Norrin is a secreted signaling molecule with structural and functional characteristics of an autocrine and/or paracrine acting growth factor. In the eye, Norrin is constitutively expressed in Müller ...cells. Norrin specifically binds to Frizzled-4 receptors and activates the canonical Wnt/β-catenin signaling pathway that is profoundly enhanced when Tspan12 is present at the Norrin/Frizzled-4 receptor complex. In the absence of Norrin or Frizzled-4, intraretinal capillaries are not formed during developmental angiogenesis. As a result there is considerable evidence that Norrin and Frizzled-4 are part of an essential signaling system that controls the formation of the retinal vasculature during eye development. Intriguingly, Norrin promotes vessel regrowth and induces the formation of intraretinal capillaries following oxygen-induced retinopathy in mice, an animal model of retinopathy of prematurity. Moreover, Norrin has pronounced neuroprotective properties on retinal ganglion cells (RGC) with the distinct potential to decrease the damaging effects of excitotoxic NMDA-induced RGC injury. The neuroprotective effects of Norrin similarly involve an activation of Wnt/β-catenin signaling and the subsequent induction of neuroprotective growth factor synthesis in Müller cells, such as that of fibroblast growth factor-2 (FGF2) or ciliary neurotrophic factor (CNTF). Overall, Norrin and the molecules involved in its signaling pathway appear to be promising targets to develop strategies that induce intraretinal vessel formation in patients suffering from ischemic retinopathies, or that increase RGC survival in glaucoma.
EVA NEXUS-Phaco performance study Feldhaus, Lukas; Kassumeh, Stefan; Priglinger, Siegfried ...
International journal of ophthalmology,
08/2024, Letnik:
17, Številka:
8
Journal Article
Recenzirano
Odprti dostop
AIM: To investigate a novel phacoemulsification system “EVA NEXUS” (D.O.R.C., Dutch Opthalmic Research Center) in comparison to the existing system “EVA” in clinical use. And to compare both ...phacoemulsification systems in terms of efficiency, safety and postoperative inflammatory activity. METHODS: In this study standardized cataract surgery was performed on both eyes of the study participant, using the “EVA system” (control group, n=20) on one eye and the “EVA NEXUS system” (intervention group, n=20) on the other eye. Only patients with cataract LOCS Grading 1-3 and no accompanying eye diseases were included in this study. A total of 20 patients were included in this study, with each treatment arm including 20 eyes. During surgery a 0.1 mL aqueous humor sample was collected 1min after phacoemulsification to measure the total prostaglanin E2 concentrations using an enzyme-linked immunosorbent assay. The endothelial cell count, visual and refractive outcomes, and anterior chamber flare were evaluated preoperatively, and 1d, 1wk, and 3mo postoperatively. RESULTS: There were no statistically significant differences between both groups regarding intraoperative safety parameters including effective phacoemulsification time (P=0.904), balanced saline solution flow (P=0.701) and total surgery time (P=0.565). Postoperative prostaglandin E2 levels, anterior chamber flare as well as endothelial cell loss tended to be lower in the NEXUS-Group, however not being statistically significant (P=0.718; 0.164; 0.486). Both systems provided similar clinical outcomes, regarding best corrected visual acuity and refractive parameters, showing no statistically significant differences between both groups. CONCLUSION: Both systems show a high level of safety and efficency with similar results in terms of safety parameters including postoperative inflammatory activity and endothelial cell loss as well as visual and refractive outcomes. Although statistically not significant, the EVA NEXUS system tends to cause less postoperative inflammation with lower prostaglandin E2 levels and lower anterior chamber flare values.
During the pathogenesis of glaucoma, optic nerve (ON) axons become continuously damaged at the optic nerve head (ONH). This often is associated with reactive astrocytes and increased transforming ...growth factor (TGF-β) 2 levels. In this study we tested the hypothesis if the presence or absence of decorin (DCN), a small leucine-rich proteoglycan and a natural inhibitor of several members of the TGF family, would affect the expression of the TGF-βs and connective tissue growth factor (CTGF/CCN2) in human ONH astrocytes and murine ON astrocytes. We found that DCN is present in the mouse ON and is expressed by human ONH and murine ON astrocytes. DCN expression and synthesis was significantly reduced after 24 h treatment with 3 nM CTGF/CCN2, while treatment with 4 pM TGF-β2 only reduced expression of DCN significantly. Conversely, DCN treatment significantly reduced the expression of TGF-β1, TGF-β2 and CTGF/CCN2 vis-a-vis untreated controls. Furthermore, DCN treatment significantly reduced expression of fibronectin (FN) and collagen IV (COL IV). Notably, combined treatment with DCN and triciribine, a small molecule inhibitor of protein kinase B (AKT), attenuated effects of DCN on CTGF/CCN2, TGF-β1, and TGF-β2 mRNA expression. We conclude (1) that DCN is an important regulator of TGF-β and CTGF/CCN2 expression in astrocytes of the ON and ONH, (2) that DCN thereby regulates the expression of extracellular matrix (ECM) components and (3) that DCN executes its negative regulatory effects on TGF-β and CTGF/CCN2 via the pAKT/AKT signaling pathway in ON astrocytes.
In the eye, an increase in galectin-1 is associated with various chorioretinal diseases, in which retinal pigment epithelium (RPE) cells play a crucial role in disease development and progression. ...Since little is known about the function of endogenous galectin-1 in these cells, we developed a galectin-1-deficient immortalized RPE cell line (ARPE-19-LGALS1−/−) using a sgRNA/Cas9 all-in-one expression vector and investigated its cell biological properties. Galectin-1 deficiency was confirmed by Western blot analysis and immunocytochemistry. Cell viability and proliferation were significantly decreased in ARPE-19-LGALS1−/− cells when compared to wild-type controls. Further on, an increased attachment of galectin-1-deficient RPE cells was observed by cell adhesion assay when compared to control cells. The diminished viability and proliferation, as well as the enhanced adhesion of galectin-1-deficient ARPE-19 cells, could be blocked, at least in part, by the additional treatment with human recombinant galectin-1. In addition, a significantly reduced migration was detected in ARPE-19-LGALS1−/− cells. In comparison to control cells, galectin-1-deficient RPE cells had enhanced expression of sm-α-actin and N-cadherin, whereas expression of E-cadherin showed no significant alteration. Finally, a compensatory expression of galectin-8 mRNA was observed in ARPE-19-LGALS1−/− cells. In conclusion, in RPE cells, endogenous galectin-1 has crucial functions for various cell biological processes, including viability, proliferation, migration, adherence, and retaining the epithelial phenotype.
Galectin-1 and -3 are β-galactoside binding lectins with varying effects on angiogenesis and apoptosis. Since in retinal pigment epithelial cells high amounts of human recombinant galectin (hr-GAL)1 ...and 3 inhibit cell adhesion, migration and proliferation, we investigated if hr-GAL1 and 3 have homologous effects on human retinal microvascular endothelial cells (HRMEC) in vitro.
To investigate the effect of galectin-1 and -3 on HRMEC, proliferation, apoptosis and viability were analyzed after incubation with 30, 60 and 120 μg/ml hr-GAL1 or 3 by BrdU-ELISA, histone-DNA complex ELISA, live/dead staining and the WST-1 assay, respectively. Further on, a cell adhesion as well as tube formation assay were performed on galectin-treated HRMEC. Migration was investigated by the scratch migration assay and time-lapse microscopy. In addition, immunohistochemical staining on HRMEC for β-catenin, galectin-1 and -3 were performed and β-catenin expression was investigated by western blot analysis.
Incubation with hr-GAL1 or 3 lead to a decrease in proliferation, migration, adhesion and tube formation of HRMEC compared to the untreated controls. No toxic effects of hr-GAL1 and 3 on HRMEC were detected. Intriguingly, after treatment of HRMEC with hr-GAL1 or 3, an activation of the proangiogenic Wnt/β-catenin signaling pathway was observed. However, incubation of HRMEC with hr-GAL1 or 3 drew intracellular galectin-1 and -3 out of the cells, respectively.
Exogenously added hr-GAL1 or 3 inhibit angiogenic properties of HRMEC in vitro, an effect that might be mediated via a loss of intracellular endogenous galectins.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Studying anterior lens capsule cutting edge profiles from femtosecond laser-assisted capsulotomy procedures performed before and after lens fragmentation. Twenty eyes (10 patients) with age-related ...cataract underwent femtosecond laser-assisted surgery (FLACS) using the Ziemer Z8 platform. First step of laser surgery was either capsulotomy (group first) or fragmentation (group second). One eye of each patient was assigned randomly, the second eye treated with the different sequence of procedures. After anterior capsule removal, tissue was fixed in cacodylate-buffered solution and cutting-edge profiles were analysed using scanning electron microscopy (SEM). All cases had cataract grade 2 and 3 based on LOCS III grading. SEM analysis showed more smooth edges in the first group, especially in cases with pseudoexfoliation (P = 0.037); more tags and bridges and a significant number of staggered cutting patterns (7 out of 10 cases) in the second group. All cases evolved the same microgroves with "valleys and mountains " as signs of the photodisruption process. Femtosecond laser capsulotomy should be performed before lens fragmentation minimizing the rate of cutting errors. Especially in eyes with advanced cataract, as intracapsular pressure may increase due to lens fragmentation without anterior capsular opening.
To date, diseases affecting vascular structures in the posterior eye are mostly treated by laser photocoagulation and multiple intraocular injections, procedures that destroy healthy tissue and can ...cause vision-threatening complications. To overcome these drawbacks, we investigate the feasibility of receptor-mediated nanoparticle targeting to capillary endothelial cells in the retina after i.v. application. Cell-binding studies using microvascular endothelial cells showed receptor-specific binding and cellular uptake of cyclo(RGDfC)-modified quantum dots via the αvβ3 integrin receptor. Conversely, Mueller cells and astrocytes, representing off-target cells located in the retina, revealed only negligible interaction with nanoparticles. In vivo experiments, using nude mice as the model organism, demonstrated a strong binding of the ligand-modified quantum dots in the choriocapillaris and intraretinal capillaries upon i.v. injection and 1-h circulation time. Nontargeted nanoparticles, in contrast, did not accumulate to a significant amount in the target tissue. The presented strategy of targeting integrin receptors in the retina could be of utmost value for future intervention in pathologies of the posterior eye, which are to date only accessible with difficulty.
Rho-associated protein kinase (ROCK) inhibitors allow for causative glaucoma therapy. Unfortunately, topically applied ROCK inhibitors suffer from high incidence of hyperemia and low intraocular ...bioavailability. Therefore, we propose the use of poly (lactide-co-glycolide) (PLGA) microspheres as a depot formulation for intravitreal injection to supply outflow tissues with the ROCK inhibitor fasudil over a prolonged time. Fasudil-loaded microspheres were prepared by double emulsion solvent evaporation technique. The chemical integrity of released fasudil was confirmed by mass spectrometry. The biological activity was measured in cell-based assays using trabecular meshwork cells (TM cells), Schlemm's canal cells (SC cells), fibroblasts and adult retinal pigment epithelium cells (ARPE-19). Cellular response to fasudil after its diffusion through vitreous humor was investigated by electric cell-substrate impedance sensing. Microspheres ranged in size from 3 to 67 µm. The release of fasudil from microspheres was controllable and sustained for up to 45 days. Released fasudil reduced actin stress fibers in TM cells, SC cells and fibroblasts. Decreased collagen gel contraction provoked by fasudil was detected in TM cells (~2.4-fold), SC cells (~1.4-fold) and fibroblasts (~1.3-fold). In addition, fasudil readily diffused through vitreous humor reaching its target compartment and eliciting effects on TM cells. No negative effects on ARPE-19 cells were observed. Since fasudil readily diffuses through the vitreous humor, we suggest that an intravitreal drug depot of ROCK inhibitors could significantly improve current glaucoma therapy particularly for patients with comorbid retinal diseases.
Choroidal neovascularizations (CNV) are partially stabilized through a coverage of pericytes leading to a partial anti-VEGF resistence. Drugs licensed for neovascular AMD (nAMD) do not take this ...mechanical and growth factor-driven CNV stability into account. The purpose of this work was to see if inhibiting the mammalian target of rapamycin (mTOR) may successfully block angiogenic cellular pathways in primary human retinal pericytes in an in vitro model of nAMD.
The mTOR inhibitor rapamycin was used to treat human retinal pericytes (HRP) at doses ranging from 0.005 to 15 g/ml. A modified metabolism-based XTT-Assay was used to assess toxicity and anti-proliferative effects. A scratch wound experiment showed the effects on migration. On Cultrex basement membrane gels, the influence of rapamycin on the development of endothelial cell capillary-like structures by human umbilical vein vascular endothelial cells (HUVEC) in the absence and presence of pericytes was investigated.
Rapamycin showed no signs of toxicity within its range of solubility. The drug showed dose dependent anti-proliferative activity and inhibited migration into the scratch wound. Endothelial cell tube formation in a HUVEC monoculture was effectively inhibited at 45%. A co-culture of HUVEC with pericytes on Cultrex induced endothelial tube stabilization but was disrupted by the addition of rapamycin leading to degradation of 94% of the tubes.
Rapamycin allows for an efficient modulation of aspects of angiogenesis in pericytes via mTOR-modulation in vitro. Further studies are needed to elucidate whether rapamycin may have an impact on CNV in nAMD in vivo.
Low energy stereotactic radiotherapy has been proposed for the treatment of neovascular age related macular degeneration. We investigated the in vitro effect of the radiotherapy on pericytes, retinal ...pigment epithelium and endothelial cells. Primary human retinal pigment epithelium cells, human umbilical vein endothelial cells and human pericytes from Placenta were cultivated. In a pairwise protocol, one plate was irradiated at a dose of 16 Gy, while the second plate served as a non-irradiated control. Thereafter, cells were cultivated either in serum-free (non-permissive) or serum-stimulated (permissive) conditions. A life/dead assay, an XTT and a BrdU assay were performed up to 7 days after irradiation. No cell death occurred at any timepoint in any cell line after treatment nor in the control. Compared to the unirradiated controls, cell viability and metabolic activity were significantly reduced in irradiated cells in the XTT assay, except for non-permissive RPE cells. In the BrdU assay, proliferation was inhibited. While no cell death was detected in vitro, viability and proliferative capacity of all cell lines were significantly reduced. Therefore, it seems that low energy stereotactic radiotherapy inhibits angiogenesis without a direct induction of apoptosis but influencing microvascular function and stability.
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