Remodeling of the Lamina Cribrosa: Mechanisms and Potential Therapeutic Approaches for Glaucoma.

Glaucomatous optic neuropathy is the leading cause of irreversible blindness in the world. The chronic disease is characterized by optic nerve degeneration and vision field loss. The reduction of intraocular pressure remains the only proven glaucoma treatment, but it does not prevent further neurodegeneration. There are three major classes of cells in the human optic nerve head (ONH): lamina cribrosa (LC) cells, glial cells, and scleral fibroblasts. These cells provide support for the LC which is essential to maintain healthy retinal ganglion cell (RGC) axons. All these cells demonstrate responses to glaucomatous conditions through extracellular matrix remodeling. Therefore, investigations into alternative therapies that alter the characteristic remodeling response of the ONH to enhance the survival of RGC axons are prevalent. Understanding major remodeling pathways in the ONH may be key to developing targeted therapies that reduce deleterious remodeling.

Association between metabolic risk factors and optic disc cupping identified by deep learning method.

PURPOSE: This study aims to investigate correlation between metabolic risk factors and optic disc cupping and the development of glaucoma. METHODS: This study is a retrospective, cross-sectional study with over 20-year-old patients that underwent health screening examinations. Intraocular pressure (IOP), fundus photographs, Body Mass Index (BMI), waist circumference (WC), serum triglycerides, serum HDL cholesterol (HDL-C), serum LDL cholesterol (LDL-C), systolic blood pressure (BP), diastolic BP, and serum HbA1c were obtained to analyse correlation between metabolic risk factors and glaucoma. Eye with glaucomatous optic neuropathy(GON) was defined as having an optic disc with either vertical cup-to-disc ratio(VCDR) ≥ 0.7 or a VCDR difference ≥ 0.2 between the right and left eyes by measuring VCDR with deep learning approach. RESULTS: The study comprised 15,585 subjects and 877 subjects were diagnosed as GON. In univariate analyses, age, BMI, systolic BP, diastolic BP, WC, triglyceride, LDL-C, HbA1c, and IOP were significantly and positively correlated with VCDR in the optic nerve head. In linear regression analysis as independent variables, stepwise multiple regression analyses revealed that age, BMI, systolic BP, HbA1c, and IOP showed positive correlation with VCDR. In multivariate logistic analyses of risk factors and GON, higher age (odds ratio [OR], 1.054; 95% confidence interval [CI], 1.046-1.063), male gender (OR, 0.730; 95% CI, 0.609-0.876), more obese (OR, 1.267; 95% CI, 1.065-1.507), and diabetes (OR, 1.575; 95% CI, 1.214-2.043) remained statistically significant correlation with GON. CONCLUSIONS: Among the metabolic risk factors, obesity and diabetes as well as older age and male gender are risk factors of developing GON. The glaucoma screening examinations should be considered in the populations with these indicated risk factors.

Activation of the NFAT-Calcium Signaling Pathway in Human Lamina Cribrosa Cells in Glaucoma.

Purpose: Optic nerve cupping in glaucoma is characterized by remodeling of the extracellular matrix (ECM) and fibrosis in the lamina cribrosa (LC). We have previously shown that glaucoma LC cells express raised levels of ECM genes and have elevated intracellular calcium ([Ca2+]i). Raised [Ca2+]i is known to promote proliferation, activation, and contractility in fibroblasts via the calcineurin-NFAT (nuclear factor of activated T-cells) signaling pathway. In this study, we examine NFAT expression in normal and glaucoma LC cells, and investigate the effect of cyclosporin A (CsA, a known inhibitor of NFAT activity) on [Ca2+]i and ECM gene expression in normal and glaucoma LC cells. Methods: [Ca2+]i was measured with dual-wavelength Ca2+ imaging and confocal microscopy using Fura-2-AM and Fluo-4 under physiological isotonic and hypotonic cell stretch treatment. Human donor LC cells were cultured under normal physiological conditions or using a glaucoma-related stimulus, oxidative stress (H2O2, 100 µM), for 6 hours with or without CsA. NFATc3 protein levels were examined using Western blot analysis. Profibrotic ECM gene transcription (including transforming growth factor-ß1 [TGFß1], collagen 1A1 [Col1A1], and periostin) was analyzed using quantitative real time RT-PCR. Results: Basal and hypotonic cell membrane stretch-induced [Ca2+]i were significantly (P < 0.05) elevated in glaucoma LC cells compared to normal controls. There was a significant delay in [Ca2+]i reuptake into internal stores in the glaucoma LC cells. NFATc3 protein levels were increased in glaucoma LC cells. CsA (10 µM) significantly inhibited the H2O2-induced expression of NFATc3 in normal and glaucoma LC cells. CsA also reduced the H2O2-induced NFATc3 dephosphorylation (and nuclear translocation), and also suppressed the H2O2-induced elevation in profibrotic ECM genes (TGFß1, Col1A1, and periostin), both in normal and in glaucoma LC cells. Conclusions: Intracellular Ca2+ and NFATc3 expression were significantly increased in glaucoma LC cells. CsA reduced the H2O2-induced enhancement in NFATc3 protein expression and nuclear translocation and the profibrotic gene expression both in normal and in glaucoma LC cells. Therefore, targeting the calcineurin-NFATc3 signaling pathway may represent a potential avenue for treating glaucoma-associated LC fibrosis.

The role of astrocytes in optic nerve head fibrosis in glaucoma.

Glaucoma is defined as a progressive optic neuropathy and is characterized by an irreversible loss of retinal ganglion cells. The main risk factor to develop glaucoma is an increased intraocular pressure (IOP). During the course of glaucoma structural changes in the optic nerve head (ONH) take place which lead to the characteristic excavation or cupping of the ONH. In this review we will focus on mechanisms and processes involved in structural alterations of the extracellular matrix in the lamina cribrosa (LC) of the ONH, which are associated with astrocytes. In glaucoma, a disordered deposition of elastic and collagen fibers and a typical pronounced thickening of the connective tissue septae surrounding the nerve fibers can be observed in the LC region. The remodeling process of the LC and the loss of ON axons are associated with a conversion of astrocytes from quiescent to a reactivated state. The extracellular matrix changes in the LC are thought to be due to a disturbed homeostatic balance of growth factors and the reactivated astrocytes are part of this process. Reactivated astrocytes, remodeling of the ECM within the LC and an elevated IOP are taking part in the retinal ganglion cell loss in glaucoma.

The Function of Matricellular Proteins in the Lamina Cribrosa and Trabecular Meshwork in Glaucoma.

PURPOSE: To review the current literature regarding the role of matricellular proteins in glaucoma, specifically in the lamina cribrosa (LC) region of the optic nerve head (ONH) and the trabecular meshwork (TM). METHODS: A literature search was performed for published articles describing the expression and function of matricellular proteins such as thrombospondin (TSP), connective tissue growth factor (CTGF), secreted protein acidic and rich in cysteine (SPARC), and periostin in glaucoma. RESULTS: In glaucoma, there are characteristic extracellular matrix (ECM) changes associated with optic disc cupping in the ONH and subsequent visual field defects. Matricellular proteins are a family of nonstructural secreted glycoproteins, which enable cells to communicate with their surrounding ECM, including CTGF, also known as CCN2, TSPs, SPARC, periostin, osteonectin, and tenascin-C and -X, and other ECM proteins. Such proteins appear to play a role in fibrosis and increased ECM deposition. Importantly, most are widely expressed in tissues particularly in the TM and ONH, and deficiency of TSP1 and SPARC has been shown to lower intraocular pressure in mouse models of glaucoma through enhanced outflow facility. CONCLUSION: This article highlights the role of matricellular proteins in glaucoma pathology. The potential role of these proteins in glaucoma is emerging as some have an association with the pathophysiology of the TM and LC region and might therefore be potential targets for therapeutic intervention in glaucoma.

Changes in lamina cribrosa and prelaminar tissue after deep sclerectomy.

AIM: To determine the response of the lamina cribrosa (LC) and prelaminar tissue to a reduction of intraocular pressure (IOP) after nonpenetrating deep sclerectomy (NPDS) using enhanced depth imaging (EDI) spectral domain optical coherence tomography (SD-OCT). METHODS: A total of 28 eyes from 28 patients presenting with primary open angle glaucoma who underwent NPDS were studied. SD-OCT scans using EDI technology were obtained before surgery and 1 week, 1 month, and 3 months postoperatively. The OCT device was set to image a 15 × 10° vertical rectangle centred on the optic disc. The scan closest to the optic nerve head (ONH) centre was selected for analysis. The vertical distances from three equidistant points on the reference line (Bruch's membrane opening) to the anterior prelaminar tissue surface and the anterior and posterior surfaces of the LC were measured. RESULTS: The IOP decreased from 18.7 ± 4.3 to 9.1 ± 4.0 at the first week, 11.4 ± 3.7 at 1 month, and 13.1 ± 3.6 mm Hg at 3 months postoperatively (P<0.001). There was a significant reduction of the ONH cupping at 1 week (22.3%, P<0.001), 1 month (13.7%, P<0.001), and 3 months (9.8%, P=0.001) after surgery. Anterior displacement of the LC was slight but statistically significant at 1 week (4.5%, P=0.003), 1 month (3.8%, P=0.014), and 3 months postoperatively (3.3%, P=0.010). IOP reduction was significantly correlated with a reduction of ONH cupping and anterior displacement of LC at the first week and first month (P<0.05). CONCLUSIONS: Cupping reversal after NPDS is mainly due to changes in prelaminar tissue thickness, whereas the LC changes in position are less pronounced.

The pathogenic role of transforming growth factor-ß2 in glaucomatous damage to the optic nerve head.

In patients with primary open angle glaucoma (POAG), the optic nerve head (ONH) shows characteristic cupping correlated with visual field defects. The progressive optic neuropathy is characterized by irreversible loss of retinal ganglion cells (RGC). The critical risk factor for axonal damage at the ONH is an elevated intraocular pressure (IOP). The increase in IOP correlates with axonal loss in the ONH, which might be due to an impaired axoplasmatic flow leading to the loss of RGCs. Damage to the optic nerve is thought to occur in the lamina cribrosa (LC) region of the ONH, which is composed of characteristic sieve-like connective tissue cribriform plates through which RGC axons exit the eye. The cupping of the optic disc, and the compression and excavation of LC are characteristic signs of glaucomatous ONH remodelling. In ONH of POAG patients a disorganized distribution and deposition of elastic fibers and a typical pronounced thickening of the connective tissue septae surrounding the optic nerve fibers is found. Transforming growth factor (TGF)-ß2 could be one of the pathogenic factors responsible for the structural alterations in POAG patients as the TGF-ß2 levels in the ONH of glaucomatous eyes are elevated as well as in the aqueous homour. TGF-ß2 leads to an increased synthesis of extracellular matrix (ECM) molecules mediated by connective tissue growth factor and to an impaired ECM degradation in cultured ONH astrocytes. Bone morphogenetic protein (BMP)-4 effectively antagonizes the effects of TGF-ß2 on matrix deposition. The BMP antagonist gremlin blocks this inhibition, allowing TGF-ß2 stimulation of ECM synthesis. Overall, the ECM in the ONH is kept in balance in the OHN by factors that augment or block the activity of TGF-ß2.

VAV2 and VAV3 as candidate disease genes for spontaneous glaucoma in mice and humans.

BACKGROUND: Glaucoma is a leading cause of blindness worldwide. Nonetheless, the mechanism of its pathogenesis has not been well-elucidated, particularly at the molecular level, because of insufficient availability of experimental genetic animal models. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that deficiency of Vav2 and Vav3, guanine nucleotides exchange factors for Rho guanosine triphosphatases, leads to an ocular phenotype similar to human glaucoma. Vav2/Vav3-deficient mice, and to a lesser degree Vav2-deficient mice, show early onset of iridocorneal angle changes and elevated intraocular pressure, with subsequent selective loss of retinal ganglion cells and optic nerve head cupping, which are the hallmarks of glaucoma. The expression of Vav2 and Vav3 tissues was demonstrated in the iridocorneal angle and retina in both mouse and human eyes. In addition, a genome-wide association study screening glaucoma susceptibility loci using single nucleotide polymorphisms analysis identified VAV2 and VAV3 as candidates for associated genes in Japanese open-angle glaucoma patients. CONCLUSIONS/SIGNIFICANCE: Vav2/Vav3-deficient mice should serve not only as a useful murine model of spontaneous glaucoma, but may also provide a valuable tool in understanding of the pathogenesis of glaucoma in humans, particularly the determinants of altered aqueous outflow and subsequent elevated intraocular pressure.

Bone morphogenetic protein 4 inhibits TGF-beta2 stimulation of extracellular matrix proteins in optic nerve head cells: role of gremlin in ECM modulation.

The characteristic cupping of the optic nerve head (ONH) in glaucoma is associated with elevated TGF-beta2 and increased synthesis and deposition of extracellular matrix (ECM) proteins. In addition to TGF-beta2, the human ONH also expresses bone morphogenetic proteins (BMPs) and BMP receptors, which are members of the TGF-beta superfamily. We examined the potential effects of BMP4 and the BMP antagonist gremlin on TGF-beta2 induction of ECM proteins in ONH cells. BMP-4 dose dependently inhibited TGF-beta2-induced fibronectin (FN) and PAI-1 expression in ONH astrocytes and lamina cribrosa (LC) cells and also reduced TGF-beta2 stimulation of collagen I, collagen VI, and elastin. Addition of gremlin blocked this BMP-4 response, increasing cellular and secreted FN as well as PAI-1 levels in both cell types. Gremlin was expressed in ONH tissues and ONH cells, and gremlin protein levels were significantly increased in the LC region of human glaucomatous ONH tissues. Interestingly, recombinant gremlin dose dependently increased ECM protein expression in cultured ONH astrocytes and LC cells. Gremlin stimulation of ECM required activation of TGF-beta receptor and R-Smad3. TGF-beta2 increased gremlin mRNA expression and protein levels in ONH cells. Inhibition of either the type I TGF-beta receptor or Smad3 phosphorylation blocked TGF-beta2-induced gremlin expression. In conclusion, BMP4 blocked the TGF-beta2 induction of ECM proteins in ONH cells. The BMP antagonist gremlin reversed this inhibition, allowing TGF-beta2 stimulation of ECM synthesis. Increased expression of gremlin in the glaucomatous ONH may further exacerbate TGF-beta2 effects on ONH ECM metabolism by inhibiting BMP-4 antagonism of TGF-beta2 signaling. Modulation of the ECM via gremlin provides a novel therapeutic target for glaucoma.

Neuroprotective effect of transpupillary thermotherapy in the optic nerve crush model of the rat.

PURPOSE: Transpupillary thermotherapy (TTT) has been shown to induce heat shock protein (Hsp) 72 in optic nerve head tissue. The neuroprotective effect of TTT was investigated in an optic nerve crush rat model. METHODS: TTT was performed onto the optic nerve head in the right eye of subject rats. After 24 h, an optic nerve crush injury using an aneurysm clip was performed at 2 mm from the optic nerve head for 60 s. At 7 and 14 days later, retrograde labelling of retinal ganglion cells (RGCs) with DTMR crystal was carried out and the density of the surviving RGCs was evaluated. Immunohistochemical staining was performed to confirm the expression of Hsp72. RESULTS: At 7 days after optic nerve crush injury, the mean density of surviving RGCs was higher in TTT group (372.7+/-149.8 per mm(2)) than in optic nerve crush group (252.9+/-96.7 per mm(2)) with borderline significance. In the retinal areas at 1 mm from the optic nerve head, a significant increase in surviving RGCs from TTT treated eyes was observed at both 7 and 14 days after optic nerve crush injury. However, no significant differences in surviving RGCs were demonstrated 2 and 3 mm from the optic nerve head. CONCLUSIONS: These results demonstrate that TTT aimed onto the optic nerve head showed a neuroprotective effect.

Pathogenesis of ganglion "cell death" in glaucoma and neuroprotection: focus on ganglion cell axonal mitochondria.

Retinal ganglion cell axons within the globe are functionally specialized being richly provided with many mitochondria. The mitochondria produce the high energy requirement for nerve conduction in the unmyelinated part of the ganglion cell axons. We have proposed that in the initiation of glaucoma, an alteration in the quality of blood flow dynamics in the optic nerve head causes a compromise in the retinal ganglion cell axon energy requirement, rendering the ganglion cells susceptible to additional insults. One secondary insult might be light entering the eye to further affect ganglion cell axon mitochondrial function. Other insults to the ganglion cells might be substances (e.g., glutamate, nitric oxide, TNF-alpha) released from astrocytes. These effects ultimately cause ganglion cell death because of the inability of mitochondria to maintain normal function. We therefore suggest that ganglion cell apoptosis in glaucoma is both receptor and mitochondrial mediated. Agents targeted specifically at enhancing ganglion cell mitochondrial energy production should therefore be beneficial in a disease like glaucoma. Ganglion cell death in glaucoma might therefore, in principle, not be unlike the pathophysiology of numerous neurological disorders involving energy dysregulation and oxidative stress. The trigger(s) for ganglion cell apoptosis in glaucoma is/are likely to be multifactorial, and the rationale for targeting impaired energy production as a possibility of improving a patient's quality of life is based on logic derived from laboratory studies where neuronal apoptosis is shown to occur via different mechanisms. Light-induced neuronal apoptosis is likely to be more relevant to ganglion cell death in glaucoma than, for example, neuronal apoptosis associated with Parkinson's disease. Logic suggests that enhancing mitochondrial function generally will slow down ganglion cell apoptosis and therefore benefit glaucoma patients. On the basis of our laboratory studies, we suggest that supplements such as creatine, alpha-lipoic acid, nicotinamide, and epigallocatechin gallate (EGCG), all of which counteract oxidative stress induced by light and other triggers, are worthy of consideration for the treatment of such patients as they can be taken orally to reach the retina without having significant side effects.

Thermal injury induces heat shock protein in the optic nerve head in vivo.

PURPOSE: To investigate the induction of heat shock protein (Hsp)70 in the optic nerve head by localized laser application in transpupillary thermotherapy (TTT). METHODS: TTT was performed on the right eye of Norwegian brown rats with an 810-nm diode laser installed on a slit lamp biomicroscope. The laser was aimed at the center of the optic nerve head with a 50-microm spot size. Various exposures (range, 60-200 mW) were used with an exposure duration of 60 seconds, and the various exposure durations (range, 1-5 minutes) were used with a power of 100 mW. Twenty hours after laser irradiation, immunohistochemical staining and Western blot analyses were performed. For morphologic analysis of the optic nerve head, confocal scanning laser ophthalmoscopy and scanning electron microscopy were performed. RESULTS: In the control eyes, Hsp70 was detected minimally in the optic nerve tissues by immunohistochemistry. After TTT, Hsp70 in the optic nerve tissue was induced more than in the control eyes. By Western blot, Hsp70 expression was found to increase progressively after TTT as the power was increased, but it also decreased slightly at powers >140 mW. The optimal setting of TTT without tissue damage was determined to be 100 mW for 60 seconds. CONCLUSIONS: Transpupillary laser irradiation of the optic nerve head induces Hsp70 expression. This result can be applied to the neuroprotective experiments in glaucoma by enhancement of a natural cytoprotective stress response.

Distribution and expression of transforming growth factor-beta and platelet-derived growth factor in the normal and glaucomatous monkey optic nerve heads.

PURPOSE: Remodeling of the extracellular matrix occurs in the lamina cribrosa in progressed glaucomatous optic nerve damage including disc cupping. We examined immunohistochemical changes in the transforming growth factor (TGF)-beta and platelet-derived growth factor (PDGF) in the optic nerve head in an experimentally induced glaucoma model. METHODS: We used 3 cynomolgus and 2 Japanese monkey eyes. Glaucoma was induced by repeated argon laser photocoagulation of the chamber angle. Eyes were enucleated after disc cupping had formed 3 to 5 months after photocoagulation. The optic nerve head was examined for expression of TGF-beta1, -beta2 and -beta3 and PDGF-A and -B in frozen sections and by the biotin ExtraAvidin-alkali phosphatase method. RESULTS: Normal monkey eyes showed TGF-beta1, -beta2 and -beta3, and PDGF-A and -B in the optic nerve head including the nerve fibers, glial cells, and vascular cells. Glaucomatous eyes showed stronger expression of TGF-beta1 and -beta2 in the glial cells around the lamina cribrosa. The staining intensities for TGF-beta3, PDGF-A and -B were the same as in normal eyes. CONCLUSIONS: Eyes with experimental glaucoma showed higher expression of TGF-beta1 and -beta2 around the lamina cribrosa. These findings may show upregulation of extracellular matrix production as related to remodeling of the lamina cribrosa in glaucoma.

Disruption of the blood-brain barrier in experimental optic neuritis: immunocytochemical co-localization of H2O2 and extravasated serum albumin.

PURPOSE: To probe the role of endogenous hydrogen peroxide (H2O2) in the pathogenesis of disruption of the blood-brain barrier (BBB) associated with experimental allergic encephalomyelitis (EAE), an animal model for primary central nervous system demyelination. METHODS: Strain-13 guinea pigs were sensitized for EAE with central myelin in complete Freund's adjuvant. Magnetic resonance imaging with Gd-DTPA was performed twice a week for 2 weeks to assess disruption of the BBB, in vivo, by the enhancement of the optic nerves. Two weeks after antigenic sensitization, ultracytochemical localization of endogenous H2O2 was performed using the cerium perhydroxide method, with co-localization of endogenous serum albumin extravasation using gold-labeled antibodies against serum albumin. Examination of blood vessels for perivascular immunogold-labeled serum albumin and H2O2 derived reaction product began in the optic nerve head and proceeded toward the retrobulbar optic nerve until a total of 20 vessels were evaluated per animal. RESULTS: Magnetic resonance imaging revealed Gd-DTPA enhancement of the optic nerves in all animals sensitized for EAE. Optic nerve ultrastructure revealed colloidal gold-labeled antibodies against serum albumin in the perivascular and adjacent interstitial spaces of capillaries and small venules in which H2O2 derived cerium perhydroxide reaction product was also simultaneously evident. Immunogold-labeled serum albumin was predominantly confined to the intravascular compartment of the optic nerve in the absence of perivascular H2O2 and/or perivascular foci of inflammatory cells. The difference between the mean percentage of blood vessels (61.8%) with co-localization of perivascular immunogold-labeled serum albumin and cerium perhydroxide reaction product, to the mean percentage of blood vessels (9.5%) with perivascular immunogold-labeled serum albumin in the absence of cerium perhydroxide, was statistically significant (P = 0.0019). CONCLUSIONS: Endogenous H2O2, found at the foci of BBB disruption, may be one of the mediators involved in the alteration of vascular permeability in experimental optic neuritis.