isolamento & purificação , Folhas de Planta , Raízes de Plantas/química , Plantas Medicinais , Ratos Wistar , Sementes/química , Fatores de Tempo , Carga Tumoral/efeitos dos fármacosRESUMO
We evaluated an anti-inflammatory effect of topical administration of tofacitinib, janus kinase (JAK) blocker, on corneal inflammation. Topical instillation of either tofacitinib or PBS was applied after wounding BALB/c mice corneas with alkali burn. Topical instillation was performed until day 14 after injury and injured eye was analyzed. The vascularized area in the alkali burned cornea was significantly reduced in the tofacitinib group compared with that in the PBS group. The immunoreactivity of Gr-1, F4/80, IFN-γ, and phosphorylated STAT(signal transducer and activator of transcription)1 in corneal stroma was diminished significantly in the tofacitinib group. Using laser capture microdissection system and quantitative PCR array analysis, the expression levels of CXCL9, CXCL5, CCL7, CCL2, MMP(matrix metalloproteinase)-9, and STAT1 in corneal stroma were down-regulated in the tofacitinib group. In in vitro study, human fibroblast pretreated by IFN-γ showed phosphorylation of STAT1, and this phosphorylation was down-regulated by adding tofacitinib to the culture medium. These results indicate the topical application of JAK inhibitor causes down-regulation of JAK- or IFN-γ-related molecules. Therefore, we deduce that application of JAK inhibitor for topical instillation may contribute to the treatment of corneal inflammation.
Assuntos
Córnea/patologia , Neovascularização da Córnea/prevenção & controle , Ceratite/tratamento farmacológico , Piperidinas/administração & dosagem , Pirimidinas/administração & dosagem , Pirróis/administração & dosagem , Administração Tópica , Animais , Células Cultivadas , Córnea/efeitos dos fármacos , Neovascularização da Córnea/etiologia , Neovascularização da Córnea/patologia , Modelos Animais de Doenças , Janus Quinase 3/antagonistas & inibidores , Ceratite/complicações , Ceratite/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Inibidores de Proteínas Quinases/administração & dosagemRESUMO
The aim of this experimental study was to compare the efficacy of topical aflibercept and topical bevacizumab in preventing corneal neovascularization. A chemical burn was created in the right central cornea of male Sprague-Dawley rats, followed immediately by instillation of one drop (25 mg/ml, 20 µl volume) of aflibercept (15 eyes), bevacizumab (14 eyes), or saline (15 eyes). Treatment was repeated twice daily for 7 days. Corneal neovascularization was determined using corneal photographs (ImageJ) on days 1, 4, 7, 10, and histological and immunofluorescence studies, on day 10. Stromal immunoreactivity was evaluated 2 days after injury in 6 rats treated singly with bevacizumab or aflibercept. Corneal neovascularization was observed clinically on day 4 in all groups. In the aflibercept group, the area of neovascularization increased from 7.38 ± 2.23% on day 4 to 21.73 ± 14.59% on day 7 and 31.0 ± 23.61% on day 10. Corresponding values in the bevacizumab group were 6.04% ± 1.81%, 51.27 ± 15.50%, and 54.4 ± 11.33%, and in the control group, 8.99 ± 1.93%, 42.6 ± 19.59%, and 55.15 ± 11.54%. The area of neovascularization was significantly smaller on days 7 and 10 in the aflibercept group than in the control and bevacizumab groups (P < 0.001, all analyses), with no significant differences between the latter two groups (day 7, P = 0.868; day 10, P = 0.213). Clinical findings were compatible with the histological data and supported by immunofluorescence and corneal flat-mount staining. Both drugs demonstrated variable penetration into the corneal stroma. Topical aflibercept effectively inhibits corneal neovascularization in a rat model of chemical burn. These findings may have important therapeutic implications for humans.
Assuntos
Inibidores da Angiogênese/farmacologia , Bevacizumab/farmacologia , Queimaduras Químicas/tratamento farmacológico , Lesões da Córnea/tratamento farmacológico , Neovascularização da Córnea/prevenção & controle , Queimaduras Oculares/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Administração Tópica , Inibidores da Angiogênese/administração & dosagem , Animais , Bevacizumab/administração & dosagem , Queimaduras Químicas/complicações , Túnica Conjuntiva/efeitos dos fármacos , Lesões da Córnea/complicações , Neovascularização da Córnea/tratamento farmacológico , Neovascularização da Córnea/etiologia , Modelos Animais de Doenças , Queimaduras Oculares/complicações , Masculino , Soluções Oftálmicas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Fatores de Crescimento do Endotélio Vascular/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagemRESUMO
OBJECTIVE: The deficiency of very low-density lipoprotein receptor resulted in Wnt signaling activation and neovascularization in the retina. The present study sought to determine whether the very low-density lipoprotein receptor extracellular domain (VLN) is responsible for the inhibition of Wnt signaling in ocular tissues. APPROACH AND RESULTS: A plasmid expressing the soluble VLN was encapsulated with poly(lactide-co-glycolide acid) to form VLN nanoparticles (VLN-NP). Nanoparticles containing a plasmid expressing the low-density lipoprotein receptor extracellular domain nanoparticle were used as negative control. MTT, modified Boyden chamber, and Matrigel (™) assays were used to evaluate the inhibitory effect of VLN-NP on Wnt3a-stimulated endothelial cell proliferation, migration, and tube formation. Vldlr(-/-) mice, oxygen-induced retinopathy, and alkali burn-induced corneal neovascularization models were used to evaluate the effect of VLN-NP on ocular neovascularization. Wnt reporter mice (BAT-gal), Western blotting, and luciferase assay were used to evaluate Wnt pathway activity. Our results showed that VLN-NP specifically inhibited Wnt3a-induced endothelial cell proliferation, migration, and tube formation. Intravitreal injection of VLN-NP inhibited abnormal neovascularization in Vldlr(-/-), oxygen-induced retinopathy, and alkali burn-induced corneal neovascularization models, compared with low-density lipoprotein receptor extracellular domain nanoparticle. VLN-NP significantly inhibited the phosphorylation of low-density lipoprotein receptor-related protein 6, the accumulation of ß-catenin, and the expression of vascular endothelial growth factor in vivo and in vitro. CONCLUSIONS: Taken together, these results suggest that the soluble VLN is a negative regulator of the Wnt pathway and has antiangiogenic activities. Nanoparticle-mediated expression of VLN may thus represent a novel therapeutic approach to treat pathological ocular angiogenesis and potentially other vascular diseases affected by Wnt signaling.
Assuntos
Córnea/irrigação sanguínea , Neovascularização da Córnea/prevenção & controle , Ácido Láctico/química , Nanopartículas , Ácido Poliglicólico/química , Receptores de LDL/metabolismo , Neovascularização Retiniana/prevenção & controle , Vasos Retinianos/metabolismo , Transfecção/métodos , Via de Sinalização Wnt , Proteína Wnt3A/metabolismo , Animais , Movimento Celular , Proliferação de Células , Células Cultivadas , Neovascularização da Córnea/genética , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/fisiopatologia , Modelos Animais de Doenças , Humanos , Injeções Intravítreas , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Interferência de RNA , Ratos Sprague-Dawley , Receptores de LDL/genética , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/fisiopatologia , Vasos Retinianos/fisiopatologia , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína Wnt3A/genética , beta Catenina/metabolismoRESUMO
Quite a number of pathological factors exist that can disturb the balance between anti-angiogenic and proangiogenic mechanisms, thus causing vascularization of the cornea. The neovessels are immature, ill-formed, and show increased permeability, which is dangerous of corneal edema, lipid deposition, and opacification. Moreover, as known, corneal neovascularization (CNV; preexisting or postoperative) may contribute to immune response against the transplant. Suppression of neovascularization is able to decrease the risk of corneal transplant rejection. In order to identify the principal strategy for struggling against CNV, we should first get a better understanding of its etiology, pathogenesis, and role in transplant immunity as well as mechanisms of action of available treatment methods.
Assuntos
Inibidores da Angiogênese/farmacologia , Córnea , Neovascularização da Córnea , Transplante de Córnea/efeitos adversos , Neovascularização Patológica/prevenção & controle , Complicações Pós-Operatórias , Córnea/irrigação sanguínea , Córnea/patologia , Córnea/cirurgia , Neovascularização da Córnea/diagnóstico , Neovascularização da Córnea/etiologia , Neovascularização da Córnea/prevenção & controle , Humanos , Neovascularização Patológica/diagnóstico , Neovascularização Patológica/etiologia , Complicações Pós-Operatórias/diagnóstico , Complicações Pós-Operatórias/prevenção & controleRESUMO
PURPOSE: To investigate the effects and mechanisms of fasudil hydrochloride (fasudil) on and in alkali burn-induced corneal neovascularization (CNV) in mice. METHODS: To observe the effect of fasudil, mice with alkali-burned corneas were treated with either fasudil eye drops or phosphate-buffered saline (PBS) four times per day for 14 consecutive days. After injury, CNV and corneal epithelial defects were measured. The production of reactive oxygen species (ROS) and heme oxygenase-1(HO-1) was measured. The infiltration of polymorphonuclear neutrophils (PMNs) and the mRNA expressions of CNV-related genes were analyzed on day 14. RESULTS: The incidence of CNV was significantly lower after treatment with 100 µM and 300 µM fasudil than with PBS, especially with 100 µM fasudil. Meanwhile, the incidences of corneal epithelial defects was lower (n=15, all p<0.01). After treatment with 100 µM fasudil, the intensity of DHE ï¬uorescence was reduced in the corneal epithelium and stroma than with PBS treatment (n=5, all p<0.01), and the number of filtrated PMNs decreased. There were significant differences between the expressions of VEGF, TNF-a, MMP-8, and MMP-9 in the 100 µM fasudil group and the PBS group (n=8, all p<0.05). The production of HO-1 protein in the 100 µM fasudil group was 1.52±0.34 times more than in the PBS group (n=5 sample, p<0.05). CONCLUSIONS: 100 µM fasudil eye drops administered four times daily can significantly inhibit alkali burn-induced CNV and promote the healing of corneal epithelial defects in mice. These effects are attributed to a decrease in inflammatory cell infiltration, reduction of ROS, and upregulation of HO-1 protein after fasudil treatment.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Queimaduras Químicas/tratamento farmacológico , Queimaduras Químicas/enzimologia , Neovascularização da Córnea/prevenção & controle , Queimaduras Oculares/tratamento farmacológico , Queimaduras Oculares/enzimologia , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Álcalis/toxicidade , Animais , Queimaduras Químicas/patologia , Neovascularização da Córnea/etiologia , Neovascularização da Córnea/patologia , Modelos Animais de Doenças , Queimaduras Oculares/patologia , Feminino , Heme Oxigenase-1/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
BACKGROUND: The purpose of this study was to investigate the effect of recombinant pigment epithelium-derived factor (rPEDF), secreted by ARPE-19 cells transfected with the human PEDF gene and transplanted subconjunctivally in normal and in rabbits in which corneal neovascularization was elicited by a chemical burn. METHODS: Twenty grey Chinchilla Bastard rabbits were randomly assigned to four groups; neovascularization was induced in groups A, B, and C by alkali cauterization. Seven days later, group A received no cell implantation, non-transfected ARPE-19 cells were implanted subconjunctivally in group B, and PEDF-transfected ARPE-19 cells were implanted subconjunctivally in groups C and D (non-cauterized). In-vivo rPEDF secretion was analyzed by immunoblotting, and ELISA of extracts of conjunctival tissue samples taken at different time points. Digital photographs acquired on days 7, 14, and 21 after cauterization were evaluated for lead vessel length, vascular invasion area, and overall neovascularization rate. RESULTS: At days 14 and 21 after cauterization, significant differences were observed between groups A, B, and C in lead vessel length (day 21: 5.91 ± 0.45, 5.11 ± 1.22, 3.79 ± 0.59 mm, repectively), vascular invasion area (day 21: 35.5 ± 8.65, 34.86 ± 4.92, 19.2 ± 5.03 mm(2) respectively), and rate of corneal neovascularization. Compared to controls, neovascularization was reduced by 37.5 % on day 14 and 47 % on day 21. Analysis of conjunctival tissue extracts showed that rPEDF was secreted by the transplanted PEDF-transfected cells. CONCLUSION: Subconjunctivally transplanted, PEDF-transfected ARPE-19 cells secrete rPEDF, which inhibits the corneal neovascularization elicited by alkali cauterization.