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Purpose: We aimed to determine the impact of artificial sweeteners (AS), especially saccharin, on the progression and treatment efficacy of patients with neovascular age-related macular degeneration (nAMD) under anti-vascular endothelial growth factor (anti-VEGF-A) treatment. Methods: In a cross-sectional study involving 46 patients with nAMD undergoing intravitreal anti-VEGF therapy, 6 AS metabolites were detected in peripheral blood using liquid chromatography - tandem mass spectrometry (LC-MS/MS). Disease features were statistically tested against these metabolite levels. Additionally, a murine choroidal neovascularization (CNV) model, induced by laser, was used to evaluate the effects of orally administered saccharin, assessing both imaging outcomes and gene expression patterns. Polymerase chain reaction (PCR) methods were used to evaluate functional expression of sweet taste receptors in a retinal pigment epithelium (RPE) cell line. Results: Saccharin levels in blood were significantly higher in patients with well-controlled CNV activity (P = 0.004) and those without subretinal hyper-reflective material (P = 0.015). In the murine model, saccharin-treated mice exhibited fewer leaking laser scars, lesser occurrence of bleeding, smaller fibrotic areas (P < 0.05), and a 40% decrease in mononuclear phagocyte accumulation (P = 0.06). Gene analysis indicated downregulation of inflammatory and VEGFR-1 response genes in the treated animals. Human RPE cells expressed taste receptor type 1 member 3 (TAS1R3) mRNA and reacted to saccharin stimulation with changes in mRNA expression. Conclusions: Saccharin appears to play a protective role in patients with nAMD undergoing intravitreal anti-VEGF treatment, aiding in better pathological lesion control and scar reduction. The murine study supports this observation, proposing saccharin's potential in mitigating pathological VEGFR-1-induced immune responses potentially via the RPE sensing saccharin in the blood stream.
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Neovascularización Coroidal , Degeneración Macular , Humanos , Ratones , Animales , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Sacarina/uso terapéutico , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Edulcorantes , Estudios Transversales , Cromatografía Liquida , Espectrometría de Masas en Tándem , Neovascularización Coroidal/metabolismo , Degeneración Macular/metabolismo , ARN Mensajero/genética , Inyecciones Intravítreas , Inhibidores de la Angiogénesis/uso terapéuticoRESUMEN
BACKGROUND: The role of CD133 und ABCB5 is discussed in treatment resistance in several types of cancer. The objective of this study was to evaluate whether CD133+/ABCB5+ colocalization differs in untreated, in beam radiation treated, and in chemotherapy treated retinoblastoma specimens. Additionally, CD133, ABCB5, sphingosine kinase 1, and sphingosine kinase 2 gene expression was analyzed in WERI-RB1 (WERI RB1) and etoposide-resistant WERI RB1 subclones (WERI ETOR). METHODS: Active human untreated retinoblastoma specimens (n = 12), active human retinoblastoma specimens pretreated with beam radiation before enucleation (n = 8), and active human retinoblastoma specimens pretreated with chemotherapy before enucleation (n = 7) were investigated for localization and expression of CD133 and ABCB5 by immunohistochemistry. Only specimens with IIRC D, but not E, were included in this study. Furthermore, WERI RB1 and WERI ETOR cell lines were analyzed for CD133, ABCB5, sphingosine kinase 1, and sphingosine kinase 2 by the real-time polymerase chain reaction (RT-PCR). RESULTS: Immunohistochemical analysis revealed the same amount of CD133+/ABCB5+ colocalization islets in untreated and treated human retinoblastoma specimens. Quantitative RT-PCR analysis showed a statistically significant upregulation of CD133 in WERI ETOR (p = 0.002). No ABCB5 expression was detected in WERI RB1 and WERI ETOR. On the other hand, SPHK1 (p = 0.0027) and SPHK2 (p = 0.017) showed significant downregulation in WERI ETOR compared to WERI RB1. CONCLUSIONS: CD133+/ABCB5+ co-localization islets were noted in untreated and treated human retinoblastoma specimens. Therefore, we assume that CD133+/ABCB5+ islets might play a role in retinoblastoma genesis, but not in retinoblastoma treatment resistance.
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Neoplasias de la Retina , Retinoblastoma , Humanos , Retinoblastoma/genética , Retinoblastoma/tratamiento farmacológico , Retinoblastoma/metabolismo , Etopósido/uso terapéutico , Neoplasias de la Retina/genéticaRESUMEN
Diabetic retinopathy is characterized by dysfunction of the retinal vascular network, combined with a persistent low-grade inflammation that leads to vision-threatening complications. Netrin-4 (NTN4) is a laminin-related secreted protein and guidance cue molecule present in the vascular basal membrane and highly expressed in the retina. A number of studies inferred that the angiogenic abilities of NTN4 could contribute to stabilize vascular networks and modulate inflammation. Analyzing human specimens, we show that NTN4 and netrin receptors are upregulated in the diabetic retina. We further evaluated a knock-out model for NTN4 undergoing experimental diabetes induced by streptozotocin. We investigated retina function and immune cells in vivo and demonstrated that NTN4 provides a protective milieu against inflammation in the diabetic retina and prevents cytokine production.
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Retinopatía Diabética/genética , Netrinas/genética , Retinitis/metabolismo , Animales , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/genética , Electrorretinografía , Regulación de la Expresión Génica , Humanos , Ratones Transgénicos , Netrinas/metabolismo , Retina/patología , Retina/fisiología , Retinitis/etiologíaRESUMEN
Tumor necrosis factor (TNF)α is an inflammatory cytokine likely to be involved in the process of corneal inflammation and neovascularization. In the present study we evaluate the role of the two receptors, TNF-receptor (TNF-R)p55 and TNF-Rp75, in the mouse model of suture-induced corneal neovascularization and lymphangiogenesis. Corneal neovascularization and lymphangiogenesis were induced by three 11-0 intrastromal corneal sutures in wild-type (WT) C57BL/6J mice and TNF-Rp55-deficient (TNF-Rp55d) and TNF-Rp75-deficient (TNF-Rp75d) mice. The mRNA expression of VEGF-A, VEGF-C, Lyve-1 and TNFα and its receptors was quantified by qPCR. The area covered with blood- or lymphatic vessels, respectively, was analyzed by immunohistochemistry of corneal flatmounts. Expression and localization of TNFα and its receptors was assessed by immunohistochemistry of sagittal sections and Western Blot. Both receptors are expressed in the murine cornea and are not differentially regulated by the genetic alteration. Both TNF-Rp55d and TNF-Rp75d mice showed a decrease in vascularized area compared to wild-type mice 14 days after suture treatment. After 21 days there were no differences detectable between the groups. The number of VEGF-A-expressing macrophages did not differ when comparing WT to TNF-Rp55d and TNF-Rp75d. The mRNA expression of lymphangiogenic markers VEGF-C or LYVE-1 does not increase after suture in all 3 groups and lymphangiogenesis showed a delayed effect only for TNF-Rp75d. TNFα mRNA and protein expression increased after suture treatment but showed no difference between the three groups. In the suture-induced mouse model, TNFα and its ligands TNF-Rp55 and TNF-Rp75 do not play a significant role in the pathogenesis of neovascularisation and lymphangiogenesis.
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Córnea/patología , Neovascularización de la Córnea/genética , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Animales , Córnea/metabolismo , Neovascularización de la Córnea/patología , Eliminación de Gen , Humanos , Linfangiogénesis , Ratones Endogámicos C57BL , ARN Mensajero/genética , Receptores Tipo I de Factores de Necrosis Tumoral/análisis , Receptores Tipo II del Factor de Necrosis Tumoral/análisisRESUMEN
PURPOSE: Recent studies indicate that FLASH irradiation, which involves ultra-high dose rates in a short time window (usually >40 Gy/s in <500 ms), might be equally efficient against tumors but less harmful to healthy tissues, compared to conventional irradiation with the same total dose. Aiming to verify the latter claim for ocular proton radiotherapy, in vivo experiments with mice are being carried out by Charité - Universitätsmedizin Berlin. This work presents the implemented setup for delivering FLASH proton radiation to a single eye of mice at the Helmholtz-Zentrum Berlin für Materialien und Energie (HZB). MATERIALS AND METHODS: The HZB cyclotron is tuned to provide a high-intensity 68 MeV focused proton beam. Outside the vacuum beamline, the protons hit a single scatterer, which also serves as range shifter, and a rotating modulator wheel, which produces a flat depth-dose distribution. Two transmission ionization chambers in between, read out by fast electronics, are used as dose monitors for triggering an in-vacuum beam shutter, which blocks the beam once the desired dose has been delivered. A collimating aperture shapes the radiation field at the isocenter, which is measured by a radioluminescent screen and a CCD camera. At the same position, a parallel-plate ionization chamber of type Advanced Markus® is used for absolute dosimetry and characterization of the spread-out Bragg peak inside a water phantom. A thin-foil mirror of adjustable tilt in the beam path assists the correct alignment of the target through side illumination. Radiochromic films of type EBT3 are used to supplement the dosimetry and assist the alignment. RESULTS: A dose rate of 75 Gy/s has been measured, delivering within 200 ms 15 Gy (RBE) with a reproducibility better than ±1%. A depth-dose curve with a range of 5.2 mm in water, 0.9 mm distal fall-off (90%-10%), and ±2.5% ripple has been demonstrated, with a PTV of 6.3 mm diameter, 1.7 mm lateral penumbra (90%-10%), 8% uniformity, and 3% symmetry. CONCLUSIONS: The implemented setup is able to accommodate ocular irradiation of narcotized mice with protons, targeting selectively the left or the right eye, under conventional and FLASH conditions. Switching between these two modes can be done within half an hour, including the calibration of the dose monitors and the verification of the dose delivery. Further upgrades are planned after the completion of the on-going experiment.
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Terapia de Protones , Animales , Ratones , Fantasmas de Imagen , Protones , Radiometría , Dosificación Radioterapéutica , Reproducibilidad de los ResultadosRESUMEN
The article "Lack of netrin-4 alters vascular remodeling in the retina".
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PURPOSE: Netrin-4 (NTN4) is a protein that plays an important role in the regulation of angiogenesis in the pathological retina. Some evidences show that it can also have a role in inflammation and vascular stability. We will explore these questions in vivo in the mature mouse retina. METHODS: We created a NTN4 knockout that expresses EGFP in mononuclear phagocytes (CSFR1-positive cells) to track inflammation in vivo in the retina by scanning laser ophthalmoscopy (SLO). Fundus angiography permitted to study blood vessels. Retinal function was assessed with electroretinography (ERG). RESULTS: Lack of NTN4 leads to an increased amount of amoeboid mononuclear phagocytes in the adult retina, and blood vessels displayed increased tortuosity when compared with the wildtype. Inner retina function also seemed affected in NTN4 null. Lack of NTN4 resulted in a higher persistence of hyaloid artery and spontaneous leakage in the adult retina. No differences were found regarding vessel bifurcation, vessel width, or vein/artery ratio. CONCLUSIONS: These in vivo data show for the first time that lack of NTN4 induces changes in the retinal vascular phenotype in a non-pathological scenario. This evidence widens the role of NTN4 as a guidance cue in vascular remodeling.
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Netrinas/metabolismo , Neovascularización Retiniana/metabolismo , Vasos Retinianos/metabolismo , Remodelación Vascular/fisiología , Animales , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Electrorretinografía , Angiografía con Fluoresceína , Fondo de Ojo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oftalmoscopía , Neovascularización Retiniana/patología , Neovascularización Retiniana/fisiopatología , Vasos Retinianos/patología , Vasos Retinianos/fisiopatologíaRESUMEN
PURPOSE: To investigate the influence of the selective Rho-kinase (ROCK) inhibitor, fasudil, on the mRNA level of proinflammatory factors and the retinal vascular development in mice with oxygen-induced retinopathy (OIR). METHODS: C57BL/6J mice underwent standard protocol for OIR induction from postnatal days 7 to 12. Subsequently, they received a daily intraperitoneal injection of fasudil or sodium chloride from P12 to P16. Analyses were performed using vascular staining on retinal flat mounts, RNA expression by qPCR, and immunohistochemistry on paraffin sections. RESULTS: On retinal flat mounts, the proportion of avascular area and tuft formation did not differ between the fasudil and NaCl group. Immunohistochemical staining revealed a less intense staining with inflammatory markers after fasudil. Nevertheless, there were no differences on RNA level between the two groups. CONCLUSIONS: In conclusion, our findings support that daily systemic application of fasudil does not decrease retinal neovascularization in rodents with oxygen-induced retinopathy. The results of our study together with the controversial results on the effects of different ROCK inhibitors from the literature makes it apparent that effects of ROCK inhibition are more complex, and further studies are necessary to analyze its potential therapeutic effects.
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1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Enfermedades de la Retina/tratamiento farmacológico , Quinasas Asociadas a rho/antagonistas & inhibidores , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Oxígeno/toxicidad , Inhibidores de Proteínas Quinasas/farmacología , Retina/efectos de los fármacos , Enfermedades de la Retina/inducido químicamente , Enfermedades de la Retina/enzimología , Resultado del Tratamiento , Quinasas Asociadas a rho/metabolismoRESUMEN
Changes in cell function occur by specific patterns of intracellular Ca2+, activating Ca2+-sensitive proteins. The anoctamin (TMEM16) protein family has Ca2+-dependent ion channel activity, which provides transmembrane ion transport, and/or Ca2+-dependent phosphatidyl-scramblase activity. Using amino acid sequence analysis combined with measurements of ion channel function, we clarified the so far unknown Ano4 function as Ca2+-dependent, non-selective monovalent cation channel; heterologous Ano4 expression in HEK293 cells elicits Ca2+ activated conductance with weak selectivity of K+ > Na+ > Li+. Endogenously expressed Ca2+-dependent cation channels in the retinal pigment epithelium were identified as Ano4 by KO mouse-derived primary RPE cells and siRNA against Ano4. Exchanging a negatively charged amino acid in the putative pore region (AA702-855) into a positive one (E775K) turns Ano4-elicited currents into Cl- currents evidencing its importance for ion selectivity. The molecular identification of Ano4 as a Ca2+-activated cation channel advances the understanding of its role in Ca2+ signaling.
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Anoctaminas/metabolismo , Canales de Calcio/metabolismo , Calcio/metabolismo , Cationes/metabolismo , Animales , Anoctaminas/genética , Canales de Calcio/genética , Células HEK293 , Humanos , Ratones , Ratones NoqueadosAsunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Isquemia/tratamiento farmacológico , Células Mieloides/efectos de los fármacos , Enfermedades de la Retina/tratamiento farmacológico , Vasos Retinianos/patología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Humanos , Isquemia/diagnóstico , Células Mieloides/patología , Enfermedades de la Retina/diagnósticoRESUMEN
PURPOSE: To assess the influence of retinal macrophages and microglia on the formation of choroidal neovascularization (CNV). Therefore, we used a transgenic mouse (CD11b-HSVTK) in which the application of ganciclovir (GCV) results in a depletion of CD11b+ cells. METHODS: We first investigated if a local depletion of CD11b+ macrophages and microglia in the retina is feasible. In a second step, the influence of CD11b+ cell depletion on CNV formation was analysed. One eye of each CD11b-HSVTK mouse was injected with GCV, and the fellow eye received sodium chloride solution (NaCl). Cell counting was performed at day 3 and 7 (one injection) or at day 14 and 21 (two injections). Choroidal neovascularization (CNV) was induced by argon laser and analysed at day 14. RESULTS: The most effective CD11b+ cell depletion was achieved 7 days after a single injection and 14 days after two injections of GCV. After two injections of GCV, we found a significant reduction of CD11b+ cells in central (52 ± 23.9 cells/mm2 ) and peripheral retina (53 ± 20.6 cells/mm2 ); compared to eyes received NaCl (216 ± 49.0 and 210 ± 50.5 cells/mm2 , p < 0.001, respectively). Regarding CNV areas, no statistical significance was found between the groups. CONCLUSION: The CD11b-HSVTK mouse is a feasible model for a local depletion of CD11b+ cells in the retina. Nevertheless, only a partial depletion of CD11b+ cells could be achieved compared to baseline data without any intravitreal injections. Our results did not reveal a significant reduction in CNV areas. In the light of previous knowledge, the potential influence of systemic immune cells on CNV formation might be more relevant than expected.
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Antígeno CD11b/fisiología , Neovascularización Coroidal/fisiopatología , Modelos Animales de Enfermedad , Macrófagos/inmunología , Microglía/inmunología , Animales , Antivirales/farmacología , Proteínas de Unión al Calcio/fisiología , Recuento de Células , Neovascularización Coroidal/metabolismo , Femenino , Ganciclovir/farmacología , Inyecciones Intravítreas , Coagulación con Láser , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos/fisiología , Microglía/patología , Reacción en Cadena de la Polimerasa , Simplexvirus/enzimología , Timidina Quinasa/genéticaRESUMEN
The dataset presented in this article complements the article entitled "Myeloid cells contribute indirectly to VEGF expression upon hypoxia via activation of Müller cells" (C. Nürnberg, N. Kociok, C. Brockmann, T. Lischke, S. Crespo-Garcia, N. Reichhart, S. Wolf, R. Baumgrass, S.A. Eming, S. Beer-Hammer, and A.M. Joussen). This complementary dataset provides further insight into the experimental validation of the VEGFfl/fl LysMCre (here named VEGFmcko) knockout model used in the main article through genomic and quantitative Real-Time PCR in various murine tissues as well as additional flow cytometry data and immunohistochemical stainings. By providing these data, we aim to enable researcher to reproduce and critically analyze our data.
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PURPOSE: The model of oxygen-induced retinopathy (OIR) is widely used to analyze pathomechanisms in retinal neovascularization. Previous studies have shown that macrophages (MP) play a key role in vessel formation in OIR, the influence of microglia (MG) having been discussed. The aim of our study was to analyze the spatial and temporal distribution and activation of MP/MG expressing CD115 and CD11b during the process of neovascularization in OIR. METHODS: We used MacGreen mice expressing the green fluorescence protein (GFP) under the promoter for CD115. CD115+ cells were investigated in vivo by scanning laser ophthalmoscopy at postnatal days (P) 17 and 21 in MacGreen mice with OIR (75% oxygen from P7 to P12), and were compared to MacGreen room-air controls. In addition MP/MG were examined ex vivo using immunohistochemistry for CD11b+ detection on retinal flatmounts at P14, P17, and P21 of wild type mice with OIR. RESULTS: In-vivo imaging revealed the highest density of activated MP/MG in tuft areas at P17 of MacGreen mice with OIR. Tufts and regions with a high density of CD115+ cells were detected close to veins, rather to arteries. In peripheral, fully vascularized areas, the distribution of CD115+ cells in MacGreen mice with OIR was similar to MacGreen room-air controls. Correspondingly, immunohistochemical analyses of retinal flatmounts from wild type mice with OIR induction revealed that the number of CD11b+ cells significantly varies between vascular, avascular, and tuft areas as well as between the retinal layers. Activated CD11b+ cells were almost exclusively found in avascular areas and tufts of wild type mice with OIR induction; here, the proportion of activated cells related to the total number of CD11b+ cells remained stable over the course of time. CONCLUSIONS: Using two different approaches to monitor MP/MG cells, our findings demonstrated that MP/MG concentrate within pathologically vascularized areas during OIR. We were able to clarify that reactive changes of CD11b+ cell distribution to OIR primarily occur in the deep retinal layers. Furthermore, we found the highest proportion of activated CD11b+ cells in regions with pathologic neovascularization processes. Our findings support previous reports about activated MP/MG guiding revascularization in avascular areas and playing a key role in the formation and regression of neovascular tufts.
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Macrófagos/metabolismo , Microglía/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/biosíntesis , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Neovascularización Retiniana/metabolismo , Animales , Animales Recién Nacidos , Recuento de Células , Modelos Animales de Enfermedad , Angiografía con Fluoresceína , Fondo de Ojo , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/patología , Oftalmoscopía , Oxígeno/toxicidad , Neovascularización Retiniana/inducido químicamente , Neovascularización Retiniana/patologíaRESUMEN
Anti-VEGF-directed therapies have been a milestone for treating retinal vascular diseases. Depletion of monocyte lineage cells suppresses pathological neovascularization in the oxygen-induced retinopathy mouse model. However, the question whether myeloid-derived VEGF-A expression is responsible for the pathogenesis in oxygen-induced retinopathy remained unknown. We analyzed LysMCre-driven myeloid cell-specific VEGF-A knockout mice as well as mice with complete depletion of circulating macrophages through clodronate-liposome treatment in the oxygen-induced retinopathy model by immunohistochemistry, qPCR, and flow cytometry. Furthermore, we analyzed VEGF-A mRNA expression in MIO-M1 cells alone and in co-culture with BV-2 cells in vitro. The myeloid cell-specific VEGF-A knockout did not change relative retinal VEGF-A mRNA levels, the relative avascular area or macrophage/granulocyte numbers in oxygen-induced retinopathy and under normoxic conditions. We observed an insignificantly attenuated pathology in systemically clodronate-liposome treated knockouts but evident VEGF-A expression in activated Müller cells on immunohistochemically stained sections. MIO-M1 cells had significantly higher expression levels of VEGF-A in co-culture with BV-2 cells compared to cultivating MIO-M1 cells alone. Our data show that myeloid-derived cells contribute to pathological neovascularization in oxygen-induced retinopathy through activation of VEGF-A expression in Müller cells.
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Células Ependimogliales/metabolismo , Hipoxia/metabolismo , Células Mieloides/metabolismo , Neovascularización Retiniana/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Noqueados , ARN Mensajero/metabolismoRESUMEN
Purpose: The cellular immune response driven by mononuclear phagocytes (MPs) is crucial for choroidal neovascularization (CNV) progression. Case reports show that a switch from pure anti-vascular endothelial growth factor-A (VEGF-A) intravitreal treatment to aflibercept, a drug with combined anti-VEGF-A and anti-placenta growth factor (PlGF) activity, can be beneficial for patients who do not respond to anti-VEGF-A alone. Since MPs harbor VEGFR1, we hypothesize that the interplay of P1GF/vascular endothelial growth factor receptor 1 (VEGFR1) in immune cells plays a pivotal role for CNV. Methods: CNV was induced with laser, and immune cells and neovascularization were analyzed in vivo and ex vivo. Immunohistochemistry was employed for protein detection. Differential expression of angiogenic factors and macrophage polarization markers were assessed by quantitative PCR (qPCR). One day after laser, intravitreal injection of aflibercept or anti-PlGF was performed. Results: In the early inflammatory phase after laser, Plgf but not Vegfa was significantly upregulated. VEGF-A upregulation is limited to the scar, whereas PlGF shows a wider distribution. M1 (proinflammatory) macrophage markers were upregulated in the early phase of CNV. However, M2 (proangiogenic) markers showed more inconsistent dynamics. We demonstrated that both aflibercept and anti-PlGF treatments decrease the overall amount of activated subretinal MPs, and especially of those expressing PlGF. These data correlated with a reduction in leakage associated to CNV. Aflibercept showed a stronger reduction in both parameters. Conclusions: The results hint at an interplay between PlGF/VEGFR1 and MPs that is important in the early phase of CNV. A combined inhibition of VEGF-A and PlGF is superior to a specific anti-PlGF treatment in terms of subretinal MP recruitment.
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Neovascularización Coroidal/inmunología , Fagocitos/inmunología , Factor de Crecimiento Placentario/antagonistas & inhibidores , Retina/inmunología , Inhibidores de la Angiogénesis/farmacología , Animales , Anticuerpos Bloqueadores/farmacología , Proteínas de Unión al Calcio , Neovascularización Coroidal/tratamiento farmacológico , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica , Factores Inmunológicos/farmacología , Inyecciones Intravítreas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos , Microscopía Fluorescente , Oftalmoscopía , Factor de Crecimiento Placentario/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes de Fusión/farmacología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Degeneración Macular Húmeda/tratamiento farmacológico , Degeneración Macular Húmeda/inmunologíaRESUMEN
PURPOSE: To investigate whether vascular differentiation marker von Willebrand factor (vWf) and proliferation marker KI67 expression correlate with MUC4 localization around stromal tumor vascularization in human conjunctival malignant melanoma (CMM). MATERIALS AND METHODS: For the purposes of this study, we analyzed samples from human CMMs (n = 4), conjunctival compound nevi (n = 7), and samples from healthy conjunctiva (n = 7) for MUC1, 4, and 16 by immunohistochemistry. To test CMM vessel association of MUC4, we investigated the co-localization of MUC4 with vWf or KI67 in human CMM specimens (n = 10) by immunohistochemistry. Also, we investigated the MUC4 localization around vessels of healthy conjunctiva (n = 10). RESULTS: The immunohistochemical analysis demonstrated membrane-associated mucin expression in epithelia of CMM, nevi and healthy conjunctiva, whereas only MUC4 was localized perivascular in CMM tissue in this preliminary analysis. Co-staining analysis with vWf and KI67 demonstrated MUC4 localization around stromal vessels in human CMM specimens. In contrast, no MUC4 localization has been seen around healthy conjunctiva stroma vessels. CONCLUSIONS: MUC4 was detected around vWf/KI67-positive CMM stromal vascular tissue, but not around healthy conjunctival stroma vessels. Therefore, we assume that MUC4 might play a role in tumor cell migration toward vessels inducing metastasis.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Conjuntiva/irrigación sanguínea , Neoplasias Hepáticas/irrigación sanguínea , Melanoma/irrigación sanguínea , Mucina 4/metabolismo , Neovascularización Patológica/metabolismo , Factor de von Willebrand/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Antígeno Ca-125/metabolismo , Terapia Combinada , Neoplasias de la Conjuntiva/patología , Neoplasias de la Conjuntiva/terapia , Femenino , Humanos , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/terapia , Metástasis Linfática , Masculino , Melanoma/secundario , Melanoma/terapia , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Persona de Mediana EdadRESUMEN
Purpose: Netrin-4, a secreted protein, is found in the basement membrane of blood vessels and acts as a key regulator of angiogenesis. Here we investigated the role of Netrin-4 in the mouse-model of suture-induced corneal hem- and lymphangiogenesis. Methods: Corneal hem- and lymphangiogenesis were induced in Netrin-4-deficient (Ntn4-/-) and wild-type (WT) mice by placing three 11-0 nylon sutures intrastromally. Fourteen days after suturing, the vascularized area was analyzed via corneal flat mount immunohistochemistry. Messenger RNA levels for VEGF-A, VEGF-C, Lyve-1, Netrin-4, Unc5H2, "deleted in colon cancer" receptor, and Neogenin in treated and nontreated mouse corneas, cultured human corneal keratocytes (HCK) and epithelial cells (HCEC+HCET) were analyzed by quantitative PCR. Results: In wild-type mice, Netrin-4 mRNA expression in the cornea decreased in growing corneal neovascularization after suturing. Correspondingly, Ntn4-/- mice showed an increased vascularized area compared to that in WT mice. Expression of VEGF-A mRNA was higher in Ntn4-/- versus WT mice. There was no Netrin-4 expression in lymphatic vessels and the area of lymphatic vascularization did not differ between Ntn4-/- and WT mice, nor did expression of VEGF-C and Lyve-1 mRNA. Human corneal epithelial cells showed mainly Netrin-4 mRNA expression, which increased after stimulation, while HCK demonstrated Unc5H2 mRNA expression. Expression of VEGF-A, Netrin-4, Unc5H2, and Neogenin mRNA in HCEC and HCK did not differ significantly between the serum-free condition and VEGF-A or Netrin-4 stimulation. Conclusions: Absence of Netrin-4 increased corneal hemangiogenesis but not lymphangiogenesis in the mouse-model of suture-induced neovascularization. Netrin-4 acted as an antiangiogenic factor in the cornea, with which the healthy cornea is enriched via its expression by corneal epithelial cells.
Asunto(s)
Córnea/irrigación sanguínea , Neovascularización de la Córnea/genética , Regulación de la Expresión Génica , Linfangiogénesis/genética , Vasos Linfáticos/patología , Factores de Crecimiento Nervioso/genética , ARN Mensajero/genética , Animales , Células Cultivadas , Córnea/metabolismo , Córnea/patología , Neovascularización de la Córnea/metabolismo , Neovascularización de la Córnea/patología , Modelos Animales de Enfermedad , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Factores de Crecimiento Nervioso/biosíntesis , Netrinas , Reacción en Cadena en Tiempo Real de la Polimerasa , Suturas/efectos adversosRESUMEN
AIMS/HYPOTHESIS: Although the renin-angiotensin system plays an important role in the progression of diabetic retinopathy, its influence therein has not been systematically evaluated. Here we test the suitability of a new translational model of diabetic retinopathy, the TetO rat, for addressing the role of angiotensin-II receptor 1 (AT1) blockade in experimental diabetic retinopathy. METHODS: Diabetes was induced by tetracycline-inducible small hairpin RNA (shRNA) knockdown of the insulin receptor in rats, generating TetO rats. Systemic treatment consisted of an AT1 blocker (ARB) at the onset of diabetes, following which, 4-5 weeks later the retina was analysed in vivo and ex vivo. Retinal function was assessed by Ganzfeld electroretinography (ERG). RESULTS: Retinal vessels in TetO rats showed differences in vessel calibre, together with gliosis. The total number and the proportion of activated mononuclear phagocytes was increased. TetO rats presented with loss of retinal ganglion cells (RGC) and ERG indicated photoreceptor malfunction. Both the inner and outer blood-retina barriers were affected. The ARB treated group showed reduced gliosis and an overall amelioration of retinal function, alongside RGC recovery, whilst no statistically significant differences in vascular and inflammatory features were detected. CONCLUSIONS/INTERPRETATION: The TetO rat represents a promising translational model for the early neurovascular changes associated with type 2 diabetic retinopathy. ARB treatment had an effect on the neuronal component of the retina but not on the vasculature.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Retinopatía Diabética/metabolismo , Receptor de Insulina/metabolismo , Animales , Western Blotting , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/genética , Retinopatía Diabética/genética , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunohistoquímica , Masculino , Reacción en Cadena de la Polimerasa , Ratas , Receptor de Insulina/genética , Receptores de Angiotensina/metabolismo , Retina/metabolismo , Retina/patología , Células Ganglionares de la RetinaRESUMEN
Severe hypertension destroys eyesight. The RAS (renin-angiotensin system) may contribute to this. This study relied on an established angiotensin, AngII (angiotensin II)-elevated dTGR (double-transgenic rat) model and same-background SD (Sprague-Dawley) rat controls. In dTGRs, plasma levels of AngII were increased. We determined the general retinal phenotype and observed degeneration of ganglion cells that we defined as vascular degeneration. We also inspected relevant gene expression and lastly observed alterations in the outer blood-retinal barrier. We found that both scotopic a-wave and b-wave as well as oscillatory potential amplitude were significantly decreased in dTGRs, compared with SD rat controls. However, the b/a-wave ratio remained unchanged. Fluorescence angiography of the peripheral retina indicated that exudates, or fluorescein leakage, from peripheral vessels were increased in dTGRs compared with controls. Immunohistological analysis of blood vessels in retina whole-mount preparations showed structural alterations in the retina of dTGRs. We then determined the general retinal phenotype. We observed the degeneration of ganglion cells, defined vascular degenerations and finally found differential expression of RAS-related genes and angiogenic genes. We found the expression of both human angiotensinogen and human renin in the hypertensive retina. Although the renin gene expression was not altered, the AngII levels in the retina were increased 4-fold in the dTGR retina compared with that in SD rats, a finding with mechanistic implications. We suggest that alterations in the outer blood-retinal barrier could foster an area of visual-related research based on our findings. Finally, we introduce the dTGR model of retinal disease.