Barbara M. Braunger

Norrin Promotes Vascular Regrowth after Oxygen-Induced Retinal Vessel Loss and Suppresses Retinopathy in Mice

Norrin is a secreted protein that is involved in retinal angiogenesis and activates the Wnt-signaling pathway. We studied the role of Norrin in microvascular endothelial cells in vitro, and in a mouse model of retinopathy characterized by oxygen-induced vascular loss followed by hypoxia-induced pathological neovascularization. Recombinant Norrin significantly increased proliferation, viability, migration, and tube formation in vitro. Two independent transgenic mouse strains with ectopic overexpression of Norrin from the lens (βB1-Crystallin-Norrin), or the retinal pigment epithelium (Rpe65-Norrin) were generated and exposed to high oxygen. Following oxygen treatment, vascular loss was significantly smaller in retinae of transgenic mice from both strains as compared to wild-type littermates. In addition, the anatomical correct regrowth of vessels was significantly increased, while pathological neovascularization was suppressed. In vitro and in vivo effects of Norrin could be blocked by adding DKK (Dickkopf)-1, an inhibitor of Wnt/β-catenin signaling. Treatment of microvascular endothelial cells with Norrin caused a substantial increase in the expression of angiopoietin-2 (Ang-2). When inhibitory antibodies against Ang-2 were added to Norrin, the proliferative effects of Norrin were significantly suppressed. We conclude that Norrin is a potent factor to induce angiogenesis in microvascular endothelial cells, which has the distinct potential to suppress the damaging effects of oxygen-induced retinopathy in vivo. The effects of Norrin appear to be mediated, at least partially, via the induction of Ang-2.

Constitutive overexpression of Norrin activates Wnt/β-catenin and endothelin-2 signaling to protect photoreceptors from light damage.

Norrin is a retinal signaling molecule which is expressed in Müller glia and binds to Frizzled-4 to activate canonical Wnt/β-catenin signaling. Norrin is part of an essential signaling system that controls the formation of retinal capillaries during development. To evaluate neuroprotective properties of Norrin independently from its function during retinal angiogenesis, we generated transgenic mice (Rpe65-Norrin) that constitutively express Norrin in the retinal pigmented epithelium. Substantial amounts of Norrin were secreted into the outer retina, which triggered retinal Wnt/β-catenin signaling in conjunction with an increase in the expression of endothelin-2 (EDN2), endothelin receptor B (EDNRB), and glial fibrillary acidic protein (GFAP). Photoreceptors of Norrin-overexpressing mice were significantly less vulnerable to light-induced damage compared to their wild-type littermates. Following light damage, we observed less apoptotic death of photoreceptors and a better retinal function than in controls. The protective effects were abolished if either Wnt/β-catenin or EDN2 signaling was blocked by intravitreal injection of Dickkopf-1 or BQ788, respectively. Light-damaged retinae from transgenic mice contained higher amounts of brain-derived neurotrophic factor (BDNF) and pAkt than those of wild-type littermates. We conclude that constitutive overexpression of Norrin protects photoreceptors from light damage, an effect that is mediated by Wnt/β-catenin and EDN2 signaling and involves neurotrophic activities of BDNF. The findings suggest that Norrin and its associated signaling pathways have strong potentials to attenuate photoreceptor death following injury.

The aqueous humor outflow pathways in glaucoma: A unifying concept of disease mechanisms and causative treatment

Intraocular pressure (IOP) is the critical risk factor for glaucoma, a neurodegenerative disease and frequent cause of blindness worldwide. As of today, all effective strategies to treat glaucoma aim at lowering IOP. IOP is generated and maintained via the aqueous humor circulation system in the anterior eye. Aqueous humor is secreted by the ciliary processes and exits the eye through the trabecular meshwork (TM) or the uveoscleral outflow pathways. The TM outflow pathways provide resistance to aqueous humor outflow and IOP builds up in response to it. In the normal eye, the resistance is localized in the inner wall region, which comprises the juxtacanalicular connective tissue (JCT) and the inner wall endothelium of Schlemms canal (SC). Outflow resistance in the inner wall region is lowered through the contraction of the ciliary muscle or the relaxation of contractile myofibroblasts in the posterior part of the TM and the adjacent scleral spur. Patients with primary open-angle glaucoma (POAG), the most frequent form of glaucoma, typically suffer from an abnormally high outflow resistance of the inner wall region. There is increasing evidence that the increase in TM outflow resistance in POAG is the result of a characteristic change in the biological properties of the resident cells in the JCT, which increasingly acquire the phenotype of contractile myofibroblasts. This scenario strengthens simultaneously both their actin cytoskeleton and their directly associated extracellular matrix fibrils, leads to overall stiffening of the tissue, and is modulated by transforming growth factor-β (TGF-β)/connective tissue growth factor (CTGF) signaling. Essentially comparable changes appear to occur in SC endothelial cells in glaucoma. Causative therapy concepts targeting the aqueous outflow pathways in glaucoma should aim at interfering with this process either by attenuating TM or SC stiffness, and/or by modulating TGF-β/CTGF signaling.

TGF-β Signaling Protects Retinal Neurons from Programmed Cell Death during the Development of the Mammalian Eye

We investigated the influence of transforming growth factor-β (TGF-β) signaling on developmental programmed cell death in the mouse retina by direct and specific molecular targeting of TGF-β type II receptor (TβRII) and Smad7 in retinal progenitor cells. Mice were generated carrying a conditional deletion of the TβRII in cells that originate from the inner layer of the optic cup. The animals showed a significant decrease of phosphorylated Smad3 in both the central and peripheral retina, which indicates the diminished activity of TGF-β signaling. TβRII deficiency significantly increased the apoptotic death of retinal neurons during embryonic and postnatal development without affecting their proliferation. In contrast, treatment with TGF-β2 inhibited cell death of retinal ganglion cells in dissociated retinal cell cultures, an effect that was blocked by inhibiting the phosphorylation of Smad3. The increase in apoptosis during development resulted in a significant reduction in the number of neurons in adult TβRII-deficient mice. The effect was most pronounced in the inner retina neurons and resulted in functional deficits as determined by electroretinography. In contrast, a conditional deletion of TGF-β-inhibiting Smad7 in retinal neurons significantly enhanced Smad3 phosphorylation and significantly decreased apoptosis of retinal neurons in embryos and pups. Moreover, the number of retinal ganglion cells was significantly higher in Smad7-deficient mice compared with control littermates. TβRII-deficient pups showed a lower level of nerve growth factor (NGF) in its mRNA; however, higher levels were observed in Smad7-deficient pups, which strongly suggests that the protective effects of TGF-β signaling on developmental cell death are mediated through NGF.

Intraocular Pressure and the Mechanisms Involved in Resistance of the Aqueous Humor Flow in the Trabecular Meshwork Outflow Pathways

Intraocular pressure (IOP), the critical risk factor for glaucoma, is generated and maintained by the aqueous humor circulation system. Aqueous humor is secreted from the epithelial layers of the ciliary body and exits the eye through the trabecular meshwork or the uveoscleral outflow pathways. IOP builds up in response to a resistance to aqueous humor flow in the trabecular outflow pathways. The trabecular outflow resistance is localized in the inner wall region, which comprises the juxtacanalicular connective tissue (JCT) and the inner wall endothelium of Schlemms canal (SC). Outflow resistance in this region is lowered through the relaxation of contractile myofibroblast-like cells in trabecular meshwork and the adjacent scleral spur, or the contraction of the ciliary muscle. In primary open-angle glaucoma, the most frequent form of glaucoma, outflow resistance of the inner wall region is typically higher than normal. There is evidence that the increase in resistance is related to characteristic biological changes in the resident cells of the JCT, which more and more acquire the structural and functional characteristics of contractile myofibroblasts. The changes involve an augmentation of their actin cytoskeleton and of their surrounding fibrillary extracellular matrix, which connects to JCT cells via integrins. This scenario leads to an overall stiffening of the inner wall region, and is modulated by transforming growth factor-β/connective tissue growth factor signaling. Essentially comparable changes appear to occur in SC endothelial cells. Stiffening of JCT and SC cells is very likely a critical causative factor for the increase in trabecular outflow resistance in POAG.

Deletion of ocular transforming growth factor β signaling mimics essential characteristics of diabetic retinopathy.

Diabetic retinopathy, a major cause of blindness, is characterized by a distinct phenotype. The molecular causes of the phenotype are not sufficiently clear. Here, we report that deletion of transforming growth factor β signaling in the retinal microenvironment of newborn mice induces changes that largely mimic the phenotype of nonproliferative and proliferative diabetic retinopathy in humans. Lack of transforming growth factor β signaling leads to the formation of abundant microaneurysms, leaky capillaries, and retinal hemorrhages. Retinal capillaries are not covered by differentiated pericytes, but by a coat of vascular smooth muscle-like cells and a thickened basal lamina. Reactive microglia is found in close association with retinal capillaries. In older animals, loss of endothelial cells and the formation of ghost vessels are observed, findings that correlate with the induction of angiogenic molecules and the accumulation of retinal hypoxia-inducible factor 1α, indicating hypoxia. Consequently, retinal and vitreal neovascularization occurs, a scenario that leads to retinal detachment, vitreal hemorrhages, neuronal apoptosis, and impairment of sensory function. We conclude that transforming growth factor β signaling is required for the differentiation of retinal pericytes during vascular development of the retina. Lack of differentiated pericytes initiates a scenario of structural and functional changes in the retina that mimics those of diabetic retinopathy strongly indicating a common mechanism.

Tg(Grm1) transgenic mice: a murine model that mimics spontaneous uveal melanoma in humans?

Although rare, uveal melanoma (UM) is the most common primary intraocular tumor in adults. About half of UM patients develop metastatic disease typically in the liver and die within a short period, due to ineffective systemic therapies. UM has unique and distinct genetic features predictive of metastasis. Animal models are required to improve our understanding of therapeutic options in disseminated UM. Since spontaneous murine UM models are lacking, our aim was to analyze the suitability of the established transgenic melanoma mouse model Tg(Grm1) as a new UM model system. We demonstrated that adult Grm1 transgenic mice develop choroidal thickening and uveal melanocytic neoplasia with expression of the melanocytic markers S100B and MelanA. Further, we showed that GRM1 is expressed in human UM, similar to skin melanoma. This study presents a new mouse model for spontaneous UM and suggests that the glutamate signaling pathway is a possible target for UM therapy.

The Different Functions of Norrin

Norrin is a secreted protein which is encoded by the NDP gene mutated in Norrie disease. In the eye, the major site of Norrin expression is the Muller glia. Norrin activates the canonical Wnt/β-catenin signaling pathway via specific binding to the frizzled (Fzd)4/low-density lipoprotein receptor-related protein (Lrp)5/6 receptor complex, and is part of an essential signaling system that controls the formation of retinal capillaries during development. Independent from its angiogenic function, Norrin has pronounced neuroprotective properties on retinal ganglion cells via activation of Wnt/β-Catenin signaling and subsequent induction of neurotrophic growth factors in Muller cells. In addition, there is evidence that the expression of Norrin in uterus and placenta is required for reproduction

Heterozygous modulation of TGF-β signaling does not influence Müller glia cell reactivity or proliferation following NMDA-induced damage

AbstractThe stimulation of progenitor or stem cells proliferation in the retina could be a therapeutic avenue for the treatment of various ocular neurodegenerative disorders. Müller glia cells have been discussed to represent a progenitor cell population in the adult retina. In the brain, TGF-β signaling regulates the fate of stem cells; however, its role in the vertebrate retina is unclear. We therefore investigated whether manipulation of the TGF-β signaling pathway is sufficient to promote Müller glia cell proliferation and subsequently their trans-differentiation into retinal neurons. To this end, we used mice with heterozygous deficiency of the essential TGF-β receptor type II or of the inhibitory protein SMAD7, in order to down- or up-regulate the activity of TGF-β signaling, respectively. Excitotoxic damage was applied by intravitreal N-methyl-d-aspartate injection, and BrdU pulse experiments were used to label proliferative cells. Although we successfully stimulated Müller glia cell reactivity, our findings indicate that a moderate modulation of TGF-β signaling is not sufficient to provoke Müller glia cell proliferation. Hence, TGF-β signaling in the retina might not be the essential causative factor to maintain mammalian Müller cells in a quiescent, non-proliferative state that prevents a stem cell-like function.

The regulation of connective tissue growth factor expression influences the viability of human trabecular meshwork cells

Connective tissue growth factor (CTGF) induces extracellular matrix (ECM) synthesis and contractility in human trabecular meshwork (HTM) cells. Both processes are involved in the pathogenesis of primary open‐angle glaucoma. To date, little is known about regulation and function of CTGF expression in the trabecular meshwork (TM). Therefore, we analysed the effects of different aqueous humour proteins and stressors on CTGF expression in HTM cells. HTM cells from three different donors were treated with endothelin‐1, insulin‐like growth factor (IGF)‐1, angiotensin‐II, H2O2 and heat shock and were analysed by immunohistochemistry, real‐time RT‐PCR and Western blotting. Viability after H2O2 treatment was measured in CTGF silenced HTM‐N cells and their controls. Latrunculin A reduced expression of CTGF by about 50% compared to untreated HTM cells, whereas endothelin‐1, IGF‐1, angiotensin‐II, heat shock and oxidative stress led to a significant increase. Silencing of CTGF resulted in a delayed expression of αB‐crystallin and in reduced cell viability in comparison to the controls after oxidative stress. Conversely, CTGF treatment led to a higher cell viability rate after H2O2 treatment. CTGF expression is induced by factors that have been linked to glaucoma. An increased level of CTGF appears to protect TM cells against damage induced by stress. The beneficial effect of CTGF for viability of TM cells is likely associated with the effects on increased ECM synthesis and higher contractility of the TM, thereby contributing to reduced aqueous humour outflow facility causing increased intraocular pressure.