Cassandra Flügel-Koch

Disruption of anterior segment development by TGF-β1 overexpression in the eyes of transgenic mice

Previous experiments showed that transgenic mice expressing a secreted self‐activating transforming growth factor (TGF) ‐β1 did not show a phenotype in the lens and cornea until postnatal day 21, when anterior subcapsular cataracts, sporadic thickening of the corneal stroma, and thinning of the corneal epithelium were noted (Srinivasan et al., 1998 ). To examine the effects of higher concentrations of TGF‐β1 on the lens and cornea, we constructed transgenic mice harboring the strong, lens‐specific chicken βB1‐crystallin promoter driving an activated porcine TGF‐β1 gene. In contrast to the earlier study, the transgenic mice had microphthalmic eyes with closed eyelids. Already at embryonic day (E) 13.5, the future cornea of the transgenic mice was threefold thicker than that of wild‐type littermates due to increased proliferation of corneal stromal mesenchyme cells. Staining of fibronectin and thrombospondin‐1 was increased in periocular mesenchyme. At E17.5, the thickened transgenic corneal stroma was vascularized and densely populated by abundant star‐shaped, neural cell adhesion molecule–positive cells of mesenchymal appearance surrounded by irregular swirls of collagen and extracellular matrix. The corneal endothelium, anterior chamber, and stroma of iris/ciliary body did not develop, and the transgenic cornea was opaque. Fibronectin, perlecan, and thrombospondin‐1 were elevated, whereas type VI collagen decreased in the transgenic corneal stroma. Stromal mesenchyme cells expressed α‐smooth muscle actin as did lens epithelial cells and cells of the retinal pigmented epithelium. By E17.5, lens fiber cells underwent apoptotic cell death that was followed by apoptosis of the entire anterior lens epithelium between E18.5 and birth. Posteriorly, the vitreous humor was essentially absent; however, the retina appeared relatively normal. Thus, excess TGF‐β1, a mitogen for embryonic corneal mesenchyme, severely disrupts corneal and lens differentiation. Our findings profoundly contrast with the mild eye phenotype observed with presumably lower levels of ectopic TGF‐β and illustrate the complexity of TGF‐β utilization and the importance of dose when assessing the effects of this growth factor.

Presence of a Contractile Cell Network in the Human Choroid

Immunohistochemical staining of choroidal whole mounts as well as serial tangential sections and electron-microscopic investigation of 21 human donor eyes revealed a network of nonvascular alpha-smooth-muscle-actin-positive cells (NVSMC) in the entire choroid most densely arranged in the posterior part of the suprachoroid of the submacular region. Ultrastructurally these cells present the typical structure of myofibroblasts. Double labeling with desmin and smooth muscle myosin showed a positive reaction only in few of these cells. Posteriorly the NVSMC net reaches the optic nerve, anteriorly it ends in the region of the vortex veins but does not continue into the ciliary muscle. In places, intimate contact between the contractile cells and the elastic fiber network of the choroidal stroma and the perivascular sheaths is seen. The elastic net of the choroid is firmly connected with the posterior elastic tendons of the ciliary muscle. In the course of accommodation, the ciliary muscle pulls this net foreward presumably influencing position and diameter of the choroidal vessels. We assume that the network of NVSMC of the choroid counteracts the ciliary body movements during accommodation, thus guaranteeing the three-dimensional architecture of the choroid and the position of the retina, particularly in the macular region.

Secreted glycoprotein myocilin is a component of the myelin sheath in peripheral nerves

The structure of the myelin sheath in peripheral nerves requires the expression of a specific set of proteins. In the present study, we report that myocilin, a member of the olfactomedin protein family, is a component of the myelin sheath in peripheral nerves. Myocilin is a secreted glycoprotein that forms multimers and contains a leucine zipper and an olfactomedin domain. Mutations in myocilin are responsible for some forms of glaucoma, a neurodegenerative disease that is characterized by a continuous loss of optic nerve axons. Myocilin mRNA was detected by Northern blotting in RNA from the rat sciatic and ophthalmic nerves. By one‐ and two‐dimensional gel electrophoresis of proteins from the rat and human sciatic nerves, myocilin was found to migrate at an isoelectric point (pI) of 5.2–5.3 and a molecular weight of 55–57 kDa. Immunohistochemistry showed immunoreactivity for myocilin in paranodal terminal loops of the nodes of Ranvier and outer mesaxons and basal/abaxonal regions of the myelin sheath. Double‐labeling experiments with antibodies against myelin basic protein showed no overlapping, while overlapping immunoreactivity was observed with antibodies against myelin‐associated glycoprotein. The expression of myocilin in the sciatic nerve became detectable at postnatal day (P) 15 and reached adult levels at P20. No or minor expression of myocilin mRNA was found in brain, spinal cord, and optic nerve. mRNA of myocilin was detected in schwannoma cells in situ, but at considerably lower levels than in myelinated nerves. Myocilin might significantly contribute to the structure of the myelin sheath in peripheral nerves.

Morphologic Indication for Proprioception in the Human Ciliary Muscle

PURPOSE To search for proprioceptive nerve terminals in human ciliary muscle. METHODS In 48 human donor eyes, histologic and ultrathin sections cut in different planes and wholemounts of the ciliary muscle were studied. Immunohistochemical staining with antibodies against pan-neuronal antigens and antigens reported as markers for sensory terminals in other organs was performed. RESULTS Among the markers for proprioceptive terminals, only calretinin was present in the ciliary body. Calretinin-immunoreactive (IR) nerve terminals surrounded the posterior and reticular ciliary muscle tips and their elastic tendons. Terminals in that region contained mitochondria and neurofilaments. At the anterior tips larger terminals with numerous membrane-filled vesicles were located between the muscle fibers. The most elaborate network of calretinin-IR nerve fibers was present in the ground plate covering the circular muscle portion. Here calretinin-IR neurons with morphologic features of mechanoreception were present. Within the circular muscle portion numerous calretinin-IR ganglion cells were found. Their processes were connected to the calretinin-IR network but also surrounded ciliary muscle cells and NADPH-diaphorase-positive ganglion cells. CONCLUSIONS These morphologic findings indicate that there are proprioreceptors in the ciliary muscle that morphologically and presumably functionally differ at different locations. At the posterior muscle tips, the receptors could measure stretch of the tendons, whereas the large receptor organs located at the anterior muscle tips morphologically resemble mechanoreceptors measuring shear stress. The presence of the numerous intrinsic nerve cells indicates that contraction of the circular muscle portion can be modulated locally via a self-contained reflex arc.

Therapeutic potential of AAV-mediated MMP-3 secretion from corneal endothelium in treating glaucoma

Abstract Intraocular pressure (IOP) is maintained as a result of the balance between production of aqueous humour (AH) by the ciliary processes and hydrodynamic resistance to its outflow through the conventional outflow pathway comprising the trabecular meshwork (TM) and Schlemm’s canal (SC). Elevated IOP, which can be caused by increased resistance to AH outflow, is a major risk factor for open-angle glaucoma. Matrix metalloproteinases (MMPs) contribute to conventional aqueous outflow homeostasis in their capacity to remodel extracellular matrices, which has a direct impact on aqueous outflow resistance and IOP. We observed decreased MMP-3 activity in human glaucomatous AH compared to age-matched normotensive control AH. Treatment with glaucomatous AH resulted in significantly increased transendothelial resistance of SC endothelial and TM cell monolayers and reduced monolayer permeability when compared to control AH, or supplemented treatment with exogenous MMP-3. Intracameral inoculation of AAV-2/9 containing a CMV-driven MMP-3 gene (AAV-MMP-3) into wild type mice resulted in efficient transduction of corneal endothelium and an increase in aqueous concentration and activity of MMP-3. Most importantly, AAV-mediated expression of MMP-3 increased outflow facility and decreased IOP, and controlled expression using an inducible promoter activated by topical administration of doxycycline achieved the same effect. Ultrastructural analysis of MMP-3 treated matrices by transmission electron microscopy revealed remodelling and degradation of core extracellular matrix components. These results indicate that periodic induction, via use of an eye drop, of AAV-mediated secretion of MMP-3 into AH could have therapeutic potential for those cases of glaucoma that are sub-optimally responsive to conventional pressure-reducing medications.

The expression of myocilin during murine eye development

PurposeTo study the expression and localization of myocilin in the developing mouse eye. Myocilin is a 55- to 57-kDa secreted glycoprotein that is mutated in some forms of primary open-angle glaucoma.MethodsThe eyes of NMRI mice were studied from embryonic day (E) 14.5 to postnatal day (P) 21, and at 2–3 months of age. Immunohistochemistry was performed with antibodies against myocilin. The specificity of the antibodies was checked by two-dimensional gel electrophoresis. RNA was isolated from eyes at various ages, and the presence of myocilin mRNA was analyzed by northern blot hybridization.ResultsNo immunostaining for myocilin was seen before E16.5. At around E17.5, a distinct positive immunoreactivity of optic nerve axons in the developing nerve fiber layer of the retina was observed. At P5–6, immunostaining appeared in perikarya of optic nerve ganglion cells. In the anterior eye, no immunoreactivity was observed until P10. At P12–14, the cells of the epithelial layers of ciliary body and iris, as well as the cells of the trabecular meshwork and iris stroma, became immunoreactive for myocilin. At that time, positive staining for myocilin was also seen in the corneal endothelium and in keratocytes of the corneal stroma. An essentially similar staining pattern was seen in adult eyes. Northern blot analysis for myocilin mRNA in RNA from developing mouse eyes was negative until P9. At P12, a distinct band was observed. A band with similar mobility, but somewhat more intense, was detected in mRNA from adult mouse eyes 2–3 months of age.ConclusionsThe onset of immunoreactivity for myocilin in the retina occurs in parallel with the maturation of optic nerve ganglion cells. In the anterior eye, the expression of myocilin is associated with the final development of those tissues that are directly involved in aqueous humor dynamics. The presence of myocilin might be important for proper function and structure of mature optic nerve ganglion cells and aqueous humor outflow.

Enhancement of Outflow Facility in the Murine Eye by Targeting Selected Tight-Junctions of Schlemm's Canal Endothelia.

The juxtacanalicular connective tissue of the trabecular meshwork together with inner wall endothelium of Schlemm’s canal (SC) provide the bulk of resistance to aqueous outflow from the anterior chamber. Endothelial cells lining SC elaborate tight junctions (TJs), down-regulation of which may widen paracellular spaces between cells, allowing greater fluid outflow. We observed significant increase in paracellular permeability following siRNA-mediated suppression of TJ transcripts, claudin-11, zonula-occludens-1 (ZO-1) and tricellulin in human SC endothelial monolayers. In mice claudin-11 was not detected, but intracameral injection of siRNAs targeting ZO-1 and tricellulin increased outflow facility significantly. Structural qualitative and quantitative analysis of SC inner wall by transmission electron microscopy revealed significantly more open clefts between endothelial cells treated with targeting, as opposed to non-targeting siRNA. These data substantiate the concept that the continuity of SC endothelium is an important determinant of outflow resistance, and suggest that SC endothelial TJs represent a specific target for enhancement of aqueous movement through the conventional outflow system.

VEGF as a Paracrine Regulator of Conventional Outflow Facility

Purpose Vascular endothelial growth factor (VEGF) regulates microvascular endothelial permeability, and the permeability of Schlemms canal (SC) endothelium influences conventional aqueous humor outflow. We hypothesize that VEGF signaling regulates outflow facility. Methods We measured outflow facility (C) in enucleated mouse eyes perfused with VEGF-A164a, VEGF-A165b, VEGF-D, or inhibitors to VEGF receptor 2 (VEGFR-2). We monitored VEGF-A secretion from human trabecular meshwork (TM) cells by ELISA after 24 hours of static culture or cyclic stretch. We used immunofluorescence microscopy to localize VEGF-A protein within the TM of mice. Results VEGF-A164a increased C in enucleated mouse eyes. Cyclic stretch increased VEGF-A secretion by human TM cells, which corresponded to VEGF-A localization in the TM of mice. Blockade of VEGFR-2 decreased C, using either of the inhibitors SU5416 or Ki8751 or the inactive splice variant VEGF-A165b. VEGF-D increased C, which could be blocked by Ki8751. Conclusions VEGF is a paracrine regulator of conventional outflow facility that is secreted by TM cells in response to mechanical stress. VEGF affects facility via VEGFR-2 likely at the level of SC endothelium. Disruption of VEGF signaling in the TM may explain why anti-VEGF therapy is associated with decreased outflow facility and sustained ocular hypertension.

Anteriorly located zonular fibres as a tool for fine regulation in accommodation.

To describe an anteriorly located system of zonular fibres that could be involved in fine‐tuning of accommodation.

Morphological Alterations Within the Peripheral Fixation of the Iris Dilator Muscle in Eyes With Pigmentary Glaucoma

PURPOSE To analyze the peripheral fixation of the iris dilator muscle in normal eyes and in eyes with pigmentary glaucoma (PG). METHODS Using 63 control eyes (age 18 months-99 years), the peripheral iris dilator was investigated by light microscopy, immunohistochemistry, and electron microscopy. Development was studied using 18 differently aged fetal eyes stained immunohistochemically against α-smooth muscle (SM) actin. The peripheral iris dilator muscle in PG was analyzed using semithin and ultrathin sections of six glutaraldehyde-fixed eyes from three donors aged 38, 62, and 74 years. RESULTS In normal eyes, the peripheral end of the iris dilator muscle is arranged in a sphincter-like manner. Arcade-shaped tendinous connections associated with myofibroblasts (iridial strands) anchor the iris dilator within the elastic-fibromuscular ciliary meshwork that also serves as fixation area for the elastic tendons of the inner ciliary muscle portions. The iridial strands are innervated and can adapt their length during accommodation. The PG eyes show incomplete circular bundles and iridial strands that are mainly anchored to the iris stroma and the flexible uveal parts of the trabecular meshwork. CONCLUSIONS The normal anchorage of the peripheral iris dilator and its presumably neuronally regulated length adaptation stabilize the peripheral iris during accommodation. Insufficient fixation in PG could promote posterior bowing of the iris with rubbing against the zonular fibers and pigment liberation from the iris pigmented epithelium.