RESUMO
Innate immune memory allows macrophages to adequately respond to pathogens to which they have been pre-exposed. To what extent different pattern recognition receptors, cytokines and resolution signals influence innate immune memory needs further elucidation. The present study assessed whether lipopolysaccharide (LPS) tolerance in monocytes and macrophages is affected by these factors. Human CD14+ cells were isolated from peripheral blood, stimulated by LPS and re-stimulated after 3 days of resting. Hereafter, immune-responsive gene 1 (IRG-1), heme oxygenase 1 (HO-1), tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) expression were assessed. Our study revealed the following findings: (1) While pre-stimulation with the Toll-like receptor 4 ligand LPS inhibits the induction of IRG-1, TNF-α and IL-6 expression, pre-stimulation with TLR 1/2 ligands only affects cytokine production but not IRG-1 expression upon subsequent TLR4 engagement. (2) Prior TNF-α stimulation does not affect LPS tolerance but rather increases LPS-mediated cytokine expression. (3) Dimethyl itaconate (DMI) inhibits the expression of IRG-1 in a dose-dependent manner but does not affect TNF-α or IL-6 expression. (4) Docosahexaenoic acid (DHA) partly inhibits IRG-1 expression in monocytes but not in M(IFNγ) and M(IL-4) polarized macrophages. LPS tolerance is not affected in these cells by DHA. The data presented in this study partly corroborate and extend previous findings on innate immune memory and warrant further studies on LPS tolerance to gain a better understanding of innate immune memory at the molecular level.
Assuntos
Lipopolissacarídeos , Monócitos , Humanos , Monócitos/metabolismo , Interleucina-6/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Macrófagos/metabolismo , Citocinas/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Tolerância ImunológicaRESUMO
Cleavage of amyloid precursor protein (APP) by ß-secretase BACE1 initiates the production and accumulation of neurotoxic amyloid-ß peptides, which is widely considered an essential pathogenic mechanism in Alzheimer's disease (AD). Here, we report that BACE1 is essential for normal auditory function. Compared with wild-type littermates, BACE1-/- mice of either sex exhibit significant hearing deficits, as indicated by increased thresholds and reduced amplitudes in auditory brainstem responses (ABRs) and decreased distortion product otoacoustic emissions (DPOAEs). Immunohistochemistry revealed aberrant synaptic organization in the cochlea and hypomyelination of auditory nerve fibers as predominant neuropathological substrates of hearing loss in BACE1-/- mice. In particular, we found that fibers of spiral ganglion neurons (SGN) close to the organ of Corti are disorganized and abnormally swollen. BACE1 deficiency also engenders organization defects in the postsynaptic compartment of SGN fibers with ectopic overexpression of PSD95 far outside the synaptic region. During postnatal development, auditory fiber myelination in BACE1-/- mice lags behind dramatically and remains incomplete into adulthood. We relate the marked hypomyelination to the impaired processing of Neuregulin-1 when BACE1 is absent. To determine whether the cochlea of adult wild-type mice is susceptible to AD treatment-like suppression of BACE1, we administered the established BACE1 inhibitor NB-360 for 6 weeks. The drug suppressed BACE1 activity in the brain, but did not impair hearing performance and, upon neuropathological examination, did not produce the characteristic cochlear abnormalities of BACE1-/- mice. Together, these data strongly suggest that the hearing loss of BACE1 knock-out mice represents a developmental phenotype.SIGNIFICANCE STATEMENT Given its crucial role in the pathogenesis of Alzheimer's disease (AD), BACE1 is a prime pharmacological target for AD prevention and therapy. However, the safe and long-term administration of BACE1-inhibitors as envisioned in AD requires a comprehensive understanding of the various physiological functions of BACE1. Here, we report that BACE1 is essential for the processing of auditory signals in the inner ear, as BACE1-deficient mice exhibit significant hearing loss. We relate this deficit to impaired myelination and aberrant synapse formation in the cochlea, which manifest during postnatal development. By contrast, prolonged pharmacological suppression of BACE1 activity in adult wild-type mice did not reproduce the hearing deficit or the cochlear abnormalities of BACE1 null mice.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Cóclea/metabolismo , Potenciais Evocados Auditivos do Tronco Encefálico , Secretases da Proteína Precursora do Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/genética , Cóclea/fisiologia , Proteína 4 Homóloga a Disks-Large/genética , Proteína 4 Homóloga a Disks-Large/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Bainha de Mielina/metabolismo , Neuregulina-1/genética , Neuregulina-1/metabolismo , Gânglio Espiral da Cóclea/metabolismo , Gânglio Espiral da Cóclea/fisiologiaRESUMO
The aim of this study was to evaluate whether damage to the neurovascular unit in diabetes depends on reactive metabolites such as methylglyoxal (MG), and to assess its impact on retinal gene expression. Male Wistar rats were supplied with MG (50 mM) by drinking water and compared with age-matched streptozotocin-diabetic animals and untreated controls. Retinal damage was evaluated for the accumulation of MG-derived advanced glycation end products, changes in hexosamine and PKC pathway activation, microglial activation, vascular alterations (pericyte loss and vasoregression), neuroretinal function assessed by electroretinogram, and neurodegeneration. Retinal gene regulation was studied by microarray analysis, and transcription factor involvement was identified by upstream regulator analysis. Systemic application of MG by drinking water increased retinal MG to levels comparable with diabetic animals. Elevated retinal MG resulted in MG-derived hydroimidazolone modifications in the ganglion cell layer, inner nuclear layer, and outer nuclear layer, a moderate activation of the hexosamine pathway, a pan-retinal activation of microglia, loss of pericytes, increased formation of acellular capillaries, decreased function of bipolar cells, and increased expression of the crystallin gene family. MG mimics important aspects of diabetic retinopathy and plays a pathogenic role in microglial activation, vascular damage, and neuroretinal dysfunction. In response to MG, the retina induces expression of neuroprotective crystallins.-Schlotterer, A., Kolibabka, M., Lin, J., Acunman, K., Dietrich, N., Sticht, C., Fleming, T., Nawroth, P., Hammes, H.-P. Methylglyoxal induces retinopathy-type lesions in the absence of hyperglycemia: studies in a rat model.
Assuntos
Retinopatia Diabética/induzido quimicamente , Hiperglicemia/fisiopatologia , Aldeído Pirúvico/farmacologia , Retina/efeitos dos fármacos , Animais , Capilares/efeitos dos fármacos , Capilares/metabolismo , Capilares/fisiopatologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Retinopatia Diabética/etiologia , Retinopatia Diabética/metabolismo , Retinopatia Diabética/fisiopatologia , Modelos Animais de Doenças , Produtos Finais de Glicação Avançada/metabolismo , Hiperglicemia/metabolismo , Masculino , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/fisiologia , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Pericitos/fisiologia , Aldeído Pirúvico/metabolismo , Ratos , Ratos Wistar , Retina/metabolismo , Retina/fisiopatologia , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/metabolismo , Vasos Retinianos/fisiopatologia , Estreptozocina/farmacologiaRESUMO
AIMS/HYPOTHESIS: Linagliptin has protective effects on the retinal neurovascular unit but, in proliferative retinopathy, dipeptidyl peptidase 4 (DPP-4) inhibition could be detrimental. The aim of this study was to assess the effect of linagliptin on ischaemia-induced neovascularisation of the retina. METHODS: C57BL/6J and glucagon-like peptide 1 (GLP-1) receptor (Glp1r)-/- mice were subjected to a model of oxygen-induced retinopathy (OIR). Both strains were subcutaneously treated with linagliptin from postnatal days 12 to 16. Non-injected OIR and non-exposed mice served as controls. Capillary proliferations and systemic levels of active GLP-1 were quantified. The effects of linagliptin on vascular endothelial growth factor (VEGF)-induced downstream signalling were assessed in human umbilical vein endothelial cells (HUVECs) using western blot for retinal phosphorylated extracellular signal-regulated kinase (ERK)1/2 and retinal gene expression analyses. RESULTS: Linagliptin treatment led to an increase in active GLP-1 and a decreased number of neovascular nuclei in OIR mice vs controls (-30%, p < 0.05). As the reduction in neovascularisation was similar in both C57BL/6J and Glp1r-/- mice, the anti-angiogenic effects of linagliptin were independent of GLP-1R status. The expression of Vegf (also known as Vegfa) and Hif1a was increased in C57BL/6J OIR mice upon linagliptin treatment (three- vs 1.5-fold, p < 0.05, p < 0.01, respectively). In HUVECs, linagliptin inhibited VEGF-induced increases in mitogen-activated protein kinase (MAPK)/ERK (-67%, p < 0.001) and MAPK/c-Jun N-terminal kinase (JNK) (-13%, p < 0.05) pathway activities. In the retinas of C57BL/6J mice, p-ERK1/2 levels were significantly reduced upon linagliptin treatment (-47%, p < 0.05). CONCLUSIONS/INTERPRETATION: Systemic treatment with linagliptin demonstrated GLP-1R-independent anti-angiogenic effects mediated by an inhibition of VEGF receptor downstream signalling. The specific effects of linagliptin on diabetic retinopathy are of potential benefit for individuals with diabetes, independent of metabolic effects.
Assuntos
Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/metabolismo , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Linagliptina/uso terapêutico , Oxigênio/efeitos adversos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Retinopatia Diabética/etiologia , Modelos Animais de Doenças , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Retina/metabolismo , Retina/patologia , Neovascularização Retiniana/tratamento farmacológico , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Background: Zebrafish (Danio rerio) have been established in recent years as a model organism to study Diabetic Retinopathy (DR). Loss of endothelial cells and pericytes is an early hallmark sign of developing DR in the mammalian retina. However, morphology, numbers, ratios, and distributions of different vascular cells in the retinal compartment in zebrafish have not yet been analyzed and compared with the mammalian retina. Methods: The retinal trypsin digest protocol was established on the zebrafish retina. Cell types were identified using the Tg(nflk:EGFP)-reporter line. Cells were quantified using quantitative morphometry. Results: Vascular cells in the zebrafish retina have distinct morphologies and locations. Nuclei of vascular mural cells appear as long and flat nuclei located near the vessel wall. Round nuclei within the vessel walls can be identified as endothelial cells. The vessel diameter decreases from central to peripheral parts of the retina. Additionally, the numbers of vascular cells decrease from central to peripheral parts of the retina. Discussion: The retinal trypsin digest protocol, which can be applied to the zebrafish retina, provides novel insights into the zebrafish retinal vascular architecture. Quantification of the different cell types shows that, in comparison to the mammalian retina, zebrafish have higher numbers of mural cells and an increased mural cell to endothelial cell ratio. This protocol enables to quantify mural cell and endothelial cell numbers, is easily adaptable to different transgenic and mutant zebrafish lines and will enable investigators to compare novel models on a single cell level.
RESUMO
AIMS: Glucagon-like peptide-1 receptor agonists are effective treatments for type 2 diabetes, effectively lowering glucose without weight gain and with low risk for hypoglycemia. However, their influence on the retinal neurovascular unit remains unclear. In this study, we analyzed the effects of the GLP-1 RA lixisenatide on diabetic retinopathy. METHODS: Vasculo- and neuroprotective effects were assessed in experimental diabetic retinopathy and high glucose-cultivated C. elegans, respectively. In STZ-diabetic Wistar rats, acellular capillaries and pericytes (quantitative retinal morphometry), neuroretinal function (mfERG), macroglia (GFAP western blot) and microglia (immunohistochemistry) quantification, methylglyoxal (LC-MS/MS) and retinal gene expressions (RNA-sequencing) were determined. The antioxidant properties of lixisenatide were tested in C. elegans. RESULTS: Lixisenatide had no effect on glucose metabolism. Lixisenatide preserved the retinal vasculature and neuroretinal function. The macro- and microglial activation was mitigated. Lixisenatide normalized some gene expression changes in diabetic animals to control levels. Ets2 was identified as a regulator of inflammatory genes. In C. elegans, lixisenatide showed the antioxidative property. CONCLUSIONS: Our data suggest that lixisenatide has a protective effect on the diabetic retina, most likely due to a combination of neuroprotective, anti-inflammatory and antioxidative effects of lixisenatide on the neurovascular unit.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Retinopatia Diabética , Ratos , Animais , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/etiologia , Retinopatia Diabética/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Caenorhabditis elegans , Cromatografia Líquida , Ratos Wistar , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Espectrometria de Massas em Tandem , Antioxidantes/farmacologia , GlucoseRESUMO
In atherosclerotic lesions, macrophages are exposed to CSFs and various microenvironmental cues, which ultimately drive their polarization state. We studied the expression of different CSFs in artery specimen and cultured vascular cells and assessed whether concurrent stimulation (CS) of monocytes with CSF1 and polarizing cytokines generated macrophages (CSM1 and CSM2) that were phenotypically and functionally different from classically polarized M1 and M2 macrophages. We also assessed the influence of acetylsalicylic acid (ASA) on the capacity of polarized macrophages to stimulate T-cell proliferation. CSF1 was the most prominent CSF expressed in arteries and cultured vascular cells. M1 and CSM1 macrophages differed in CD86 and CD14 expression, which was up-regulated respectively down-regulated by LPS. M2 and CSM2 macrophages were phenotypically similar. Cyclooxygenase expression was different in CSM1 (COX-1- and COX-2+ after LPS stimulation) and CSM2 (COX-1+ and COX-2- ) macrophages. TNFα production was more pronounced in CSM1 macrophages, whereas IL-10 was produced at higher levels by CSM2 macrophages. Proliferation of allogeneic T cells was strongly supported by CSM2, but not by CSM1 polarized macrophages. Although ASA did not affect anti-CD3/CD28-mediated proliferation, it significantly reduced CSM2 and CSM1-mediated T-cell proliferation. Supernatants of LPS-stimulated CSM2 but not of CSM1 macrophages could overcome the inhibition by ASA. Hence, we demonstrate that CSM1 and CSM2 macrophages are phenotypically and to some extent functionally distinct from classically polarized M1 and M2 macrophages. CSM2 macrophages produce a COX-1-dependent soluble factor that supports T-cell proliferation, the identity hereof is still elusive and warrants further studies.
Assuntos
Citocinas , Monócitos , Diferenciação Celular , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Monócitos/metabolismoRESUMO
Rationale: Vasoregression secondary to glial activation develops in various retinal diseases, including retinal degeneration and diabetic retinopathy. Photoreceptor degeneration and subsequent retinal vasoregression, characterized by pericyte loss and acellular capillary formation in the absence diabetes, are also seen in transgenic rats expressing the polycystic kidney disease (PKD) gene. Activated Müller glia contributes to retinal vasodegeneration, at least in part via the expression of the soluble epoxide hydrolase (sEH). Given that an increase in sEH expression triggered vascular destabilization in diabetes, and that vasoregression is similar in diabetic mice and PKD rats, the aim of the present study was to determine whether sEH inhibition could prevent retinal vasoregression in the PKD rat. Methods: One-month old male homozygous transgenic PKD rats were randomly allocated to receive vehicle or a sEH inhibitor (sEH-I; Sar5399, 30 mg/kg) for four weeks. Wild-type Sprague-Dawley (SD) littermates received vehicle as controls. Retinal sEH expression and activity were measured by Western blotting and LC-MS, and vasoregression was quantified in retinal digestion preparations. Microglial activation and immune response cytokines were assessed by immunofluorescence and quantitative PCR, respectively. 19,20-dihydroxydocosapentaenoic acid (19,20-DHDP) mediated Notch signaling, microglial activation and migration were assessed in vivo and in vitro. Results: This study demonstrates that sEH expression and activity were increased in PKD retinae, which led to elevated production of 19,20-DHDP and the depression of Notch signaling. The latter changes elicited pericyte loss and the recruitment of CD11b+/CD74+ microglia to the perivascular region. Microglial activation increased the expression of immune-response cytokines, and reduced levels of Notch3 and delta-like ligand 4 (Dll4). Treatment with Sar5399 decreased 19,20-DHDP generation and increased Notch3 expression. Sar5399 also prevented vasoregression by reducing pericyte loss and suppressed microglial activation as well as the expression of immune-response cytokines. Mechanistically, the activation of Notch signaling by Dll4 maintained a quiescent microglial cell phenotype, i.e. reduced both the surface presentation of CD74 and microglial migration. In contrast, in retinal explants, 19,20-DHDP and Notch inhibition both promoted CD74 expression and reversed the Dll4-induced decrease in migration. Conclusions: Our data indicate that 19,20-DHDP-induced alterations in Notch-signaling result in microglia activation and pericyte loss and contribute to retinal vasoregression in polycystic kidney disease. Moreover, sEH inhibition can ameliorate vasoregression through reduced activity of inflammatory microglia. sEH inhibition is thus an attractive new therapeutic approach to prevent retinal vasoregression.
Assuntos
Epóxido Hidrolases/antagonistas & inibidores , Doenças Renais Policísticas/complicações , Degeneração Retiniana/tratamento farmacológico , Vasos Retinianos/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Epóxido Hidrolases/metabolismo , Ácidos Graxos Insaturados/metabolismo , Humanos , Masculino , Microglia/efeitos dos fármacos , Microglia/imunologia , Doenças Renais Policísticas/genética , Ratos , Ratos Transgênicos , Retina/citologia , Retina/efeitos dos fármacos , Retina/imunologia , Retina/patologia , Degeneração Retiniana/genética , Degeneração Retiniana/imunologia , Degeneração Retiniana/patologia , Vasos Retinianos/patologia , Canais de Cátion TRPP/genéticaRESUMO
AIMS: Low levels of reactive oxygen species and resulting oxidative protein modifications may play a beneficial role in cellular function under stress conditions. Here we studied the influence of age-dependent protein carbonylation on expression and activity of the anti-oxidative selenoenzyme glutathione peroxidase (GPx) in insulin-deficient Ins2Akita mice and type 2 diabetic obese db/db mice in context of diabetic nephropathy. METHODS: Protein carbonylation, GPx expression and activity were examined in kidney tissue and lysates by common histological and protein biochemical methods. RESULTS: In kidneys of Ins2Akita mice, carbonylated proteins, GPx-1 and GPx-4 expression were mainly detected in podocytes and mesangial cells. GPx activity was increased in kidney cortex homogenates of these mice. Remarkably, young animals did not show a concomitant increase in GPx expression but enhanced GPx carbonylation. No carbonylation-dependent modification of GPx activity was detected in db/db mice. In cultured podocytes hyperglycemia induced an increase in GPx expression but had no effect on activity or carbonylation. In kidney tissue sections of type 1 or type 2 diabetes patients, GPx-1 and GPx-4 expression but not overall protein carbonylation was significantly decreased. CONCLUSIONS: These results indicate the existence of a threshold for beneficial carbonylation-dependent redox signaling during the progression of diabetic nephropathy.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Glutationa Peroxidase/metabolismo , Glomérulos Renais/metabolismo , Podócitos/metabolismo , Carbonilação Proteica/fisiologia , Fatores Etários , Idoso , Animais , Estudos de Casos e Controles , Células Cultivadas , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Glomérulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Estresse Oxidativo/fisiologia , Podócitos/patologiaRESUMO
OBJECTIVE: Diabetic retinopathy (DR) is induced by an accumulation of reactive metabolites such as ROS, RNS, and RCS species, which were reported to modulate the activity of cation channels of the TRPC family. In this study, we use Trpc1/4/5/6-/- compound knockout mice to analyze the contribution of these TRPC proteins to diabetic retinopathy. METHODS: We used Nanostring- and qPCR-based analysis to determine mRNA levels of TRPC channels in control and diabetic retinae and retinal cell types. Chronic hyperglycemia was induced by Streptozotocin (STZ) treatment. To assess the development of diabetic retinopathy, vasoregression, pericyte loss, and thickness of individual retinal layers were analyzed. Plasma and cellular methylglyoxal (MG) levels, as well as Glyoxalase 1 (GLO1) enzyme activity and protein expression, were measured in WT and Trpc1/4/5/6-/- cells or tissues. MG-evoked toxicity in cells of both genotypes was compared by MTT assay. RESULTS: We find that Trpc1/4/5/6-/- mice are protected from hyperglycemia-evoked vasoregression determined by the formation of acellular capillaries and pericyte drop-out. In addition, Trpc1/4/5/6-/- mice are resistant to the STZ-induced reduction in retinal layer thickness. The RCS metabolite methylglyoxal, which represents a key mediator for the development of diabetic retinopathy, was significantly reduced in plasma and red blood cells (RBCs) of STZ-treated Trpc1/4/5/6-/- mice compared to controls. GLO1 is the major MG detoxifying enzyme, and its activity and protein expression were significantly elevated in Trpc1/4/5/6-deficient cells, which led to significantly increased resistance to MG toxicity. GLO1 activity was also increased in retinal extracts from Trpc1/4/5/6-/- mice. The TRPCs investigated here are expressed at different levels in endothelial and glial cells of the retina. CONCLUSION: The protective phenotype in diabetic retinopathy observed in Trpc1/4/5/6-/- mice is suggestive of a predominant action of TRPCs in Müller cells and microglia because of their central position in the retention of a proper homoeostasis of the neurovascular unit.
Assuntos
Retinopatia Diabética/metabolismo , Lactoilglutationa Liase/metabolismo , Aldeído Pirúvico/metabolismo , Canais de Cátion TRPC/genética , Animais , Células Cultivadas , Retinopatia Diabética/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Aldeído Pirúvico/sangue , Retina/metabolismo , Canais de Cátion TRPC/metabolismoRESUMO
Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.
Assuntos
Diabetes Mellitus Experimental/diagnóstico , Diabetes Mellitus Experimental/fisiopatologia , Microdissecção/métodos , Retina/patologia , Neovascularização Retiniana/patologia , Animais , Modelos Animais de Doenças , Humanos , Peixe-ZebraRESUMO
BACKGROUND/AIMS: Dipeptidyl peptidase 4 (DPP4) inhibitors improve glycemic control in type 2 diabetes, however, their influence on the retinal neurovascular unit remains unclear. METHODS: Vasculo- and neuroprotective effects were assessed in experimental diabetic retinopathy and high glucose-cultivated C. elegans, respectively. In STZ-diabetic Wistar rats (diabetes duration of 24 weeks), DPP4 activity (fluorometric assay), GLP-1 (ELISA), methylglyoxal (LC-MS/MS), acellular capillaries and pericytes (quantitative retinal morphometry), SDF-1a and heme oxygenase-1 (ELISA), HMGB-1, Iba1 and Thy1.1 (immunohistochemistry), nuclei in the ganglion cell layer, GFAP (western blot), and IL-1beta, Icam1, Cxcr4, catalase and beta-actin (quantitative RT-PCR) were determined. In C. elegans, neuronal function was determined using worm tracking software. RESULTS: Linagliptin decreased DPP4 activity by 77% and resulted in an 11.5-fold increase in active GLP-1. Blood glucose and HbA1c were reduced by 13% and 14% and retinal methylglyoxal by 66%. The increase in acellular capillaries was diminished by 70% and linagliptin prevented the loss of pericytes and retinal ganglion cells. The rise in Iba-1 positive microglia was reduced by 73% with linagliptin. In addition, the increase in retinal Il1b expression was decreased by 65%. As a functional correlate, impairment of motility (body bending frequency) was significantly prevented in C. elegans. CONCLUSION: Our data suggest that linagliptin has a protective effect on the microvasculature of the diabetic retina, most likely due to a combination of neuroprotective and antioxidative effects of linagliptin on the neurovascular unit.
Assuntos
Retinopatia Diabética/prevenção & controle , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Hipoglicemiantes/uso terapêutico , Linagliptina/uso terapêutico , Animais , Glicemia/metabolismo , Caenorhabditis elegans/efeitos dos fármacos , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Hipoglicemiantes/farmacocinética , Linagliptina/farmacocinética , Masculino , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Aldeído Pirúvico/metabolismo , Ratos , Ratos Wistar , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismoRESUMO
Obese Zucker diabetic fatty (ZDF) rats are used as a type-2 diabetes model for microvascular complications. In order to study retinopathy in this model, changes in retinal vasculature were analyzed by quantitative morphometry and related to retinal expression of 46 selected genes that were analyzed by microfluidic card PCR technology. At 3 months of age, obese animals had developed stable hyperglycemia (20.7 ± 1.3 mmol/L plasma glucose vs. 6.5 ± 0.1 mmol/L in lean). Hyperinsulinemia initially presented in obese rats at 2 months (10.5 ± 0.7 µg/L plasma insulin vs. 0.2 ± 0.04 µg/L in lean) and decreased at 3 months (3.9 ± 0.6 vs. 0.5 ± 0.09 µg/ml in lean). At 8 months of age, animals had developed microvascular complications. An increased number of acellular capillaries in obese (24 ± 5/mm(2)) versus lean (15 ± 4/mm(2)) and a decreased number of retinal pericytes in obese (2,270 ± 250/mm(2)) versus lean animals (1,620 ± 243/mm(2)) could be observed. VEGFa, MIF, and HIF-1α were the most abundantly expressed and inflammatory genes such as TNFα and IL-6 are the least abundantly expressed genes. None of these genes were differentially regulated. Surprisingly, specific growth factors such as bFGF (FGF2) and placental growth factor, and adhesion molecules such as ICAM-1 were abundantly expressed and up-regulated in diabetic versus non-diabetic ZDF rats. In summary, we observed in type-2 diabetic ZDF rats retinopathy with retinal vasoregression along with a simultaneous up-regulation of specific growth factors such as bFGF and adhesion molecules, but only minor changes in key inflammatory genes.
Assuntos
Diabetes Mellitus Tipo 2/complicações , Retinopatia Diabética/genética , Retinopatia Diabética/imunologia , Retina/imunologia , Animais , Retinopatia Diabética/etiologia , Modelos Animais de Doenças , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/imunologia , Humanos , Interleucina-6/genética , Interleucina-6/imunologia , Masculino , Ratos , Ratos Zucker , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
Diabetes induces vasoregression, neurodegeneration and glial activation in the retina. Formation of advanced glycation endoproducts (AGEs) is increased in diabetes and contributes to the pathogenesis of diabetic retinopathy. CD74 is increased in activated microglia in a rat model developing both neurodegeneration and vasoregression. In this study, we aimed at investigating whether glucose and major AGE precursor methylglyoxal induce increased CD74 expression in the retina. Expression of CD74 in retinal microglia was analyzed in streptozotocin-diabetic rats by wholemount immunofluorescence. Nondiabetic mice were intravitreally injected with methylglyoxal. Expression of CD74 was studied by retinal wholemount immunofluorescence and quantitative real-time PCR, 48 h after the injection. CD74-positive cells were increased in diabetic 4-month retinas. These cells represented a subpopulation of CD11b-labeled activated microglia and were mainly located in the superficial vascular layer (13.7-fold increase compared to nondiabetic group). Methylglyoxal induced an 9.4-fold increase of CD74-positive cells in the superficial vascular layer and elevated gene expression of CD74 in the mouse retina 2.8-fold. In summary, we identified CD74 as a microglial activation marker in the diabetic retina. Exogenous methylglyoxal mimics the response in normoglycemic retina. This suggests that methylglyoxal is important in mediating microglial activation in the diabetic retina.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Retinopatia Diabética/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Microglia/metabolismo , Aldeído Pirúvico/metabolismo , Retina/metabolismo , Animais , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos CD11/genética , Antígenos CD11/metabolismo , Retinopatia Diabética/genética , Feminino , Glucose/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-DawleyRESUMO
Early retinal vascular changes in the development of diabetic retinopathy (DR) include capillary basal lamina (BL) thickening, pericyte loss and the development of acellular capillaries. Expression of the CCN (connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed) family member CCN2 or connective tissue growth factor (CTGF), a potent inducer of the expression of BL components, is upregulated early in diabetes. Diabetic mice lacking one functional CTGF allele (CTGFâº/â») do not show this BL thickening. As early events in DR may be interrelated, we hypothesized that CTGF plays a role in the pathological changes of retinal capillaries other than BL thickening. We studied the effects of long-term (6-8 months) streptozotocin-induced diabetes on retinal capillary BL thickness, numbers of pericytes and the development of acellular capillaries in wild type and CTGFâº/â» mice. Our results show that an absence of BL thickening of retinal capillaries in long-term diabetic CTGFâº/â» mice is associated with reduced pericyte dropout and reduced formation of acellular capillaries. We conclude that CTGF is involved in structural retinal vascular changes in diabetic rodents. Inhibition of CTGF in the eye may therefore be protective against the development of DR.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Diabetes Mellitus Experimental/patologia , Vasos Retinianos/patologia , Animais , Capilares/patologia , Fator de Crescimento do Tecido Conjuntivo/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Células Endoteliais/patologia , Feminino , Haploinsuficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pericitos/patologia , Vasos Retinianos/metabolismo , Fatores de TempoRESUMO
Retinal digestion is a commonly used method for studying experimental diabetic retinopathy in animal models. The method allows to assess qualitatively and quantitatively the morphology of the retinal vasculature, including characteristics of endothelial cells and pericytes. The digestion method uses the enzyme trypsin and enables the precise evaluation of venolar and arteriolar diameters, endothelial cell and pericyte numbers, and the formation of acellular capillaries.