Enhanced Optic Nerve Expansion and Altered Ultrastructure of Elastic Fibers Induced by Lysyl Oxidase Inhibition in a Mouse Model of Marfan Syndrome.

Two major constituents of exfoliation material, fibrillin-1 and lysyl oxidase-like 1 (encoded by FBN1 and LOXL1), are implicated in exfoliation glaucoma, yet their individual contributions to ocular phenotype are minor. To test the hypothesis that a combination of FBN1 mutation and LOXL1 deficiency exacerbates ocular phenotypes, the pan-LOX inhibitor ß-aminopropionitrile (BAPN) was used to treat adult wild-type (WT) and Fbn1C1041G/+ mice for 8 weeks and their eyes were examined. Although intraocular pressure did not change and exfoliation material was not detected in the eyes, BAPN treatment worsened optic nerve and axon expansion in Fbn1C1041G/+ mice, an early sign of axonal damage in rodent models of glaucoma. Disruption of elastic fibers was detected only in Fbn1C1041G/+ mice, which increased with BAPN treatment, as shown by histologic and immunohistochemical staining of the optic nerve pia mater. Transmission electron microscopy showed that Fbn1C1041G/+ mice had fewer microfibrils, smaller elastin cores, and a lower density of elastic fibers compared with WT mice in control groups. BAPN treatment led to elastin core expansion in both WT and Fbn1C1041G/+ mice, but an increase in the density of elastic fiber was confined to Fbn1C1041G/+ mice. LOX inhibition had a stronger effect on optic nerve and elastic fiber parameters in the context of Fbn1 mutation, indicating the Marfan mouse model with LOX inhibition warrants further investigation for exfoliation glaucoma pathogenesis.

Optic neuropathy associated with TGFß dysregulation in mice with a glaucoma-causative mutation of ADAMTS10.

Glaucoma is a neurodegenerative disease that causes irreversible blindness due to loss of retinal ganglion cells (RGCs) and their axons. We previously identified a G661R mutation of ADAMTS10 (A Disintegrin And Metalloproteinase with ThromboSpondin type 1 motif 10) as the disease-causing mutation in a beagle model of glaucoma. ADAMTS10 is a secreted matrix metalloproteinase that belongs to the ADAMTS family which is involved in extracellular matrix (ECM) turnover. Previous studies have shown that ADAMTS10 binds fibrillin microfibrils, promotes their formation, and influences their fibrillin isoform composition. Here, we established a mouse model carrying the G661R mutation of ADAMTS10 (ADAMTS10G661R/G661R) to investigate its ocular phenotypes related to glaucoma and to explore possible functions of ADAMTS10. We found that ADAMTS10 was expressed in the inner retina and along RGC axons in the optic nerve. However, ADAMTS10 was not colocalized with fibrillin microfibrils in these tissues, suggesting fibrillin-independent function for ADAMTS10. In electroretinogram experiments, we found that ADAMTS10G661R/G661R mice had reduced amplitude of retinal responses to dim light stimulus, indicating RGC dysfunction. The reduced RGC function coincided with RGC axon structural changes manifested as smaller optic nerves and fewer optic nerve axons, which may contribute to glaucoma. The reduced number of optic nerve axons found for ADAMTS10G661R/G661R mice occurred early, suggesting developmental deficits. Subsequent experiments found increased apoptosis in the retina of ADAMTS10G661R/G661R mice during postnatal development, which could result in fewer RGCs produced, accounting for fewer optic nerve axons in adulthood. Consistent with a protective effect of transforming growth factor ß (TGFß) signaling against apoptosis during retinal development as shown previously by others, we found increased apoptosis accompanied by decreased TGFß signaling in the developing retina of ADAMTS10G661R/G661R mice, suggesting a novel role for ADAMTS10 in regulating TGFß signaling which could involve direct interaction between ADAMTS10 and latent TGFß.

Lysyl oxidase-like 1 deficiency alters ultrastructural and biomechanical properties of the peripapillary sclera in mice.

Lysyl oxidase-like 1 encoded by the LOXL1 gene is a member of the lysyl oxidase family of enzymes that are important in the maintenance of extracellular matrix (ECM)-rich tissue. LOXL1 is important for proper elastic fiber formation and mice lacking LOXL1 (Loxl1-/- ) exhibit systemic elastic fiber disorders, such as pelvic organ prolapse, a phenotype associated with exfoliation syndrome (XFS) in humans. Patients with XFS have a significant risk of developing exfoliation glaucoma (XFG), a severe form of glaucoma, which is a neurodegenerative condition leading to irreversible blindness if not detected and treated in a timely fashion. Although Loxl1-/- mice have been used extensively to investigate mechanisms of pelvic organ prolapse, studies of eyes in those mice are limited and some showed inconsistent ocular phenotypes. In this study we demonstrate that Loxl1-/- mice have significant anterior segment biometric abnormalities which recapitulate some human XFS features. We then focused on the peripapillary sclera (PPS), a critical structure for maintaining optic nerve health. We discovered quantitative and qualitive changes in ultrastructure of PPS, such as reduced elastic fibers, enlarged collagen fibrils, and transformed collagen lamella organization detected by transmission electron microscopy (TEM). Importantly, these changes corelate with altered tissue biomechanics detected by Atomic Force Microscopy (AFM) of PPS in mice. Together, our results support a crucial role for LOXL1 in ocular tissue structure and biomechanics, and Loxl1-/- mice could be a valuable resource for understanding the role of scleral tissue biomechanics in ocular disease.

Adamts10 controls transforming growth factor ß family signaling that contributes to retinal ganglion cell development.

Although mutations in ADAMTS10 have long been known to cause autosomal recessive Weill-Marchesani Syndrome which is characterized by short stature and ocular abnormalities, more recent work has shown that certain mutations in ADAMTS10 cause glaucoma in dogs. In humans, glaucoma is the leading cause of irreversible vision loss that affects tens of millions of people world-wide. Vision loss in glaucoma is a result of neurodegeneration of retinal ganglion cells that form the inner-most layer of the retina and whose axons form the optic nerve which relays visual information to the brain. ADAMTS10 contributes to the formation of microfibrils which sequester latent transforming growth factor ß (TGFß). Among its many biological functions, TGFß promotes the development of retinal ganglion cells and is also known to play other roles in glaucoma pathogenesis. The aim of this study was to test the hypothesis that ADAMTS10 plays a role in retinal ganglion cell development through regulation of TGFß signaling. To this end, Adamts10 expression was targeted for reduction in zebrafish embryos carrying either a fluorescent reporter that labels retinal ganglion cells, or a fluorescent reporter of pSmad3-mediated TGFß family signaling. Loss of adamts10 function in zebrafish embryos reduced retinal ganglion cell reporter fluorescence and prevented formation of an ordered retinal ganglion cell layer. Targeting adamts10 expression also drastically reduced constitutive TGFß signaling in the eye. Direct inhibition of the TGFß receptor reduced retinal ganglion cell reporter fluorescence similar to the effect of targeting adamts10 expression. These findings unveil a previously unknown role for Adamts10 in retinal ganglion cell development and suggest that the developmental role of Adamts10 is mediated by active TGFß family signaling. In addition, our results show for the first time that Adamts10 is necessary for pSmad3-mediated constitutive TGFß family signaling.

Altered Ocular Fibrillin Microfibril Composition in Mice With a Glaucoma-Causing Mutation of Adamts10.

Purpose: Previously, we identified a G661R mutation of ADAMTS10 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif 10) as being disease causative in a colony of Beagles with inherited primary open-angle glaucoma (POAG). Mutations in ADAMTS10 are known to cause Weill-Marchesani syndrome (WMS), which is also caused by mutations in the fibrillin-1 gene (FBN1), suggesting functional linkage between ADAMTS10 and fibrillin-1, the principal component of microfibrils. Here, we established a mouse line with the G661R mutation of Adamts10 (Adamts10G661R/G661R) to determine if they develop features of WMS and alterations of ocular fibrillin microfibrils. Methods: Intraocular pressure (IOP) was measured using a TonoLab rebound tonometer. Central cornea thickness (CCT), anterior chamber depth (ACD) and axial length (AL) of the eye were examined by spectral-domain optical coherence tomography. Sagittal eye sections from mice at postnatal day 10 (P10) and at 3 and 24 months of age were stained with antibodies against fibrillin-1, fibrillin-2, and ADAMTS10. Results: IOP was not elevated in Adamts10G661R/G661R mice. Adamts10G661R/G661R mice had smaller bodies, thicker CCT, and shallower ACD compared to wild-type mice but normal AL. Adamts10G661R/G661R mice displayed persistent fibrillin-2 and enhanced fibrillin-1 immunofluorescence in the lens zonules and in the hyaloid vasculature and its remnants in the vitreous. Conclusions: Adamts10G661R/G661R mice recapitulate the short stature and ocular phenotypes of WMS. The altered fibrillin-1 and fibrillin-2 immunoactivity in Adamts10G661R/G661R mice suggests that the G661R mutation of Adamts10 perturbs regulation of the fibrillin isotype composition of microfibrils in the mouse eye.

Increased Susceptibility to Glaucomatous Damage in Microfibril Deficient Mice.

Purpose: To test whether mice with microfibril deficiency due to the Tsk mutation of fibrillin-1 (Fbn1Tsk/+) have increased susceptibility to pressure-induced retinal ganglion cell (RGC) degeneration. Methods: Intraocular pressure (IOP) elevation was induced in Fbn1Tsk/+ and wild type (wt) mice by injecting microbeads into the anterior chamber. Mice were then followed up for four months, with IOP measurements every three to six days. Retinas were stained for Brn3a to determine RGC number. Optic nerve cross-sections were stained with p-phenylene diamine to determine nerve area, axon number, and caliber and thickness of the pia mater. Results: Microbead injection induced significant IOP elevation that was significantly less for Fbn1Tsk/+ mice compared with wt. The optic nerves and optic nerve axons were larger, and the elastic fiber-rich pia mater was thinner in Fbn1Tsk/+ mice. Microbead injection resulted in reduced optic nerve size, thicker pia mater, and a slight decrease in axon size. Fbn1Tsk/+ mice had significantly greater loss of RGCs and optic nerve axons compared with wt (14.8% vs. 5.8%, P = 0.002, and 17.0% vs. 7.5%, P = 0.002, respectively). Conclusions: Fbn1Tsk/+mice had altered optic nerve structure as indicated by larger optic nerves, larger optic nerve axons and thinner pia mater, consistent with our previous findings. Despite lower IOP elevation, Fbn1Tsk/+mice had greater loss of RGCs and optic nerve axons, suggesting increased susceptibility to IOP-induced optic nerve degeneration in microfibril-deficient mice.

Telmisartan Reduces Axon Degeneration in Mice With Experimental Glaucoma.

Purpose: The purpose of this study was to determine if treatment with telmisartan, an angiotensin II type 1 receptor blocker (ARB), protects against retinal ganglion cell (RGC) degeneration in a mouse glaucoma model with induced elevation of intraocular pressure (IOP). Methods: IOP elevation was induced by injection of polystyrene microbeads into the anterior chamber of the right eye of 3-month-old C57BL/6J mice, with the left eye serving as contralateral control. Starting the day of microbead injection, mice were maintained on solid food pellets with or without incorporated telmisartan. IOP was measured by Tono Lab tonometry prior to and weekly after microbead injection. Twelve weeks postinjection, mice were euthanized to obtain optic nerves for analysis of RGC axons. The total numbers of optic nerve axons were determined manually and automatedly using AxonJ. Degenerating axons were counted manually. Results: IOP elevation induced by microbead injection was similar in magnitude and duration in vehicle and telmisartan-fed mice, although IOP was reduced 5.8% in uninjected mice treated with telmisartan (P = 0.0027). Axon loss determined by manual and automated methods was greater in vehicle compared to telmisartan-treated mice (manual: 9.5% vs. 1.8%, P = 0.044; automated: 14.2% vs. 2.9%, P = 0.0375). An increase in the percent of axons undergoing degeneration was observed in nerves from microbead-injected eyes that was greater in vehicle-treated compared to telmisartan-treated mice (49.0% vs. -0.58%, P = 0.0019). Conclusions: Elevation of IOP by microbead injection led to loss of RGC axons in vehicle-treated mice that was largely prevented by telmisartan treatment, suggesting a neuroprotective effect of telmisartan.

Enlarged Optic Nerve Axons and Reduced Visual Function in Mice with Defective Microfibrils.

Glaucoma is a leading cause of irreversible vision loss due to retinal ganglion cell (RGC) degeneration that develops slowly with age. Elevated intraocular pressure (IOP) is a significant risk factor, although many patients develop glaucoma with IOP in the normal range. Mutations in microfibril-associated genes cause glaucoma in animal models, suggesting the hypothesis that microfibril defects contribute to glaucoma. To test this hypothesis, we investigated IOP and functional/structural correlates of RGC degeneration in mice of either sex with abnormal microfibrils due to heterozygous Tsk mutation of the fibrilin-1 gene (Fbn1Tsk/+). Although IOP was not affected, Fbn1Tsk/+ mice developed functional deficits at advanced age consistent with glaucoma, including reduced RGC responses in electroretinogram (ERG) experiments. While RGC density in the retina was not affected, the density of RGC axons in the optic nerve was significantly reduced in Fbn1Tsk/+ mice. However, reduced axon density correlated with expanded optic nerves, resulting in similar numbers of axons in Fbn1Tsk/+ and control nerves. Axons in the optic nerves of Fbn1Tsk/+ mice were significantly enlarged and axon diameter was strongly correlated with optic nerve area, as has been reported in early pathogenesis of the DBA/2J mouse model of glaucoma. Our results suggest that microfibril abnormalities can lead to phenotypes found in early-stage glaucomatous neurodegeneration. Thinning of the elastic fiber-rich pia mater was found in Fbn1Tsk/+ mice, suggesting mechanisms allowing for optic nerve expansion and a possible biomechanical contribution to determination of axon caliber.

Dopamine D1 receptor-mediated upregulation of BKCa currents modifies Müller cell gliosis in a rat chronic ocular hypertension model.

Müller cell gliosis is a common response in many retinal pathological conditions. We previously demonstrated that downregulation of Kir channels contributes to Müller cell gliosis in a rat chronic ocular hypertension (COH) model. Here, the possible involvement of outward K+ currents in Müller cell gliosis was investigated. Outward K+ current densities in Müller cells isolated from COH rats, as compared with those in normal rats, showed a significant increase, which was mainly contributed by large-conductance Ca2+ -activated K+ (BKCa ) channels. The involvement of BKCa channels in Müller cell gliosis is suggested by the fact that glial fibrillary acidic protein (GFAP) levels were augmented in COH retinas when these channels were suppressed by intravitreal injections of iberiotoxin. In COH retinas an increase in dopamine (DA) D1 receptor (D1R) expression in Müller cells was revealed by both immunohistochemistry and Western blotting. Moreover, protein levels of tyrosine hydroxylase were also increased, and consistent to this, retinal DA contents were elevated. SKF81297, a selective D1R agonist, enhanced BKCa currents of normal Müller cells through intracellular cAMP-PKA signaling pathway. Furthermore, GFAP levels were increased by the D1R antagonist SCH23390 injected intravitreally through eliminating the BKCa current upregulation in COH retinas, but partially reduced by SKF81297. All these results strongly suggest that the DA-D1R system may be activated to a stronger extent in COH rat retinas, thus increasing BKCa currents of Müller cells. The upregulation of BKCa channels may antagonize the Kir channel inhibition-induced depolarization of Müller cells, thereby attenuating the gliosis of these cells.

Hydrogen Sulfide Targets the Cys320/Cys529 Motif in Kv4.2 to Inhibit the Ito Potassium Channels in Cardiomyocytes and Regularizes Fatal Arrhythmia in Myocardial Infarction.

AIMS: The mechanisms underlying numerous biological roles of hydrogen sulfide (H2S) remain largely unknown. We have previously reported an inhibitory role of H2S in the L-type calcium channels in cardiomyocytes. This prompts us to examine the mechanisms underlying the potential regulation of H2S on the ion channels. RESULTS: H2S showed a novel inhibitory effect on Ito potassium channels, and this effect was blocked by mutation at the Cys320 and/or Cys529 residues of the Kv4.2 subunit. H2S broke the disulfide bridge between a pair of oxidized cysteine residues; however, it did not modify single cysteine residues. H2S extended action potential duration in epicardial myocytes and regularized fatal arrhythmia in a rat model of myocardial infarction. H2S treatment significantly increased survival by ∼1.4-fold in the critical 2-h time window after myocardial infarction with a protection against ventricular premature beats and fatal arrhythmia. However, H2S did not change the function of other ion channels, including IK1 and INa. INNOVATION AND CONCLUSION: H2S targets the Cys320/Cys529 motif in Kv4.2 to regulate the Ito potassium channels. H2S also shows a potent regularizing effect against fatal arrhythmia in a rat model of myocardial infarction. The study provides the first piece of evidence for the role of H2S in regulating Ito potassium channels and also the specific motif in an ion channel labile for H2S regulation.