John Danias

Pseudoexfoliation syndrome, a systemic disorder with ocular manifestations

Pseudoexfoliation syndrome (PXS) is a systemic condition with eye manifestations. In the eye, pseudoexfoliation material deposits on various structures of the anterior segment. The nature of this material is mostly fibrillar with fibers made up of microfibrils and coated with amorphous material. The composition of these fibrils is diverse and includes basement membrane components as well as enzymes involved in extracellular matrix maintenance. Pseudoexfoliation is the most common cause of secondary open-angle glaucoma (pseudoexfoliation glaucoma, PXG) worldwide. The goal of this review is to summarize our knowledge on the genetics of this systemic disorder and its resultant ocular manifestations. PXS familial aggregation suggests genetic inheritance. PXS has been strongly associated with single nucleotide polymorphisms (SNPs) of the lysyl oxidase-like 1 (LOXL1) gene on chromosome 15q24.1. Two of these SNPs confer a higher than 99% population attributable risk for PXS and PXG in the Nordic population; however, they carry different risks in different populations. The high risk haplotypes also vary among different populations. LOXL1 is one of group of the enzymes involved in the cross-linking of collagen and elastin in the extracellular matrix. Its function in connective tissue maintenance has been confirmed in mice; however, its actual role in PXS remains unclear. Contactin-associated protein-like 2 also has a strong genetic association with PXS in a German cohort and is an attractive candidate molecule. It encodes for a protein involved in potassium channel trafficking. Other candidate genes linked to PXS include lysosomal trafficking regulator, clusterin, adenosine receptors, matrix metalloproteinase-1 (MMP1), and glutathione transferase. These genes may be modifying genes for development of PXS and PXG.

Morphologic changes in the outflow pathways of bovine eyes treated with corticosteroids.

PURPOSEnTo analyze morphologic changes in the trabecular meshwork (TM) of bovine eyes treated with topical prednisolone and exhibiting elevated intraocular pressure for 4 weeks.nnnMETHODSnThe TM of four adult Braford cow eyes treated with 0.5% prednisolone eye drops three times daily for 7 weeks and their contralateral eyes treated with artificial tear preparation and that of two adult untreated Braford cows and untreated young calves eyes were analyzed with light and electron microscopy. Increased extracellular matrix (ECM) under the outflow loops was evaluated quantitatively. Additionally, deparaffinized tissue of treated eyes was labeled with an antibody against type VI collagen for immunocytochemistry.nnnRESULTSnIn steroid-treated eyes ECM (plaques) accumulated under the endothelium of the inner wall of the outflow loops. On electron microscopy, this material contained fine fibrils that labeled for type VI collagen. Plaques were also seen in the contralateral controls of the treated animals but here they were significantly less in amount. In the untreated Braford controls and in untreated calf eyes, plaques were nearly absent. In the TM cells of the treated eyes there was a loss of glycogen from the cytoplasm and an increase in basement membrane-like material. These changes were not seen in contralateral eyes or eyes of untreated animals.nnnCONCLUSIONSnAccumulations of ECM in the treated eyes resembled morphologic changes in human eyes with primary open-angle glaucoma and steroid-induced glaucoma. This animal model, therefore, provides a good tool in which to further study the pathogenesis of TM changes in glaucoma.

Triamcinolone Acetonide Decreases Outflow Facility in C57BL/6 Mouse Eyes

PURPOSEnTo determine the effect of triamcinolone acetonide (TA) on outflow facility in mice.nnnMETHODSnAnimals received 20 μL of TA (40 mg/mL) suspension subconjunctivally either bilaterally or unilaterally and were euthanized after either 1 week or 3 weeks. Before mice were killed, IOP was measured with a rebound tonometer. Outflow facility was determined using simultaneous pressure and flow measurements. Another set of animals received bilateral injection of anecortave acetate (AA) with or without bilateral TA injection and their outflow facility was also determined. Myocilin expression was investigated in a subset of eyes using quantitative PCR (qPCR).nnnRESULTSnOutflow facility of eyes in animals receiving bilateral TA injection (TA(BL)) and TA-treated eyes of animals receiving unilateral injection (TA(UL)) was significantly decreased compared to naïve control eyes (C(naive)) after 1 week and 3 weeks of TA treatment (ANOVA P < 0.01, P < 0.001, respectively). Eyes treated with AA (with or without TA) had higher outflow facility than animals treated with TA (P < 0.05). IOP data did not show any significant difference between groups. qPCR analysis revealed significant decrease in myocilin expression in eyes receiving AA compared to naïve control and TA-treated eyes (ANOVA P < 0.001).nnnCONCLUSIONSnSteroid treatment significantly decreases outflow facility in C57BL/6 mice despite having small effect on IOP. This animal model can be useful for studying the pathogenesis of steroid-induced glaucoma.

Oral Microbiome Link to Neurodegeneration in Glaucoma

Background Glaucoma is a progressive optic nerve degenerative disease that often leads to blindness. Local inflammatory responses are implicated in the pathology of glaucoma. Although inflammatory episodes outside the CNS, such as those due to acute systemic infections, have been linked to central neurodegeneration, they do not appear to be relevant to glaucoma. Based on clinical observations, we hypothesized that chronic subclinical peripheral inflammation contributes to neurodegeneration in glaucoma. Methods Mouthwash specimens from patients with glaucoma and control subjects were analyzed for the amount of bacteria. To determine a possible pathogenic mechanism, low-dose subcutaneous lipopolysaccharide (LPS) was administered in two separate animal models of glaucoma. Glaucomatous neurodegeneration was assessed in the retina and optic nerve two months later. Changes in gene expression of toll-like receptor 4 (TLR4) signaling pathway and complement as well as changes in microglial numbers and morphology were analyzed in the retina and optic nerve. The effect of pharmacologic blockade of TLR4 with naloxone was determined. Findings Patients with glaucoma had higher bacterial oral counts compared to control subjects (p<0.017). Low-dose LPS administration in glaucoma animal models resulted in enhancement of axonal degeneration and neuronal loss. Microglial activation in the optic nerve and retina as well as upregulation of TLR4 signaling and complement system were observed. Pharmacologic blockade of TLR4 partially ameliorated the enhanced damage. Conclusions The above findings suggest that the oral microbiome contributes to glaucoma pathophysiology. A plausible mechanism by which increased bacterial loads can lead to neurodegeneration is provided by experiments in animal models of the disease and involves activation of microglia in the retina and optic nerve, mediated through TLR4 signaling and complement upregulation. The finding that commensal bacteria may play a role in the development and/or progression of glaucomatous pathology may also be relevant to other chronic neurodegenerative disorders.

Gene Expression Changes in Steroid-Induced IOP Elevation in Bovine Trabecular Meshwork

PURPOSEnTo determine whether gene expression changes occur in the trabecular meshwork (TM) of cow eyes with steroid-induced intraocular pressure (IOP) elevation.nnnMETHODSnAdult female Braford cows (n = 4) were subjected to uniocular prednisolone acetate treatment for 6 weeks. IOP was monitored with an applanation tonometer. At the conclusion of the experiment, animals were euthanized, eyes were enucleated, and the TM was dissected and stored in an aqueous nontoxic tissue storage reagent. RNA was extracted and subjected to microarray analysis using commercial oligonucleotide bovine arrays. Some of the genes differentially expressed between control and experimental eyes were confirmed by quantitative RT-PCR and some of the respective proteins were studied by immunoblotting.nnnRESULTSnIOP began to increase after 3 weeks of treatment, reaching a peak 2 weeks later. IOP differences between corticosteroid-treated and fellow control eyes were 6 ± 1 mm Hg (mean ± SD) at the conclusion of the study. Microarray analysis revealed that expression of 258 genes was upregulated, whereas expression of 187 genes was downregulated in the TM of eyes with steroid-induced IOP elevation. Genes identified to be differentially expressed include genes coding for cytoskeletal proteins, enzymes, growth and transcription factors, as well as extracellular matrix proteins and immune response proteins. A number of relevant gene networks were detected by bioinformatic analysis.nnnCONCLUSIONSnSteroid-induced IOP elevation alters gene expression in the bovine TM. Identification of genes with changing expression in this model of open-angle glaucoma may help elucidate the primary changes occurring at the molecular level in this condition.

A Role for Complement in Glaucoma

Chronic open angle glaucoma is a degenerative optic neuropathy that can lead to blindness. We have shown that one of the major genes with altered expression in the glaucomatous retina is complement component C1q in both animal models of the disease as well as in humans. These observations together with evidence of upregulation of other complement components within the retina suggest a role for complement in the pathogenesis of this disease. We review the current evidence that supports such a role and discuss possible mechanisms through which complement may act. A thorough understanding of these mechanisms is important in allowing us to rationally design new therapeutic approaches.

Tissue Plasminogen Activator in Trabecular Meshwork Attenuates Steroid Induced Outflow Resistance in Mice

Tissue plasminogen activator, a serine protease encoded by the PLAT gene is present in the trabecular meshwork (TM) and other ocular tissues and has been reported to be downregulated by treatment with steroids in vitro. Steroids are known to cause changes in outflow facility of aqueous humor in many species. In the present study, we tested whether overexpression of PLAT can prevent and/or reverse the outflow facility of mouse eyes treated with steroids. Animals received bilateral injection with 20 µl of triamcinolone acetonide (TA) (40mg/ml) suspension subconjunctivally to induce outflow facility changes. Some animals received unilateral intracameral injection with 2 µl of adenoviral suspension [3-4x1012 virus genomes per milliliter (vg/ml)] carrying sheep PLAT cDNA (AdPLAT) either concurrently with TA injection or one week after TA injection, whereas others received bilateral intracameral injection with 2µl of adenoviral suspension (9x1012 vg/ml) carrying no transgene (AdNull) concurrently with TA injection. Animals were sacrificed one week after AdPLAT or AdNull treatment. Endogenous mRNA expression levels of mouse PAI-1 and MMP-2, -9 and -13 were also measured using qRT-PCR. Outflow facility one week after AdPLAT administration was increased by 60% and 63% respectively for animals that had not or had been pretreated with steroids. Overexpression of PLAT significantly upregulated expression of PAI-1, MMP-2, -9 and -13 compared to the levels found in TA only treated eyes. These findings suggest that overexpression of PLAT in TM of mouse eyes can both prevent and reverse the decrease in outflow facility caused by steroid treatment and is associated with upregulation of MMPs.

Retinal Gene Expression Changes Related to IOP Exposure and Axonal Loss in DBA/2J Mice

PURPOSEnTo determine the effects of cumulative IOP exposure and axonal damage on retinal gene expression in DBA/2 mice.nnnMETHODSnDBA/2J, DBA/2J(pe) (pearl), and C57BL/6 mice from 3 to 12 months of age were used. IOP was measured with a rebound tonometer, and optic nerve (ON) damage was determined by grading of ON sections. Retinal RNA was subjected to microarray analysis. Comparisons explored the effects of cumulative IOP exposure (cIOPx) as well as ON damage (ONd) in the DBA/2J animals compared with that in the C57BL/6 and pearl mice. RT-PCR was performed to confirm some of the genes and bioinformatic analysis to identify affected gene networks.nnnRESULTSnMicroarrays revealed that an increasing number of genes were up- or downregulated in 9- and 12-month DBA/2J mice with various degrees of ONd. A smaller number of genes were expressed differentially between eyes with different cIOPx at the same age, from 6 months on. Expression of 1385 and 1133 genes differed between DBA/2J animals and C57BL/6 or pearl mice, respectively, and some them were confirmed by RT-PCR. Bioinformatics analysis identified functional gene networks, including members of the complement system, that appeared to be related to cIOPx, ONd, or both.nnnCONCLUSIONSnGene expression changes occur in retinas of DBA/2 mice with various amounts of cIOPx as well as ONd. Genes involved, code for proteins with diverse cellular functions and include among others the complement system. cIOPx and ONd affect common as well as unique gene sets.

Recreating a human trabecular meshwork outflow system on microfabricated porous structures

Glaucoma is the leading cause of irreversible blindness, resulting from an increase in intraocular pressure (IOP). IOP is the only modifiable risk factor of glaucoma and is controlled by the outflow of the aqueous humor through the human trabecular meshwork (HTM). Currently, the lack of a proper in vitro HTM model impedes advances in understanding outflow physiology and discovering effective IOP‐lowering anti‐glaucoma therapeutics. Therefore, we designed and constructed an in vitro HTM model using micropatterned, porous SU‐8 scaffolds, which support cells to recapitulate functional HTM morphology and allow the study of outflow physiology. The pore size of SU‐8 scaffolds, surface coating, cell seeding density, and culture duration were evaluated for HTM cell growth. The bioengineered HTM was characterized by F‐actin staining and immunocytochemistry of HTM markers. A stand‐alone perfusion chamber with an integrated pressure sensing system was further constructed and used for the investigation of the outflow facility of the bioengineered HTM treated with latrunculin B—an IOP lowering agent. Cells in the in vitro model exhibited HTM‐like morphology, expression of α‐smooth muscle actin, myocilin, and αB‐crystallin, outflow characteristics and drug responsiveness. Altogether, we have developed an in vitro HTM model system for understanding HTM cell biology and screening of pharmacological or biological agents that affect trabecular outflow facility, expediting discovery of IOP‐lowering, anti‐glaucoma therapeutics. Biotechnol. Bioeng. 2013;110: 3205–3218.

Reduction of Steroid-Induced Intraocular Pressure Elevation in Sheep by Tissue Plasminogen Activator

PURPOSEnTo investigate whether tissue plasminogen activator (tPA) can prevent and/or reverse steroid-induced IOP elevation in an ovine model.nnnMETHODSnThree animal groups were subjected to bilateral steroid-induced IOP elevation using thrice daily topical ocular prednisolone administration. In the first group (N = 8), one eye each of two sheep was injected intravitreally with 100 μg, 200 μg, 500 μg, or 1 mg human recombinant tPA, while contralateral eyes received vehicle. In the second group (N = 2), one eye was injected intravitreally with tPA (100 μg), while contralateral eyes received vehicle containing L-arginine. In the third group (N = 4), each animal received intravitreal tPA in one eye concurrently with initiation of bilateral steroid administration. IOP was monitored for the duration of the experiment. Tissues from eyes of the third group were used to determine relative gene expression.nnnRESULTSnIn the first and second groups, IOP decreased by 9.7 (±2.8) and 9.7 (±1.6) mm Hg, respectively, 24 hours after tPA administration. In the third group, tPA-treated eyes did not develop IOP elevation with ΔIOP of 11.8 (±1.3) mm Hg 8 days later. In all tPA-treated eyes, IOP remained low until the end of the study. mRNA levels in the trabecular meshwork were decreased for plasminogen activator tissue (PLAT), increased for matrix-metalloproteinase 1 (MMP-1), and stable for plasminogen activator inhibitor 1 (PAI-1), MMP-2, MMP-9, and MMP-13 in tPA-treated eyes compared with contralateral controls. PAI-1 mRNA levels in ciliary processes also remained similar.nnnCONCLUSIONSnRecombinant human tPA is effective in both preventing and reversing steroid-induced IOP elevation in sheep. Tissue plasminogen activator may be useful as a therapeutic agent in steroid-induced glaucoma.