Cross-Inhibition of Norrin and TGF-beta Signaling Modulates Development of Retinal and Choroidal Vasculature

PURPOSE. Norrin is essential for the formation of the retinal vasculature during development and promotes its repair after damage via activation of Wnt/beta-catenin signaling. Since retinal TGF-beta signaling has essentially opposite effects on the retinal vasculature we investigated if and how Norrin inhibits TGF-beta signaling, and vice versa. METHODS. Eyes from transgenic mice with an overexpression of Norrin (beta Bl-Norrin) and/or active TGF-beta (beta B1-TGF-beta 1) in the lens were generated and analyzed by light microscopy, immunohistochemistry, and TUNEL. Further on, protein as well as mRNA levels were investigated by Western blot analyses and real-time RT-PCR, respectively. RESULTS. In beta B1-TGF-beta l mice, the lack of retinal vascular development and choriocapillaris maintenance was rescued when transgenic Norrin was additionally overexpressed in the eye. In addition, retinal Wnt/beta-catenin signaling and the levels of SMAD7, an inhibitor of the canonical TGF-beta pathway, were substantially suppressed in retinae of beta B1-TGF-beta 1 mice. In contrast, Norrin normalized Wnt/beta-catenin signaling and SMAD7 levels in double transgenic mice. Moreover, in retinae of beta B1-TGF-beta 1 mice, the amounts of phosphorylated SMAD3, a downstream mediator of TGF-beta signaling, were increased compared to those of beta B1-Norrin/ beta B1-TGF-beta 1 mice. In vitro, Norrin substantially reduced the TGF-beta-mediated induction of target genes, an effect that was blocked by Dickkopf-1, a specific inhibitor of Wnt/beta-catenin signaling. CONCULSIONS. High amounts of TGF-beta in the eye cause a substantial reduction in the activity of Wnt/beta-catenin signaling. This effect is inhibited in the presence of high amounts of Norrin, which further induce the expression of SMAD7 to inhibit TGF-beta signaling

CCN2/CTGF promotor activity in the developing and adult mouse eye

CCN2/CTGF is a matricellular protein that is known to enhance transforming growth factor-β signaling and to induce a myofibroblast-like phenotype in a variety of cell types. Here, we investigated Ccn2/Ctgf promotor activity during development and in the adult mouse eye, using CTGFLacZ/+ mice in which the β-galactosidase reporter gene LacZ had been inserted into the open reading frame of Ccn2/Ctgf. Promotor activity was assessed by staining for β-galactosidase activity and by immunolabeling using antibodies against β-galactosidase. Co-immunostaining using antibodies against glutamine synthetase, glial fibrillary acidic protein, choline acetyltransferase, and CD31 was applied to identify specific cell types. Ccn2/Ctgf promotor activity was intense in neural crest-derived cells differentiating to corneal stroma and endothelium, and to the stroma of choroid, iris, ciliary body, and the trabecular meshwork during development. In the adult eye, a persistent and very strong promotor activity was present in the trabecular meshwork outflow pathways. In addition, endothelial cells of Schlemm’s canal, and of retinal and choroidal vessels, retinal astrocytes, Müller glia, and starburst amacrine cells were stained. Very strong promoter activity was seen in the astrocytes of the glial lamina at the optic nerve head. We conclude that CCN2/CTGF signaling is involved in the processes that govern neural crest morphogenesis during ocular development. In the adult eye, CCN2/CTGF likely plays an important role for the trabecular meshwork outflow pathways and the glial lamina of the optic nerve head

Angiopoietin-1 Mimetic Nanoparticles for Restoring the Function of Endothelial Cells as Potential Therapeutic for Glaucoma

A root cause for the development and progression of primary open-angle glaucoma might be the loss of the Schlemm’s canal (SC) cell function due to an impaired Angiopoietin-1 (Angpt-1)/Tie2 signaling. Current therapeutic options fail to restore the SC cell function. We propose Angpt-1 mimetic nanoparticles (NPs) that are intended to bind in a multivalent manner to the Tie2 receptor for successful receptor activation. To this end, an Angpt-1 mimetic peptide was coupled to a poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) block co-polymer. The modified polymer allowed for the fabrication of Angpt-1 mimetic NPs with a narrow size distribution (polydispersity index < 0.2) and the size of the NPs ranging from about 120 nm (100% ligand density) to about 100 nm (5% ligand density). NP interaction with endothelial cells (HUVECs, EA.hy926) as surrogate for SC cells and fibroblasts as control was investigated by flow cytometry and confocal microscopy. The NP–cell interaction strongly depended on the ligand density and size of NPs. The cellular response to the NPs was investigated by a Ca2+ mobilization assay as well as by a real-time RT-PCR and Western blot analysis of endothelial nitric oxide synthase (eNOS). NPs with a ligand density of 25% opposed VEGF-induced Ca2+ influx in HUVECs significantly which could possibly increase cell relaxation and thus aqueous humor drainage, whereas the expression and synthesis of eNOS was not significantly altered. Therefore, we suggest Angpt-1 mimetic NPs as a first step towards a causative therapy to recover the loss of SC cell function during glaucoma

TGF-beta(2)- and H2O2-Induced Biological Changes in Optic Nerve Head Astrocytes Are Reduced by the Antioxidant Alpha-Lipoic Acid

Background/Aims: The goal of the present study was to determine whether transforming growth factor-beta(2) (TGF-beta(2))- and oxidative stress-induced cellular changes in cultured human optic nerve head (ONH) astrocytes could be reduced by pretreatment with the antioxidant alpha-lipoic acid (LA). Methods: Cultured ONH astrocytes were treated with 1.0 ng/ml TGF-beta(2) for 24 h or 200 mu M hydrogen peroxide (H2O2) for 1 h. Lipid peroxidation was measured by a decrease in cis-pari-naric acid fluorescence. Additionally, cells were pretreated with different concentrations of LA before TGF-beta 2 or H2O2 exposure. Expressions of the heat shock protein (Hsp) alpha B-crystallin and Hsp27, the extracellular matrix (ECM) component fibronectin and the ECM-modulating protein connective tissue growth factor (CTGF) were examined with immunohistochemistry and real-time PCR analysis. Results: Both TGF-beta(2) and H2O2 increased lipid peroxidation. Treatment of astrocytes with TGF-beta(2) and H2O2 upregulated the expression of alpha B-crystallin, Hsp27, fibronectin and CTGF. Pretreatment with different concentrations of LA reduced the TGF-beta(2)- and H2O2-stimulated gene expressions. Conclusion: We showed that TGF-beta(2)- and H2O2-stimulated gene expressions could be prevented by pretreatment with the antioxidant LA in cultured human ONH astrocytes. Therefore, it is tempting to speculate that the use of antioxidants could have protective effects in glaucomatous optic neuropathy. Copyright (C) 2012 S. Karger AG, Base

Altered mechanobiology of Schlemm’s canal endothelial cells in glaucoma

Increased flow resistance is responsible for the elevated intraocular pressure characteristic of glaucoma, but the cause of this resistance increase is not known. We tested the hypothesis that altered biomechanical behavior of Schlemm’s canal (SC) cells contributes to this dysfunction. We used atomic force microscopy, optical magnetic twisting cytometry, and a unique cell perfusion apparatus to examine cultured endothelial cells isolated from the inner wall of SC of healthy and glaucomatous human eyes. Here we establish the existence of a reduced tendency for pore formation in the glaucomatous SC cell—likely accounting for increased outflow resistance—that positively correlates with elevated subcortical cell stiffness, along with an enhanced sensitivity to the mechanical microenvironment including altered expression of several key genes, particularly connective tissue growth factor. Rather than being seen as a simple mechanical barrier to filtration, the endothelium of SC is seen instead as a dynamic material whose response to mechanical strain leads to pore formation and thereby modulates the resistance to aqueous humor outflow. In the glaucomatous eye, this process becomes impaired. Together, these observations support the idea of SC cell stiffness—and its biomechanical effects on pore formation—as a therapeutic target in glaucoma

Transglutaminases (TGs) in Ocular and Periocular Tissues: Effect of Muscarinic Agents on TGs in Scleral Fibroblasts

10.1371/journal.pone.0018326PLoS ONE64

Advanced glycation end products cause increased CCN family and extracellular matrix gene expression in the diabetic rodent retina

Aims/hypothesis Referred to as CCN, the family of growth factors consisting of cystein-rich protein 61 (CYR61, also known as CCN1), connective tissue growth factor (CTGF, also known as CCN2), nephroblastoma overexpressed gene (NOV, also known as CCN3) and WNT1-inducible signalling pathway proteins 1, 2 and 3 (WISP1, -2 and -3; also known as CCN4, -5 and -6) affects cellular growth, differentiation, adhesion and locomotion in wound repair, fibrotic disorders, inflammation and angiogenesis. AGEs formed in the diabetic milieu affect the same processes, leading to diabetic complications including diabetic retinopathy. We hypothesised that pathological effects of AGEs in the diabetic retina are a consequence of AGE-induced alterations in CCN family expression. Materials and methods CCN gene expression levels were studied at the mRNA and protein level in retinas of control and diabetic rats using real-time quantitative PCR, western blotting and immunohistochemistry at 6 and 12 weeks of streptozotocin-induced diabetes in the presence or absence of aminoguanidine, an AGE inhibitor. In addition, C57BL/6 mice were repeatedly injected with exogenously formed AGE to establish whether AGE modulate retinal CCN growth factors in vivo. Results After 6 weeks of diabetes, Cyr61 expression levels were increased more than threefold. At 12 weeks of diabetes, Ctgf expression levels were increased twofold. Treatment with aminoguanidine inhibited Cyr61 and Ctgf expression in diabetic rats, with reductions of 31 and 36%, respectively, compared with untreated animals. Western blotting showed a twofold increase in CTGF production, which was prevented by aminoguanidine treatment. In mice infused with exogenous AGE, Cyr61 expression increased fourfold and Ctgf expression increased twofold in the retina. Conclusion/interpretation CTGF and CYR61 are downstream effectors of AGE in the diabetic retina, implicating them as possible targets for future intervention strategies against the development of diabetic retinopath

Preventive Effects of Omega-3 and Omega-6 Fatty Acids on Peroxide Mediated Oxidative Stress Responses in Primary Human Trabecular Meshwork Cells

Pathologic processes in glaucoma include increased apoptosis, accumulation of extracellular material in the trabecular meshwork and optic nerve, condensations of the cytoskeleton and precocious cellular senescence. Oxidative stress was shown to generate these alterations in primary ocular cells. Fatty acids omega-3 and -6 are alleged to constitute a prophylaxis against these deleterious effects. Here, we tested actual preventive effects omega-3 and -6 against peroxide induced stress responses in primary human trabecular meshwork cells. Changes of mitochondrial activity, proliferation, heat shock proteins, extracellular matrix components, and inflammatory markers were evaluated. Alterations of the cytoskeleton were evaluated by phalloidin labeling. Here we report a repressive effect of omega-6 on metabolic activity and proliferation, which was not detected for omega-3. Both agents were able to prevent the anti-proliferative effect of H2O2, but only omega-3 prevented metabolic repression. Expression of heat shock protein 27 was unaltered by both fatty acids, whereas heat shock protein 90 was significantly induced by both. Omega-6 increased fibronectin and connective tissue growth factor synthesis, as well as the amount of secreted fibronectin. Omega-3, instead, induced plasminogen activator inhibitor 1 synthesis. H2O2 further increased fibronectin production in omega-6 supplemented cells, which was not the case in omega-3 treated cells. H2O2 stimulation of plasminogen activator inhibitor 1 and connective tissue growth factor was repressed by both fatty acids. Both fatty acids appeared to abolish H2O2 mediated stimulation of nuclear factor κB and IL-6, but not IL-1α and IL-8. H2O2 induced formation of cross-linked actin networks and stress fibers, which was reduced by preemptive application of omega-3. Omega-6, in contrast, had no protective effect on that, and even seemed to promote condensation. Based on the observed side effects of omega-6, omega-3 appears to be the more beneficial fatty acid in respect of prophylactic intake for prevention of a glaucomatous disease

Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings

A common variant near TGFBR3 is associated with primary open angle glaucoma

Primary open angle glaucoma (POAG), a major cause of blindness worldwide, is a complex disease with a significant genetic contribution. We performed Exome Array (Illumina) analysis on 3504 POAG cases and 9746 controls with replication of the most significant findings in 9173 POAG cases and 26 780 controls across 18 collections of Asian, African and European descent. Apart from confirming strong evidence of association at CDKN2B-AS1 (rs2157719 [G], odds ratio [OR] = 0.71, P = 2.81 × 10−33), we observed one SNP showing significant association to POAG (CDC7–TGFBR3 rs1192415, ORG-allele = 1.13, Pmeta = 1.60 × 10−8). This particular SNP has previously been shown to be strongly associated with optic disc area and vertical cup-to-disc ratio, which are regarded as glaucoma-related quantitative traits. Our study now extends this by directly implicating it in POAG disease pathogenesis