Ernst R. Tamm

Gene Targeting Reveals a Widespread Role for the High-Mobility-Group Transcription Factor Sox11 in Tissue Remodeling

ABSTRACT The high-mobility-group domain-containing transcription factor Sox11 is expressed transiently during embryonic development in many tissues that undergo inductive remodeling. Here we have analyzed the function of Sox11 by gene deletion in the mouse. Sox11-deficient mice died at birth from congenital cyanosis, likely resulting from heart defects. These included ventricular septation defects and outflow tract malformations that ranged from arterial common trunk to a condition known as double outlet right ventricle. Many other organs that normally express Sox11 also exhibited severe developmental defects. We observed various craniofacial and skeletal malformations, asplenia, and hypoplasia of the lung, stomach, and pancreas. Eyelids and the abdominal wall did not close properly in some Sox11-deficient mice. This phenotype suggests a prime function for Sox11 in tissue remodeling and identifies SOX11 as a potentially mutated gene in corresponding human malformation syndromes.

The trabecular meshwork outflow pathways: Structural and functional aspects

The major drainage structures for aqueous humor (AH) are the conventional or trabecular outflow pathways, which are comprised of the trabecular meshwork (made up by the uveal and corneoscleral meshworks), the juxtacanalicular connective tissue (JCT), the endothelial lining of Schlemms canal (SC), the collecting channels and the aqueous veins. The trabecular meshwork (TM) outflow pathways are critical in providing resistance to AH outflow and in generating intraocular pressure (IOP). Outflow resistance in the TM outflow pathways increases with age and primary open-angle glaucoma. Uveal and corneoscleral meshworks form connective tissue lamellae or beams that are covered by flat TM cells which rest on a basal lamina. TM cells in the JCT are surrounded by fibrillar elements of the extracellular matrix (ECM) to form a loose connective tissue. In contrast to the other parts of the TM, JCT cells and ECM fibrils do not form lamellae, but are arranged more irregularly. SC inner wall endothelial cells form giant vacuoles in response to AH flow, as well as intracellular and paracellular pores. In addition, minipores that are covered with a diaphragm are observed. There is considerable evidence that normal AH outflow resistance resides in the inner wall region of SC, which is formed by the JCT and SC inner wall endothelium. Modulation of TM cell tone by the action of their actomyosin system affects TM outflow resistance. In addition, the architecture of the TM outflow pathways and consequently outflow resistance appear to be modulated by contraction of ciliary muscle and scleral spur cells. The scleral spur contains axons that innervate scleral spur cells or that have the ultrastructural characteristics of mechanosensory nerve endings.

Myocilin and glaucoma: facts and ideas

Mutations in the MYOC gene that encodes for myocilin are causative for some forms of juvenile and adult-onset primary open-angle glaucoma (POAG). Myocilin is a secreted 55-57kDa glycoprotein that forms dimers and multimers. Characteristic structural motifs include a myosin-like domain, a leucine zipper region and an olfactomedin domain. Most of the mutations that have been identified in patients with POAG are localized in the olfactomedin domain, which is highly conserved among species. In the eye, myocilin is expressed in high amounts in the trabecular meshwork (TM), sclera, ciliary body and iris, and at considerable lower amounts in retina and optic nerve head. Secreted myocilin is present in the aqueous humor. In the TM, myocilin is found within the cytoplasm of TM cells and in the juxtacanalicular region in association with fibrillar extracellular matrix components. Since patients with mutations in myocilin may have high intraocular pressures, the role of myocilin for aqueous humor outflow has been investigated and conflicting results have been obtained. Recombinant myocilin increases outflow resistance in perfused anterior segment organ cultures, while overexpression of myocilin after viral gene transfer appears to reduce outflow resistance. In TM cells, the expression of myocilin is induced upon treatment with dexamethasone at a time course similar to that observed in steroid-induced glaucoma. Other factors that induce myocilin expression are transforming growth factor-beta and mechanical stretch. Promoter elements that are important for the glucocorticoid induction have not been identified, but it has been shown that upstream stimulatory factor is critical for the basal promoter activity of MYOC. Mice with a targeted disruption of the myocilin gene do not express a phenotype, indicating that the glaucomatous phenotype in humans is not because of a loss-of-function effect. Experimental studies show that mutated myocilin is not secreted, but appears to accumulate in the cells. Such an accumulation might interfere with TM function and lead to impaired outflow resistance, but, so far, experimental evidence for such a scenario is lacking. In addition, the normal function(s) of myocilin is (are) still elusive.

Morphological study of the anterior segment of cynomolgus monkey eyes following treatment with prostaglandin F2α

Topically applied prostaglandin F2 alpha has been shown to lower intraocular pressure in cynomolgus monkeys. In this study the morphological changes, following topical treatment with 4-50 micrograms of prostaglandin F2 alpha for 4-8 days, were investigated. Semiquantitation of (1) accumulation of white blood cells as one sign of inflammation, (2) edema and (3) enlarged spaces between ciliary muscle cells were performed. Eighty sections per eye encompassing the whole circumference were investigated. No accumulation of white blood cells was seen in any of the eyes. Slight edema in the most anterior part of the ciliary processes occurred in most eyes, but only in part of the circumference. These changes could be either directly induced by the prostaglandin treatment or secondary to the decrease in intraocular pressure. The most pronounced change was the dilatation of the intramuscular spaces. These enlarged spaces could explain the physiologically shown increase in uveoscleral outflow.

Inactivation of the murine X-linked juvenile retinoschisis gene, Rs1h, suggests a role of retinoschisin in retinal cell layer organization and synaptic structure

Deleterious mutations in RS1 encoding retinoschisin are associated with X-linked juvenile retinoschisis (RS), a common form of macular degeneration in males. The disorder is characterized by a negative electroretinogram pattern and by a splitting of the inner retina. To gain further insight into the function of the retinoschisin protein and its role in the cellular pathology of RS, we have generated knockout mice deficient in Rs1h, the murine ortholog of the human RS1 gene. We show that pathologic changes in hemizygous Rs1h−/Y male mice are evenly distributed across the retina, apparently contrasting with the macula-dominated features in human. Similar functional anomalies in human and Rs1h−/Y mice, however, suggest that both conditions are a disease of the entire retina affecting the organization of the retinal cell layers as well as structural properties of the retinal synapse.

Sox10 is required for Schwann cell identity and progression beyond the immature Schwann cell stage

The Sox10 transcription factor is required to maintain as well as specify glial identity, adding new causes for the neuropathies associated with SOX10 mutations.

The role of TGF-β in the pathogenesis of primary open-angle glaucoma

Transforming growth factor-β2 (TGF-β2) is found in increasing amounts in aqueous humor and reactive optic nerve astrocytes of patients with primary open-angle glaucoma (POAG), a major cause of blindness worldwide. The available data strongly indicate that TGF-β2 is a key player contributing to the structural changes in the extracellular matrix (ECM) of the trabecular meshwork and optic nerve head as characteristically seen in POAG. The changes involve an induction in the expression of various ECM molecules and are remarkably similar in trabecular meshwork cells and optic nerve head astrocytes. The ECM changes in the trabecular meshwork most probably play a role in the increase of aqueous humor outflow resistance causing higher intraocular pressure (IOP). In the optic nerve head, TGF-β2-induced changes might contribute to deformation of the optic nerve axons causing impairment of axonal transport and neurotrophic supply and leading to their continuous degeneration. The increase in IOP further adds mechanical stress and strain to optic nerve axons and accelerates degenerative changes. In addition, high IOP might induce the expression of activated TGF-β1 in trabecular meshwork cells and optic nerve head astrocytes; this again might significantly lead to the progress of axonal degeneration. The action of TGF-β2 in POAG is largely mediated through the connective tissue growth factor, whereas the activities of TGF-β1 and -β2 are modulated by the blocking effects of bone morphogenetic protein-4 (BMP-4) and BMP-7, by gremlin that inhibits BMP signaling and by several species of microRNAs.

Connective Tissue Growth Factor Causes Glaucoma by Modifying the Actin Cytoskeleton of the Trabecular Meshwork

The most critical risk factor for optic nerve damage in cases of primary open-angle glaucoma (POAG) is an increased intraocular pressure (IOP) caused by a resistance to aqueous humor outflow in the trabecular meshwork (TM). The molecular pathogenesis of this increase in outflow resistance in POAG has not yet been identified, but it may involve transforming growth factor TGF-β2, which is found in higher amounts in the aqueous humor of patients with POAG. Connective tissue growth factor (CTGF) is a TGF-β2 target gene with high constitutive TM expression. In this study, we show that either adenoviral-mediated or transgenic CTGF overexpression in the mouse eye increases IOP and leads to optic nerve damage. CTGF induces TM fibronectin and α-SMA in animals, whereas actin stress fibers and contractility are both induced in cultured TM cells. Depletion of CTGF by RNA interference leads to a marked attenuation of the actin cytoskeleton. Rho kinase inhibitors cause a reversible decline in the IOP of CTGF-overexpressing mice to levels seen in control littermates. Overall, the effects of CTGF on IOP appear to be caused by a modification of the TM actin cytoskeleton. CTGF-overexpressing mice provide a model that mimics the essential functional and structural aspects of POAG and offer a molecular mechanism to explain the increase of its most critical risk factor.

Norrin mediates neuroprotective effects on retinal ganglion cells via activation of the Wnt/beta-catenin signaling pathway and the induction of neuroprotective growth factors in Muller cells.

Norrin is a secreted protein that binds to frizzled 4 and controls development of capillaries in retina and inner ear. We provide evidence that Norrin has distinct neuroprotective properties that are independent from its effects on vascular development. The function of Norrin was investigated in a mouse model of excitotoxic retinal ganglion cell (RGC) damage after intravitreal injection of NMDA, and in cultured Müller glia or immortalized RGC-5 cells. Intravitreal injection of Norrin significantly increased the number of surviving RGC axons in the optic nerve and decreased apoptotic death of retinal neurons following NMDA-mediated damage. This effect could be blocked by adding dickkopf (DKK)-1, an inhibitor of the Wnt/β-catenin signaling pathway. Treatment of eyes with combined Norrin/NMDA activated Wnt/β-catenin signaling and increased the retinal expression of leukemia inhibitory factor and endothelin-2, as well as that of neurotrophic growth factors such as fibroblast growth factor-2, brain-derived neurotrophic factor, lens epithelium-derived growth factor, and ciliary neurotrophic factor. A similar activation of Wnt/β-catenin signaling and an increased expression of neurotrophic factors was observed in cultured Müller cells after treatment with Norrin, effects that again could be blocked by adding DKK-1. In addition, conditioned cell culture medium of Norrin-treated Müller cells increased survival of differentiated RGC-5 cells. We conclude that Norrin has pronounced neuroprotective properties on retinal neurons with the distinct potential to decrease the damaging effects of NMDA-induced RGC loss. The effects of Norrin involve activation of Wnt/β-catenin signaling and subsequent induction of neurotrophic growth factors in Müller cells.

Connective tissue growth factor induces extracellular matrix deposition in human trabecular meshwork cells

The major structural change in the human trabecular meshwork (TM) of eyes with primary open-angle glaucoma (POAG) is an increase in extracellular matrix (ECM) in the juxtacanalicular region of the TM. There is evidence that treatment with TGF-beta2 causes an induction of ECM deposition in cultured human TM cells and that TGF-beta2 is causatively involved in the JCT ECM increase in POAG. In the present study, we investigated the effects of connective tissue growth factor (CTGF) on the biology of cultured human TM cells. CTGF is a downstream mediator of TGF-beta2-signaling, which is expressed at high amounts in the human TM in situ. HEK293 cells were transfected with an eukaryotic expression plasmid containing the coding sequences of human CTGF. Secreted CTGF was isolated and purified by chromatography. Primary human TM cells were incubated for 24 h with CTGF at concentrations of 2.5-100 ng/ml. Following treatment with CTGF, the expression of various ECM components that are expressed in the JCT, matrix metalloproteinases (MMPs) and integrins was investigated by real-time RT-PCR and western blot analyses. In addition, the activity of MMPs was investigated by gelatine zymography. The effect of CTGF silencing on TGF-beta2-induced gene expression was investigated by transfection of immortalized HTM cells with CTGF-specific small interfering (si)RNA before TGF-beta2 treatment. CTGF-treated human TM cells showed an increase in the expression of fibronectin, collagen types I, III, IV and VI, as well as in the integrin subunits aV and beta1. Lower concentrations of CTGF caused an autoinduction of CTGF expression. No effects were observed on the expression and activity of MMP-2, MMP-9 and plasminogen activator inhibitor-1 (PAI-1). Transfection with CTGF-specific siRNA inhibited the TGF-beta2-induced upregulation of CTGF and fibronectin. Our results indicate that treatment of human TM cells with recombinant CTGF causes distinct changes in gene expression and that CTGF is a critical mediator of the effects of TGF-beta2 on ECM synthesis in human TM cells. An intriguing aspect supported by the data of the present work is that the pharmacologic modulation of CTGF might be a useful approach to develop novel therapeutic strategies to prevent or to reverse the structural changes that occur in the TM of eyes with POAG.