RESUMO
Caffeic acid phenylethyl ester (CAPE) is an antioxidative agent originally derived from propolis. Oxidative stress is a significant pathogenic factor in most retinal diseases. Our previous study revealed that CAPE suppresses mitochondrial ROS production in ARPE-19 cells by regulating UCP2. The present study explores the ability of CAPE to provide longer-term protection to RPE cells and the underlying signal pathways involved. ARPE-19 cells were given CAPE pretreatment followed by t-BHP stimulation. We used in situ live cell staining with CellROX and MitoSOX to measure ROS accumulation; Annexin V-FITC/PI assay to evaluate cell apoptosis; ZO-1 immunostaining to observe tight junction integrity in the cells; RNA-seq to analyze changes in gene expression; q-PCR to validate the RNA-seq data; and Western Blot to examine MAPK signal pathway activation. CAPE significantly reduced both cellular and mitochondria ROS overproduction, restored the loss of ZO-1 expression, and inhibited apoptosis induced by t-BHP stimulation. We also demonstrated that CAPE reverses the overexpression of immediate early genes (IEGs) and activation of the p38-MAPK/CREB signal pathway. Either genetic or chemical deletion of UCP2 largely abolished the protective effects of CAPE. CAPE restrained ROS generation and preserved the tight junction structure of ARPE-19 cells against oxidative stress-induced apoptosis. These effects were mediated via UCP2 regulation of p38/MAPK-CREB-IEGs pathway.
Assuntos
Ácidos Cafeicos , Estresse Oxidativo , Álcool Feniletílico , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Ácidos Cafeicos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Álcool Feniletílico/farmacologia , Espécies Reativas de Oxigênio/metabolismo , HumanosRESUMO
The purpose of this study was to identify proteins that regulate vascular remodeling in an ROP mouse model. Pups were subjected to fluctuating oxygen levels and retinas sampled during vessel regression (PN12) or neovascularization (PN17) for comparative SWATH-MS proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We developed a human retinal endothelial cell (HREC) ROP correlate to validate the expression of retina neovascular-specific markers. A total of 5191 proteins were identified in OIR retinas with 498 significantly regulated in elevated oxygen and 345 after a return to normoxia. A total of 122 proteins were uniquely regulated during vessel regression and 69 during neovascularization (FC ≥ 1.5; p ≤ 0.05), with several validated by western blot analyses. Expressions of 56/69 neovascular-specific proteins were confirmed in hypoxic HRECs with 23 regulated in the same direction as OIR neovascular retinas. These proteins control angiogenesis-related processes including matrix remodeling, cell migration, adhesion, and proliferation. RNAi and transfection overexpression studies confirmed that VASP and ECH1, showing the highest levels in hypoxic HRECs, promoted human umbilical vein (HUVEC) and HREC cell proliferation, while SNX1 and CD109, showing the lowest levels, inhibited their proliferation. These proteins are potential biomarkers and exploitable intervention tools for vascular-related disorders. The proteomics data set generated has been deposited to the ProteomeXchange/iProX Consortium with the Identifier:PXD029208.
Assuntos
Retinopatia da Prematuridade , Animais , Animais Recém-Nascidos , Cromatografia Líquida , Modelos Animais de Doenças , Humanos , Recém-Nascido , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio/metabolismo , Proteômica , Retina , Retinopatia da Prematuridade/metabolismo , Espectrometria de Massas em Tandem , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Retinal diseases are often accompanied by inflammation, vascular abnormalities, and neurodegeneration that decrease vision. Treatment with exogenous PEDF is widely shown to alleviate these conditions leading us to hypothesize that loss of function of the PEDF gene disrupts these pathways and leads to visual loss. Measurements were carried out by detailed phenotyping of PEDF null mice to assess expression of immunomodulators, glia activation, systemic inflammation, vascular disturbances, and visual sensitivity often associated with retinal pathologies. With a deletion of the Pedf gene, there was increased expression of several immune modulators in Pedf-/- retinas and serum with IL-2 and GM-CSF upregulated in both. Increases in retina glia activation and macrophage infiltration, levels of serum c-reactive protein (CRP), numbers of white and red blood cells and platelets and decreased blood glucose levels were all features associated with PEDF null mice. With PEDF gene deletion, there was also a notable increase in apoptosis in early developing retinas (PN3), reduced thickness of the photoreceptor layer, swelling of the inner plexiform layer, reduced retinal sensitivity and steady-state reduced activation of Erk and Akt, two signaling pathways used by PEDF. There is a substantial body of animal data emphasizing utility of PEDF treatment in homeostatic regulation of retinal diseases, including diabetic retinopathy and age-related macular degeneration but there is little agreement or evidence on the role of endogenous PEDF in retinal diseases. Our findings strongly support the concept that a deletion of the PEDF gene makes the retina vulnerable to diseases, and argue that endogenous PEDF plays a critical role in limiting pathological events in the retina.
Assuntos
Retinopatia Diabética , Proteínas do Olho , Fatores de Crescimento Neural , Serpinas , Animais , Apoptose , Retinopatia Diabética/genética , Proteínas do Olho/genética , Deleção de Genes , Inflamação/genética , Camundongos , Camundongos Knockout , Fatores de Crescimento Neural/genética , Retina/patologia , Serpinas/genéticaRESUMO
Mitochondrial dysfunction is associated with several retinal degenerative diseases including Age-related Macular Degeneration (AMD). Human mitochondrial DNA (mtDNA) haplogroups are inherited from a common ancestral clan and are defined by specific sets of genetic differences. The purpose of this study was to determine and compare the effects of mtDNA haplogroups H and J on transcriptome regulation and cellular resilience to oxidative stress in human RPE cytoplasmic hybrid (cybrid) cell lines in vitro. ARPE-19 cybrid cell lines containing mtDNA haplogroups H and J were created by fusing platelets obtained from normal individuals containing H and J haplogroups with mitochondria-deficient (Rho0) ARPE-19 cell lines. These cybrids were exposed to oxidative stress using 300 µM hydrogen peroxide (H2O2), following which mitochondrial structural dynamics was studied at varying time points using the mitochondrial markers - TOMM20 (Translocase of Outer Mitochondrial Membrane 20) and Mitotracker. To evaluate mitochondrial function, levels of ROS, ΔΨm and [Ca2+]m were measured using flow cytometry, and ATP levels were measured using luminescence. The H and J cybrid cell transcriptomes were compared using RNAseq to determine how changes in mtDNA regulate gene expression. Inflammatory and angiogenic markers were measured using Luminex assay to understand how these mtDNAs influenced cellular response to oxidative stress. Actin filaments' morphology was examined using confocal microscopy. Following exposure to H2O2 stress, the J cybrids showed increased mitochondrial swelling and perinuclear localization, disturbed fission and fusion, increased calcium uptake (p < 0.05), and higher secreted levels of TNF-α and VEGF (p < 0.001), compared to the H cybrids. Calcium uptake by J cybrids was reduced using an IP3R inhibitor. Thirteen genes involved in mitochondrial complex I and V function, fusion/fission events, cellular energy homeostasis, antioxidant defenses, and inflammatory responses, were significantly downregulated with log2 fold changes ranging between -1.5 and -5.1. Actin levels were also significantly reduced in stressed J cybrids (p ≤ 0.001) and disruption in actin filaments was observed. Thirty-eight genes involved in mitochondrial and cellular support functions, were upregulated with log2 fold changes of +1.5 to +5.9 in J cybrids compared to H cybrids. Our results demonstrate significant structural and functional differences between mtDNA haplogroups H vs. J -containing cybrid cells. Our study suggests that the J mtDNA haplogroup can alter the transcriptome to increase cellular susceptibility to stress and retinal degenerations.
Assuntos
DNA Mitocondrial , Degeneração Macular , Cálcio/metabolismo , DNA Mitocondrial/genética , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Degeneração Macular/genética , Degeneração Macular/metabolismo , Mitocôndrias/metabolismoRESUMO
Most of the major retinal degenerative diseases are associated with significant levels of oxidative stress. One of the major sources contributing to the overall level of stress is the reactive oxygen species (ROS) generated by mitochondria. The driving force for ROS production is the proton gradient across the inner mitochondrial membrane. This gradient can be modulated by members of the uncoupling protein family, particularly the widely expressed UCP2. The overexpression and knockout studies of UCP2 in mice have established the ability of this protein to provide neuroprotection in a number of animal models of neurological disease, including retinal diseases. The expression and activity of UCP2 are controlled at the transcriptional, translational and post-translational levels, making it an ideal candidate for therapeutic intervention. In addition to regulation by a number of growth factors, including the neuroprotective factors LIF and PEDF, small molecule activators of UCP2 have been found to reduce mitochondrial ROS production and protect against cell death both in culture and animal models of retinal degeneration. Such studies point to the development of new therapeutics to combat a range of blinding retinal degenerative diseases and possibly other diseases in which oxidative stress plays a key role.
Assuntos
Doenças Neurodegenerativas , Animais , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas de Desacoplamento Mitocondrial/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Desacopladora 2/genética , Proteína Desacopladora 2/metabolismoRESUMO
Pigment epithelium derived factor (PEDF), an endogenous inhibitor of angiogenesis, targets the growth of aberrant blood vessels in many tissues, including the eye. In this study we show that PEDF prevented early mitogenic signals of vascular endothelial growth factor (VEGF-A) in primate retinal endothelial cells, blocking proliferation, migration and tube formation. PEDF inhibited the phosphorylation and activation of five major downstream VEGF-A signaling partners, namely phosphoinositide-3-OH Kinase (PI3K), AKT, FAK, Src (Y416), and PLC-γ. It did so by binding to the extracellular domain of VEGF-R2, blocking VEGF-A-induced tyrosine phosphorylation (Tyr 951 and Tyr 1175), and inhibiting VEGF-R2 receptor kinase activity. PEDF had no effect on the transcription or translation of VEGF-R2 in cultured HUVECs. PEDF also bound to the extracellular domain of VEGF-R1. We conclude that PEDF blocks the growth of new blood vessels, in part, by reducing VEGF-A activation of its key mitogenic receptor, VEGF-R2, and by preventing its downstream signals in endothelial cells.
Assuntos
Inibidores da Angiogênese/fisiologia , Células Endoteliais/efeitos dos fármacos , Proteínas do Olho/fisiologia , Fatores de Crescimento Neural/fisiologia , Vasos Retinianos/citologia , Serpinas/fisiologia , Transdução de Sinais/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Vasos Sanguíneos/efeitos dos fármacos , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Primatas , Reação em Cadeia da Polimerase em Tempo Real , Tirosina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Mutations in Serpinf1 gene which encodes pigment epithelium-derived factor (PEDF) lead to osteogenesis imperfecta type VI whose hallmark is defective matrix mineralization. We reported previously that PEDF reduced expression and synthesis of Sost/Sclerostin as well as other osteocytes genes encoding proteins that regulate matrix mineralization [1]. To determine whether PEDF had an effect on osteocyte gene expression in bone, we used bone explant cultures. First, osteocytes were isolated from surgical waste of bone fragments obtained from patients undergoing elective foot surgeries under approved IRB protocol by Penn State College of Medicine IRB committee. Primary osteocytes treated with PEDF reduced expression and synthesis of Sost/Sclerostin and matrix phosphoglycoprotein (MEPE) as well as dentin matrix protein (DMP-1). On the whole, PEDF reduced osteocyte protein synthesis by 50% and by 75% on mRNA levels. For bone explants, following collagenase digestion, bone fragments were incubated in alpha-MEM supplemented with 250 ng/ml of PEDF or BSA. After 7 days of incubation in a medium supplemented with PEDF, analysis of mRNA by PCR and protein by western blotting of encoded osteocyte proteins showed reduced Sclerostin synthesis by 39% and MEPE by 27% when compared to fragments incubated in medium supplemented with BSA. mRNA expression levels of osteocytes in bone fragments treated with PEDF were reduced by 50% for both SOST and MEPE when compared to BSA-treated bone fragments. Taken together, the data indicate that PEDF has an effect on osteocyte gene expression in bone and encourage further studies to examine effect of PEDF on bone formation indices in animal models and its effect on osteocyte gene expression in vivo following PEDF administration.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Osso e Ossos/metabolismo , Proteínas do Olho/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores de Crescimento Neural/farmacologia , Osteócitos/metabolismo , Serpinas/farmacologia , Técnicas de Cultura de Tecidos , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Morfogenéticas Ósseas/genética , Células Cultivadas , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Marcadores Genéticos/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Pessoa de Meia-Idade , Osteócitos/efeitos dos fármacos , Fosfoproteínas/genética , Fosfoproteínas/metabolismoRESUMO
BACKGROUND: Diabetic nephropathy (DN) is a major complication of type1 and type 2 diabetes. Understanding how diabetes regulate transcriptome dynamics in DN is important for understanding the biology of the disease and for guiding development of new treatments. RESULTS: We analyzed the kidney transcriptome of a DN mouse model, D2.B6-Ins2 Akita /MatbJ, before/after treatment with P78-PEDF. Age, weight, and gender-matched mice and wild-type (wt) littermates were treated at 6 weeks (early treatment) or 12 weeks (late treatment) of age for the duration of 6 weeks. Animals were implanted with an osmotic mini pump delivering 0.3 ug/g/day P78-PEDF or vehicle. Using RNA-seq, we identified14,316 transcripts (12,328 coding;1,988 non-coding) that were significant and reliably expressed (FPKM > =1) in diabetic kidneys. Expression of 1,129 (7.9%) including 901 coding genes was altered by diabetes with log2 fold changes (FC) between -86.2 and +86.0 (q < 0.05) compared to wt. Of these, 164 (14.5%) showed increased and 965 (85.5%) decreased expression with FC > 1.5. Coding genes with highest FC in diabetic kidneys include Nhej1 (32.04), Ept1 (8.6), Srd5a2 (-6.55), Aif1 (-6.05), and Angptl7 (-4.71). Early and late stage diabetic groups receiving continuous infusion of P78 showed altered expression of 316/14,316 (2.2%) transcripts, including 121 coding genes compared to non-treated diabetic controls. Of these, 183 were upregulated and 133 downregulated with FC +50.9--93.3 (q < 0.05). P78 reversed diabetes-induced changes in 138/1129 (12.2%) transcripts, including 49/901 (5.44%) coding genes. Nhej1 (-37.94), Tceanc2 (5.76), Ept1 (-4.45), Ugt1a2 (3.03), and Tmsb15l (-3.0) showed the highest FC with treatment. The DNA repair gene, Nhej1 with the greatest FC in diabetic kidneys was completely restored to control levels by both early and late P78 treatments. Expression of other coding genes regulated by diabetes with FC > =(+/-) 1.5 and completely reversed by P78 include Mamdc4, Kdm4b, Tmem252, Selm, and Hpd. RT and QRT-PCR validated expression of gene with FC > (+/-)2.0. Transcriptome changes were also observed between early and late-stage treatments. Precursor non-coding miRNAs showed the highest fold changes in expression in the diabetic and P78 treatment groups. Several diabetic-induced changes were reversed in direction of expression by treatment including Gm24083, GM25953, miR1905, Gm25535, Gm27903, and miR196a1 with FC > =(+/-)20. From Ingenuity pathway analysis (IPA), mitochondrial dysfunction, Nrf-2- mediated oxidative stress and renal injury pathways emerged as key mechanisms in DN. DN-enriching genes in these pathways were reduced in number or regulated in the opposite direction by treatment. CONCLUSIONS: Unique biomarkers and canonical pathways identified in this study may hold the key to understanding mechanisms of DN pathobiology with value for clinical translation. Our data suggest that mitochondrial dysfunction, genotoxicity and oxidative stress are principal events in DN and that P78-PEDF holds promise for its management.
Assuntos
Nefropatias Diabéticas/genética , Proteínas do Olho/química , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores de Crescimento Neural/química , Peptídeos/farmacologia , Serpinas/química , Transcriptoma , Animais , Análise por Conglomerados , Diabetes Mellitus Experimental , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/metabolismo , Modelos Animais de Doenças , Descoberta de Drogas , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/metabolismo , Peptídeos/química , Mapeamento de Interação de Proteínas , Transdução de SinaisRESUMO
Pigment epithelium-derived factor (PEDF) encoded by serpinf1 is a potent antiangiogenic factor found in a wide variety of fetal and adult tissues. Several reports have shown that lack of PEDF leads to osteogenesis imperfecta (OI) type VI whose hallmark is a defect in mineralization that leads to excessive osteoid build up that fails to mineralize. Because PEDF is antiangiogenic factor it would pose serious consequences on bone development and healing of fractures. To understand possible mechanisms by which PEDF plays a role in bone development and regulation of matrix mineralization, we determined the effects of exogenous PEDF on vascular endothelial growth factor (VEGF) expression by human mesenchymal stem cells (hMSCs) and mechanisms of its regulation by PEDF. Human MSCs incubated in normal medium supplemented with exogenous PEDF increased VEGF expression; this increase was also seen when PEDF was added to hMSCs undergoing osteogenic differentiation. MSCs maintained in osteogenic medium increased synthesis of both VEGF and PEDF but both factors were maintained relatively in balance during differentiation. To understand mechanisms by which exogenous PEDF regulated VEGF expression, hMSCs exposed to PEDF activated Erk signaling pathway in MSCs; inhibition of Erk signaling reduced VEGF mRNA expression as well as protein production suggesting that PEDF regulates VEGF expression in MSCs via Erk signaling pathway. In conclusion, PEDF increases VEGF expression by MSCs suggesting that regulation of VEGF by PEDF may be part of the mechanisms by which PEDF regulates osteoblastic mineralization.
Assuntos
Matriz Óssea/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Proteínas do Olho/farmacologia , Células-Tronco Mesenquimais/metabolismo , Fatores de Crescimento Neural/farmacologia , Serpinas/farmacologia , Regulação para Cima/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Idoso , Matriz Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Meios de Cultura/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Pessoa de Meia-Idade , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismoRESUMO
Mutations in Serpinf1 gene which encodes pigment epithelium derived factor (PEDF) lead to osteogenesis imperfecta type VI whose hallmark is defective mineralization. Mechanisms by which PEDF regulates matrix mineralization remain unknown. We examined effect of exogenous PEDF on expression of osteoblastic and osteocytic related genes and proteins in mineralizing osteoblast culture. Mineralizing human osteoblasts supplemented with exogenous PEDF for 14 days deposited 47% more mineral than cells cultured without PEDF. Analysis of selected gene expression by cells in mineralizing cultures supplemented with exogenous PEDF showed reduction in expression of Sclerostin (Sost) by 70%, matrix extracellular phosphoglycoprotein (MEPE) by 75% and dentin matrix protein (DMP-1) by 20% at day 14 of culture. Phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) expression was not affected. Western blotting and immunoprecipitation showed that sclerostin and MEPE synthesis by osteocytes were reduced by 50% and 60% respectively in mineralizing osteoblasts containing exogenous PEDF. Primary osteocytes exposed to PEDF also reduced synthesis of Sost/sclerostin by 50% within 24 h. For osteoblastic genes, Bone sialoprotein (BSP) was expressed at 75% higher by day 7 in cultures containing exogenous PEDF while Col1A1 expression remained high at all-time points. Total beta-catenin was increased in mineralizing osteoblastic cells suggesting increased Wnt activity. Taken together, the data indicate that PEDF suppressed expression of factors that inhibit mineralization while enhancing those that promote mineralization. The findings also suggest that PEDF may regulate Sost expression by osteocytes leading to enhanced osteoblastic differentiation and increased matrix mineralization.
Assuntos
Matriz Óssea/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Calcificação Fisiológica , Proteínas do Olho/metabolismo , Fatores de Crescimento Neural/metabolismo , Osteócitos/metabolismo , Serpinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Idoso , Calcificação Fisiológica/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Marcadores Genéticos , Humanos , Pessoa de Meia-Idade , beta Catenina/metabolismoRESUMO
Pigment epithelium-derived factor (PEDF) is a potent antiangiogenic factor found in a wide variety of tissues. Recent findings indicated that lack of PEDF leads to osteogenesis imperfecta type VI whose hallmark is a defect in mineralization. We investigated the effects of PEDF on human mesenchymal stem cells (hMSCs) and signaling pathways through which PEDF displays its activities in hMSCs. hMSCs incubated in a medium supplemented with PEDF induced expression of osteoblastic-related genes. In addition, PEDF induced alkaline phosphatase (ALP) activity in MSCs at 14 days of incubation in maintenance medium; hMSCs incubated in osteogenic medium in presence of PEDF expressed 19% more ALP activity (35.655 ± 1.827 U/mg protein, p = .041 than cells incubated in the same medium without PEDF supplementation (29.956 ± 2.100 U/µg protein). hMSCs incubated in osteogenic medium in presence of PEDF deposited 50% more mineral (2.108 ± 0.306 OD/ml per well per 1 × 10(4) cells per square centimeter, p = .017) than MSCs incubated in absence of the protein (1.398 ± 0.098 OD/ml per well per 1 × 10(4) cells per square centimeter) as determined by Alizarin Red quantitation. Reduction in PEDF expression in MSCs by siRNA led to decreased ALP activity (33.552 ± 2.009 U/ng protein of knockdown group vs. 39.269 ± 3.533 U/ng protein of scrambled siRNA group, p = .039) and significant reduction in mineral deposition (0.654 ± 0.050 OD/ml per well per 1 × 10(4) cells per square centimeter of knockdown group vs. 1.152 ± 0.132 OD/ml per well per 1 × 10(4) cells per square centimeter of wild-type group, p = .010). Decreased ALP activity and mineral deposition were restored by supplementation with exogenous PEDF protein. PEDF activated ERK and AKT signaling pathways in MSCs to induce expression of osteoblastic-related genes. These data suggest that PEDF is involved in MSCs osteoblastic differentiation.
Assuntos
Calcificação Fisiológica/fisiologia , Proteínas do Olho/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Fatores de Crescimento Neural/metabolismo , Serpinas/metabolismo , Idoso , Animais , Osso e Ossos/citologia , Osso e Ossos/enzimologia , Osso e Ossos/metabolismo , Diferenciação Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Células Cultivadas , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/enzimologia , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Osteoblastos/citologia , Osteoblastos/enzimologia , Osteoblastos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
PURPOSE: The study investigated effectiveness of a novel PEDF peptide mimetic to alleviate dry eye-like pathologies in a Type I diabetic mouse model established using streptozotocin. METHODS: Mice were treated topically for 3-6 weeks with Ppx (a 17-mer PEDF mimetic) 2x/day or vehicle. Corneal sensitivity, tear film, epithelial and endothelial injury were measured using Cochet-Bonnet esthesiometer, phenol red cotton thread wetting, fluorescein sodium staining, and ZO1 expression, respectively. Inflammatory and parasympathetic nerve markers and activation of the MAPK/JNK pathways in the lacrimal glands were measured. RESULTS: Diabetic mice exhibited features of dry eye including reduced corneal sensation and tear secretion and increased corneal epithelium injury, nerve degeneration, and edema. Ppx reversed these pathologies and restored ZO1 expression and morphological integrity of the endothelium. Upregulation of IL-1ß and TNFα, increased activation of P-38, JNK, and ERK, and higher levels of M3ACHR in diabetic lacrimal glands were also reversed by the peptide treatment. CONCLUSION: The study demonstrates that topical application of a synthetic PEDF mimetic effectively alleviates diabetes-induced dry eye by restoring corneal sensitivity, tear secretion, and endothelial barrier and lacrimal gland function. These findings have significant implications for the potential treatment of dry eye using a cost-effective and reproducible approach with minimal invasiveness and no obvious side effects.
Assuntos
Córnea , Diabetes Mellitus Experimental , Síndromes do Olho Seco , Proteínas do Olho , Aparelho Lacrimal , Fatores de Crescimento Neural , Serpinas , Lágrimas , Animais , Camundongos , Proteínas do Olho/metabolismo , Síndromes do Olho Seco/tratamento farmacológico , Síndromes do Olho Seco/patologia , Serpinas/farmacologia , Serpinas/uso terapêutico , Serpinas/administração & dosagem , Fatores de Crescimento Neural/farmacologia , Fatores de Crescimento Neural/uso terapêutico , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Lágrimas/metabolismo , Lágrimas/efeitos dos fármacos , Córnea/efeitos dos fármacos , Córnea/patologia , Córnea/metabolismo , Aparelho Lacrimal/efeitos dos fármacos , Aparelho Lacrimal/metabolismo , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , MasculinoRESUMO
Pigment epithelium-derived factor (PEDF) is a multifunctional protein with antiangiogenic, antioxidative, and anti-inflammatory properties. PEDF is involved in the pathogenesis of diabetic retinopathy, but its direct role in the kidneys remains unclear. We hypothesize that a PEDF fragment (P78-PEDF) confers kidney protection in diabetic nephropathy (DN). The localization of the full-length PEDF protein were determined in DBA mice following multiple low doses of streptozotocin. Using immunohistochemistry, PEDF was localized in the kidney vasculature, interstitial space, glomeruli, tubules, and renal medulla. Kidney PEDF protein and mRNA expression were significantly reduced in diabetic mice. Continuous infusion of P78-PEDF for 6 wk resulted in protection from diabetic neuropathy as indicated by reduced albuminuria and blood urea nitrogen, increased nephrin expression, decreased kidney macrophage recruitment and inflammatory cytokines, and reduced histological changes compared with vehicle-treated diabetic mice. In vitro, P78-PEDF blocked the increase in podocyte permeability to albumin and disruption of the actin cytoskeleton induced by puromycin aminonucleoside treatment. These findings highlight the importance of P78-PEDF peptide as a potential therapeutic modality in early phase diabetic renal injury.
Assuntos
Nefropatias Diabéticas/prevenção & controle , Proteínas do Olho/uso terapêutico , Fatores de Crescimento Neural/uso terapêutico , Fragmentos de Peptídeos/uso terapêutico , Serpinas/uso terapêutico , Albuminúria/prevenção & controle , Animais , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/patologia , Proteínas do Olho/fisiologia , Rim/metabolismo , Masculino , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos DBA , Fatores de Crescimento Neural/fisiologia , Podócitos/efeitos dos fármacos , Serpinas/fisiologiaRESUMO
Inflammation, neurodegeneration and microvascular irregularities are included in the spectrum of defects associated with diabetic retinopathy. Here, we evaluated intraocular deliverability features of two pigment epithelium-derived factor (PEDF) derivatives given as eye drops and their efficacy in modulating diabetes-induced retinal complications. The antiangiogenic PEDF60-77 (P60) and neuroprotective PEDF78-121 (P78) derivatives were applied to Ins2(Akita) mouse eyes once a week for 15 wks at the onset of hyperglycemia. Peptides, labeled with Alexa Fluor 488, were observed penetrating the cornea by 1-4 h and gained access to the ciliary body, retinal pigment epithelium (RPE)-choroid complex, retina microvasculature and vitreous. Peak vitreous levels were 0.2 µg/mL for P60 and 0.9 µg/mL for P78 after 0.5 and 4 h, respectively. Both peptides reduced vascular leakage by ~60% and increased zona occludens 1 (ZO1) and occludin expression in the microvasculature to nondiabetic levels. P60 induced pERK1/2 and P78 promoted pAKT in Muller glia, two signals that were dampened in diabetic conditions. Pharmacologically inhibiting AKT signaling in the retina blocked effects of the peptides on ZO1 and occludin expression. P78 reduced levels of 9/20 cytokines in diabetic vitreous including interferon (IFN)-γ, interleukin (IL)-6, IL-3 and tumor necrosis factor (TNF)-α. P60 lowered levels of 6/20 cytokines but was less effective than P78. Neuroprotective P78 prevented diabetes-induced microglia activation by ~60%, retinal ganglion cell (RGC) death by ~22% and inner plexiform layer thinning by ~13%. In summary, we provide evidence that PEDF bioactive derivatives gained access to the retina by topical delivery and validated their efficacy in reducing diabetic retinopathy complications. Our findings argue for glia regulation of microvascular leakage and an early root cause for RGC degeneration embedded in microglia activation.
Assuntos
Retinopatia Diabética/patologia , Proteínas do Olho/química , Inflamação/tratamento farmacológico , Insulina/metabolismo , Fatores de Crescimento Neural/química , Soluções Oftálmicas/uso terapêutico , Peptídeos/uso terapêutico , Vasos Retinianos/patologia , Serpinas/química , Animais , Morte Celular/efeitos dos fármacos , Retinopatia Diabética/tratamento farmacológico , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Inflamação/complicações , Inflamação/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Maleimidas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neuroglia/efeitos dos fármacos , Neuroglia/enzimologia , Neuroglia/patologia , Soluções Oftálmicas/farmacologia , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Peptídeos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Vasos Retinianos/efeitos dos fármacos , Proteínas de Junções Íntimas/metabolismo , Distribuição Tecidual/efeitos dos fármacos , Corpo Vítreo/efeitos dos fármacos , Corpo Vítreo/metabolismoRESUMO
PURPOSE: To investigate the antioxidative effect and mechanism of pigment epithelium-derived factor (PEDF) on the ocular surface damage in diabetic mice. METHODS: C57BL/6 mice were injected intraperitoneally with streptozocin to generate diabetic models and then 50 nM PEDF or artificial tears were used to treat the diabetic mice. Treatment was given three times a day for eight weeks. Corneal epithelial damage, corneal sensitivity, and tear volume were quantified by fluorescein staining, esthesiometer, and phenol red cotton thread, respectively. Animals were sacrificed at 16 weeks after diabetes and the whole globe specimens were subjected to histochemical staining. Reactive oxygen species (ROS) generation was detected by 2',7-dichlorodihydrofluorescein probe. The levels of receptor for advanced glycation end products (RAGE) and superoxide dismutase 1 (SOD1) were examined by quantitative real-time PCR and western blotting. RESULTS: Topical application of PEDF improved corneal epithelial damage, increased corneal sensitivity, and tear volume in diabetic mice. ROS levels in the cornea were significantly higher in the diabetic mice than in the normal mice. Moreover, PEDF attenuated the accumulation of ROS, decreased the expression of RAGE, and elevated SOD1 expression in the cornea. CONCLUSIONS: Topical application of PEDF can alleviate diabetes-related ocular surface damage and increase tear volume, along with the improvement of oxidative stress status.
Assuntos
Antioxidantes/metabolismo , Córnea/efeitos dos fármacos , Doenças da Córnea/tratamento farmacológico , Diabetes Mellitus Experimental/complicações , Proteínas do Olho/farmacologia , Fatores de Crescimento Neural/farmacologia , Serpinas/farmacologia , Lágrimas/metabolismo , Animais , Córnea/diagnóstico por imagem , Córnea/metabolismo , Doenças da Córnea/etiologia , Doenças da Córnea/metabolismo , Diabetes Mellitus Experimental/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Inibidores de Proteases/farmacologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Purpose: The cornea is richly innervated by the trigeminal ganglion (TG) and its function supported by secretions from the adjacent lacrimal (LG) and meibomian glands (MG). In this study we examined how pigment epithelium-derived factor (PEDF) gene deletion affects the cornea structure and function. Methods: We used PEDF hemizygous and homozygous knockout mice to study effects of PEDF deficiency on corneal innervation assessed by beta tubulin staining, mRNA expression of trophic factors, and PEDF receptors by adjacent supporting glands, corneal sensitivity measured using a Cochet-Bonnet esthesiometer, and tear production using phenol red cotton thread wetting. Results: Loss of PEDF was accompanied by reduced corneal innervation and sensitivity, increased corneal surface injury and tear production, thinning of the corneal stroma and loss of stromal cells. PEDF mRNA was expressed in the cornea and its supporting tissues, the TG, LG, and MG. Deletion of one or both PEDF alleles resulted in decreased expression of essential trophic support in the TG, LG, and MG including nerve growth factor, brain-derived neurotrophic growth factor, and GDNF with significantly increased levels of NT-3 in the LG and decreased EGF expression in the cornea. Decreased transcription of the putative PEDF receptors, adipose triglyceride lipase, lipoprotein receptor-related protein 6, laminin receptor, PLXDC1, and PLXDC2 was also evident in the TG, LG and MG with the first three showing increased levels in corneas of the Pedf+/- and Pedf-/- mice compared to wildtype controls. Constitutive inactivation of ERK1/2 and Akt was pronounced in the TG and cornea, although their protein levels were dramatically increased in Pedf-/- mice. Conclusions: This study highlights an essential role for PEDF in corneal structure and function and confirms the reported rescue of exogenous PEDF treatment in corneal pathologies. The pleiotropic effects of PEDF deletion on multiple trophic factors, receptors and signaling molecules are strong indications that PEDF is a key coordinator of molecular mechanisms that maintain corneal function and could be exploited in therapeutic options for several ocular surface diseases.
Assuntos
Córnea , Doenças da Córnea , Proteínas do Olho , Fatores de Crescimento Neural , Serpinas , Lágrimas/fisiologia , Gânglio Trigeminal , Animais , Córnea/inervação , Córnea/patologia , Córnea/fisiopatologia , Doenças da Córnea/metabolismo , Doenças da Córnea/fisiopatologia , Doenças da Córnea/terapia , Lesões da Córnea/metabolismo , Lesões da Córnea/fisiopatologia , Proteínas do Olho/genética , Proteínas do Olho/farmacologia , Deleção de Genes , Humanos , Camundongos , Camundongos Knockout , Fatores de Crescimento Neural/deficiência , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/farmacologia , Inibidores de Proteases/farmacologia , Receptores de Neuropeptídeos/metabolismo , Serpinas/deficiência , Serpinas/genética , Serpinas/farmacologia , Gânglio Trigeminal/metabolismo , Gânglio Trigeminal/fisiopatologia , Tubulina (Proteína)/metabolismo , Percepção Visual/fisiologiaRESUMO
Oxidative stress due to mitochondrial produced reactive oxygen species is a major cause of damage seen in many retinal degenerative diseases. Caffeic acid phenylethyl ester ï¼CAPEï¼ is protective agent in multiple tissues and is reported to have anti-oxidant properties. Systemically applied CAPE protected retinal ganglion cells from ischemic injury induced by increased intraocular pressure. CAPE provided complete protection for ARPE19 retinal pigment epithelial cells against tert-butyl hydrogen peroxide and reduced both basal and LPS-stimulated ROS production. The major effect of CAPE was mediated by the mitochondrial uncoupling protein UCP2 since both pharmacological inhibition of UCP2 and siRNA-induced knockdown removed the ability of CAPE to block ROS production. Based on common structural features, CAPE may be acting as a mimetic of the natural UCP2 homeostatic regulator 4-hydroxy-2-nonenal. CAPE may provide a valuable tool to treat oxidative stress-related damage in retinal and other degenerative diseases.
Assuntos
Ácidos Cafeicos/farmacologia , Mitocôndrias/efeitos dos fármacos , Neuroproteção/efeitos dos fármacos , Células Ganglionares da Retina/efeitos dos fármacos , Proteína Desacopladora 2/efeitos dos fármacos , Animais , Ésteres/metabolismo , Ésteres/farmacologia , Feminino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Álcool Feniletílico/farmacologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
In the eye, the retinal pigment epithelium (RPE) is exposed to a highly oxidative environment, partly due to elevated oxygen partial pressure from the choriocapillaris and to digestion of polyunsaturated fatty acid laden photoreceptor outer segments. Here we examined the vulnerability of RPE cells to stress and changes in their mitochondria with increased chronological aging and showed that there is greater sensitivity of the cells to oxidative stress, alterations in their mitochondrial number, size, shape, matrix density, cristae architecture, and membrane integrity as a function of age. These features correlate with reduced cellular levels of ATP, ROS, and [Ca(2+)](c), lower Deltapsim, increased [Ca(2+)](m) sequestration and decreased expression of mtHsp70, UCP2, and SOD3. Mitochondrial decay, bioenergetic deficiencies, and weakened antioxidant defenses in RPE cells occur as early as age 62. With increased severity, these conditions may significantly reduce RPE function in the retina and contribute to age related retinal anomalies.
Assuntos
Envelhecimento/patologia , Antioxidantes/metabolismo , Senescência Celular , Mitocôndrias/patologia , Epitélio Pigmentado Ocular/metabolismo , Epitélio Pigmentado Ocular/patologia , Trifosfato de Adenosina/biossíntese , Idoso , Envelhecimento/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/genética , Cálcio/metabolismo , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Criança , Citometria de Fluxo , Fluoresceínas/metabolismo , Fluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/toxicidade , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , Tamanho das Organelas/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Epitélio Pigmentado Ocular/efeitos dos fármacosRESUMO
PURPOSE: Zinc is an essential cofactor for normal cell function. Altered expression and function of zinc transporters may contribute to the pathogenesis of neurodegenerative disorders including macular degeneration. The expression and regulation of zinc transporters in the RPE and the toxicity of zinc to these cells were examined. METHODS: Zinc transporters were identified in a human RPE cell line, ARPE19, using a 28K human array, and their expression was confirmed by PCR, immunocytochemistry, and Western blot analysis in primary human RPE cultures and ARPE19. Zinc toxicity to ARPE19 was determined using monotetrazolium, propidium iodide, and TUNEL assays, and Zn(2+) uptake was visualized with Zinquin ethyl ester. The effect of various growth factors on zinc transporter expression also was examined. RESULTS: Transcripts for 20 of 23 zinc transporters are expressed in fetal human RPE, 16 of 23 in adult human RPE, and 21 of 23 in ARPE19. Zn transporter proteins were also detected in ARPE19. ZnT5 expression was not observed, whereas ZnT6, ZIP1, and ZIP13 were the most abundantly expressed in all RPE samples. The addition of low concentrations of Zn(2+) to cultures resulted in a dose-dependent increase in intracellular Zn(2+) content in ARPE19, and >30 nM Zn(2+) induced necrosis with an LC(50) of 117.4 nM. Brain-derived neurotrophic factor, ciliary neurotrophic factor, glial-derived neurotrophic factor (GDNF), and pigment epithelial-derived neurotrophic factor (PEDF) increased ZIP2 expression, GDNF and PEDF increased ZnT2 expression, and PEDF increased ZnT3 and ZnT8 expression. These neurotrophic factors also promoted Zn(2+) uptake in the RPE. CONCLUSIONS: The array of zinc transporters expressed by the RPE may play a key role in zinc homeostasis in the retina and in ocular health and diseases.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Fatores de Crescimento Neural/farmacologia , Epitélio Pigmentado Ocular/efeitos dos fármacos , Epitélio Pigmentado Ocular/metabolismo , Adulto , Western Blotting , Proteínas de Transporte/genética , Proteínas de Transporte de Cátions/genética , Células Cultivadas , Relação Dose-Resposta a Droga , Técnica Indireta de Fluorescência para Anticorpo , Perfilação da Expressão Gênica , Idade Gestacional , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Microscopia de Fluorescência , Necrose , Análise de Sequência com Séries de Oligonucleotídeos , Quinolonas/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Compostos de Tosil/metabolismo , Zinco/metabolismo , Compostos de Zinco/toxicidadeRESUMO
PURPOSE: There is growing evidence that oxidative stress contributes to the progression of primary open-angle glaucoma (POAG), a leading cause of irreversible blindness worldwide. The authors provide evidence that mitochondrial dysfunction is a possible mechanism for the loss of trabecular meshwork (TM) cells in persons with POAG. METHODS: TM from patients with POAG (GTM) and age-matched subjects without disease (NTM) were obtained by standard surgical trabeculectomy. Primary TM cultures were treated with one of the following mitochondrial respiratory chain inhibitors: rotenone (ROT, complex I inhibitor), thenoyltrifluoroacetone (TTFA, complex II inhibitor), myxothiazol or antimycin A (MYX, AM-complex III inhibitors); mitochondrial permeability transition (MPT) inhibitor cyclosporine A (CsA); and antioxidants vitamin E (Vit E) or N-acetylcysteine (NAC). Mitochondrial function was determined by changes in mitochondrial membrane potential (DeltaPsim) and adenosine triphosphate (ATP) production with the fluorescent probes 5,5',6,6'-tetrachloro-1,1'3,3'-tetraethylbenzimid azolocarbocyanine iodide (JC-1) and a luciferin/luciferase-based ATP assay, respectively. Reactive oxygen species (ROS) level, determined by H(2)-DCF-DA, and cell death, measured by lactate dehydrogenase activity and Annexin V-FITC labeling, were also examined. RESULTS: GTM cells have higher endogenous ROS levels, lower ATP levels, and decreased Delta Psi m and they are more sensitive to mitochondrial complex I inhibition than their normal counterparts. ROT induces a further increase in ROS production, the release of cytochrome c, and decreases in ATP level and Delta Psi m in GTM cells, eventually leading to apoptosis. Complex II and III inhibition had little effect on the cells. Antioxidants protect against ROT-induced death by inhibiting ROS generation and cytochrome c release. CONCLUSIONS: The authors propose that a mitochondrial complex I defect is associated with the degeneration of TM cells in patients with POAG, and antioxidants and MPT inhibitors can reduce the progression of this condition.