Article

Oxidativer Stress im Trabekelwerk beim POWG

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

Das primäre Offenwinkelglaukom (POWG) wird als eine Optikusneuropathie definiert, bei der es zu einem Verlust von Axonen des Nervus opticus und der dazugehörigen Ganglienzellen kommt. Die Ursache dieser Schädigung ist bis heute nicht geklärt. In der Pathogenese und Progression des POWG werden verschiedene Faktoren wie ein erhöhter intraokulärer Druck und eine verminderte okuläre Durchblutung verantwortlich gemacht. Aus morphologischen Untersuchungen ist bekannt, dass die Druckerhöhung beim POWG auf einer Erhöhung des Abflusswiderstandes im Bereich des Trabekelwerkes beruht. Das Trabekelwerk (TW) des POWG-Patienten zeichnet sich unter anderem durch folgende spezifische morphologische und biochemische Veränderungen aus: Akkumulation von extrazellulärer Matrix, beschleunigte Alterung, Apoptose, subklinische chronische Entzündungen und Veränderungen im Zytoskelett. Die Ursache und die Vorgänge, die zu diesen Veränderungen führen, sind bis heute nicht geklärt. In der Pathogenese des POWG wird zunehmend oxidativer Stress diskutiert. Wissenschaftliche Untersuchungen deuten darauf hin, dass im TW von POWG-Patienten ein vermehrter oxidativer Stress vorliegt. Die Behandlung von kultivierten TW-Zellen führt zu den genannten glaukomtypischen Veränderungen. Durch die Vorbehandlung mit Antioxidantien aber auch mit Wirkstoffen von modernen Antiglaukomatosa können diese glaukomtypischen Veränderungen in TW reduziert werden. Zusammenfassend können in vitro POWG-typische Veränderungen im TW durch oxidativen Stress induziert werden. Somit könnte eine Reduktion des oxidativen Stresses im TW ein wirkungsvoller Ansatz sein, die Progression des POWG zu reduzieren.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... 3,[5][6][7] In glaucoma, the TM cells are presumed to die by apoptosis though several other mechanisms of cell death are possible. [6][7][8][9][10][11][12] The human TM cell lines, and ex-vivo dissected TM tissue obtained from normal donors express several modulators of apoptosis along with an abundance of heat shock proteins (HSPs). [10][11][12] Apoptotic modulators have been expressed in ex-vivo TM specimens or TM cells in culture and have also been shown to be upregulated upon stress on in-vitro TM cell lines. ...
... [6][7][8][9][10][11][12] The human TM cell lines, and ex-vivo dissected TM tissue obtained from normal donors express several modulators of apoptosis along with an abundance of heat shock proteins (HSPs). [10][11][12] Apoptotic modulators have been expressed in ex-vivo TM specimens or TM cells in culture and have also been shown to be upregulated upon stress on in-vitro TM cell lines. Oxidative stress markers have also been found to be very highly expressed in aqueous humour and anterior segment tissue in glaucoma eyes. ...
... Oxidative stress markers have also been found to be very highly expressed in aqueous humour and anterior segment tissue in glaucoma eyes. 12 Several studies have reported increased expression of proapoptotic molecules in the anterior segment or ocular tissues including TM in glaucoma, human TM (HTM) cell lines, and ex-vivo dissected TM tissue obtained from normal donors, and express several modulators of apoptosis (Fas, Bcl-2, Bcl-Xl, Bax, and caspases). 10,[12][13][14] Literature on the autophagic process in glaucoma is limited and focus on expression of autophagy genes in tissue of the anterior segment or in-vitro experiments. ...
Article
Full-text available
Purpose: The key differences in cell death mechanisms in the trabecular meshwork (TM) in adult moderate and severe primary glaucoma remain still unanswered. This study explored key differences in cell death mechanisms in the trabecular meshwork (TM) in adult moderate and severe primary glaucoma. Design: In-vitro laboratory study on surgical specimens and primary cell lines. Methods: Select cell death-related proteins differentially expressed on mass spectrometric analysis in ex-vivo dissected TM specimens patients with severe adult primary open-angle (POAG) or angle-closure glaucoma (PACG) compared to controls (cadaver donor cornea) were validated for temporal changes in cell death-related gene expression on in-vitro primary human TM cell culture after 48 hours (moderate) or 72 hours (severe) oxidative stress with H2O2 (400-1000 uM concentration). These were compared with histone modifications after oxidative stress in human TM (HTM) culture and peripheral blood of patients with moderate and severe glaucoma. Results: Autophagy-related proteins seemed to be the predominant cell-death mechanism over apoptosis in ex-vivo dissected TM specimens in severe glaucoma. Analyzing HTM cell gene expression at 48 hours and 72 hours of oxidative stress, autophagy genes were up-regulated at 48-72 hours of exposure in contrast to apoptosis-related genes, showing down-regulation at 72 hours. There was associated increased expression of H3K14ac in HTM after 72 hours of oxidative stress and in peripheral blood of severe POAG and PACG. Conclusion: A preference of autophagy over apoptosis may underlie stage transition from moderate to severe glaucoma in the trabecular meshwork or peripheral blood, which may be tightly regulated by epigenetic modulators.
... ROS can also induce cytoskeleton remodeling, apoptosis, and release of in ammatory factors in the trabecular meshwork, resulting in abnormal extracellular matrix deposition and cell necrosis. 17 The aging of the trabecular meshwork cytoskeleton changes the cytoskeleton composition and induces an abnormal deposition of the extracellular matrix that further affects the ltration of aqueous humor. 18 These mechanisms work together to make patients with diabetes more prone to elevated IOP. ...
Preprint
Full-text available
Purpose: This study aimed to analyze the risk factors for high intraocular pressure (IOP) after phacoemulsification and intraocular lens implantation (PEI). Setting: Wuhan Aier Eye Hospital, China. Design: Retrospective case series. Methods: The medical recordsof consecutive patients who underwent uneventful PEI from December 1, 2020, to April 1, 2021, by the same surgeon were reviewed. Results:A total of 548 eyes of 416 patients were analyzed. The younger the age, the longer the axial length (AXL)and the larger the CDE; patients with diabetes were more likely to have high IOP after the surgery.For every 0.1-mm increase in the AXL, the likelihood of a 5-mm Hg increase in early postoperative IOP increased 1.167-fold and that of a 10-mm Hg increase in IOP increased 1.227-fold. The IOP increased by 0.1 mmHg for each 0.1 increase in CDE(P<0.001). Conclusions: Age is an independent protective factor for early high IOP after PEI. High myopia, anterior chamber distance >4 mm, diabetes mellitus, and high CDE are risk factors for postoperative high IOP. AL and CDE could predict the peak IOP of 5 mm Hg and 10 mm Hg after the surgery.
... The morphological analysis of glaucomatous human TM cells showed POAG-typical molecular changes, including ECM accumulation, cell death, disarrangement of the cytoskeleton, advanced senescence, NF-κB activation and the release of inflammatory markers [49,50,52]. These findings suggest that the IOP elevation, which occurs in glaucomatous patients, is related to oxidative degenerative processes affecting the human TM endothelial cells (hTMEs). ...
Article
Full-text available
Primary open-angle glaucoma (POAG) is the second leading cause of irreversible blindness worldwide. Increasing evidence suggests oxidative damage and immune response defects are key factors contributing to glaucoma onset. Indeed, both the failure of the trabecular meshwork tissue in the conventional outflow pathway and the neuroinflammation process, which drives the neurodegeneration, seem to be linked to the age-related over-production of free radicals (i.e., mitochondrial dysfunction) and to oxidative stress-linked immunostimulatory signaling. Several previous studies have described a wide range of oxidative stress-related makers which are found in glaucomatous patients, including low levels of antioxidant defences, dysfunction/activation of glial cells, the activation of the NF-κB pathway and the up-regulation of pro-inflammatory cytokines, and so on. However, the intraocular pressure is still currently the only risk factor modifiable by medication or glaucoma surgery. This present review aims to summarize the multiple cellular processes, which promote different risk factors in glaucoma including aging, oxidative stress, trabecular meshwork defects, glial activation response, neurodegenerative insults, and the altered regulation of immune response.
... It includes chronic neurodegenerative diseases of the optic nerve such as apoptosis of retinal ganglion cells (RGCs), progressive loss of optic nerve axons, and visual fields defects [1][2]. Increased intraocular pressure is considered a major risk factor [3][4][5][6][7], although numerous studies have suggested that glaucomatous neuropathy actually involves multiple factors, including impaired intraocular blood circulation [8][9][10][11], excitotoxic reactions caused by excess accumulation of glutamate [11][12][13], free radical production and oxidative stress [14][15][16][17], increased NO levels [18][19][20], and immunological factors. ...
Article
Full-text available
The mechanistic study of glaucoma pathogenesis has shifted to seeking to understand the effects of immune responses on retinal ganglion cell damage and protection. Cytokines mediate the biological effects of the immune system, and our previous study revealed an imbalance of T-helper (Th) 1-derived and Th2-derived cytokines in the serum of patients with glaucoma. In this study, we collected irises from normal individuals and patients with primary open-angle closure (POAG) or chronic angle-closure glaucoma (CACG). We used realtime polymerase chain reaction (PCR) to measure the expression of Th1 (interleukin (IL)-2, interferon-gamma (IFN-γ)), Th2 (IL-4, IL-6, IL-10), and Th3 (transforming growth factor-beta (TGF-β)) cytokines. We then performed immunohistochemical staining to characterize the localization of the upregulated cytokines in iris cryosections. We observed an upward trend in the expression of IL-2 and IFN-γ and a downward trend in IL-6 expression in the iris of POAG and CACG patients. Expression of TGF-β also increased. Immunohistochemistry revealed that IL-2 expression in POAG and CACG patients was localized in the anterior surface of the blood vessel wall in the stroma of the iris, in the cytoplasm of some cells, in the anterior epithelium, and in the posterior pigment epithelium. These findings indicate that immune status differed between the iris tissues of POAG and CACG patients and those of normal individuals. A T-helper cytokine imbalance may modulate the immune microenvironment in glaucomatous eyes and thus influence optic neuropathy.
Article
Functional deficits in glaucomatous optic neuropathy are, apart from other disturbances in the visual field, typically detected with achromatic perimetry as a well accepted gold standard. With the development of new perimetric devices and strategies (e.g. short wave perimetry, frequency doubling perimetry and flicker perimetry) individually different patterns of scotomas in the different perimetric devices could be recognized. The reasons for this could be a different sensitivity reaction of the ganglion cell subpopulations to an increased intraocular pressure as well as an influence of the underlying systemic diseases. To obtain a differentiated detection of the functional loss in the visual field in glaucoma, the use of different perimetric methods seems to be reasonable and helpful.
Article
Full-text available
Pathologic processes in glaucoma include increased apoptosis, accumulation of extracellular material in the trabecular meshwork and optic nerve, condensations of the cytoskeleton and precocious cellular senescence. Oxidative stress was shown to generate these alterations in primary ocular cells. Fatty acids omega-3 and -6 are alleged to constitute a prophylaxis against these deleterious effects. Here, we tested actual preventive effects omega-3 and -6 against peroxide induced stress responses in primary human trabecular meshwork cells. Changes of mitochondrial activity, proliferation, heat shock proteins, extracellular matrix components, and inflammatory markers were evaluated. Alterations of the cytoskeleton were evaluated by phalloidin labeling. Here we report a repressive effect of omega-6 on metabolic activity and proliferation, which was not detected for omega-3. Both agents were able to prevent the anti-proliferative effect of H₂O₂, but only omega-3 prevented metabolic repression. Expression of heat shock protein 27 was unaltered by both fatty acids, whereas heat shock protein 90 was significantly induced by both. Omega-6 increased fibronectin and connective tissue growth factor synthesis, as well as the amount of secreted fibronectin. Omega-3, instead, induced plasminogen activator inhibitor 1 synthesis. H₂O₂ further increased fibronectin production in omega-6 supplemented cells, which was not the case in omega-3 treated cells. H₂O₂ stimulation of plasminogen activator inhibitor 1 and connective tissue growth factor was repressed by both fatty acids. Both fatty acids appeared to abolish H₂O₂ mediated stimulation of nuclear factor κB and IL-6, but not IL-1α and IL-8. H₂O₂ induced formation of cross-linked actin networks and stress fibers, which was reduced by preemptive application of omega-3. Omega-6, in contrast, had no protective effect on that, and even seemed to promote condensation. Based on the observed side effects of omega-6, omega-3 appears to be the more beneficial fatty acid in respect of prophylactic intake for prevention of a glaucomatous disease.
Article
Full-text available
Glaucoma is one of the leading causes of visual impairment and blindness. Improved knowledge of the pathogenesis of this disease has allowed the exploration of new therapeutic methods. In general, elevated intraocular pressure (IOP), oxidative stress, and vascular insufficiency are accepted as the major risk factors for the progression of glaucoma. Many natural compounds have been found beneficial for glaucoma. Nutritional therapies are now emerging as potentially effective in glaucomatous therapy. One nutritional supplement with potential therapeutic value is cod liver oil, a dietary supplement that contains vitamin A and omega-3 polyunsaturated fatty acids (PUFAs). Vitamin A is important for preserving normal vision and it is a well-known antioxidant that prevents the oxidative damage that contributes to the etiology and progression of glaucoma. Vitamin A is also a crucial factor for maintaining the integrity of conjunctival and corneal ocular surfaces, and preventing the impairment of ocular epithelium caused by topical antiglaucomatous drugs. Omega-3 fatty acids are beneficial for glaucoma patients as they decrease IOP, increase ocular blood flow, and improve optic neuroprotective function. In this article, we propose that cod liver oil, as a combination of vitamin A and omega-3 fatty acids, should be beneficial for the treatment of glaucoma. However, further studies are needed to explore the relationship between cod liver oil and glaucoma.
Article
Full-text available
To determine protein regulation following activation of human, optic nerve head (ONH), lamina cribrosa (LC) cells in response to mechanical strain. LC cells were isolated and grown from donor tissue in specific media at 37°C and 5% CO(2) humidified incubator. Cells were grown to confluence on collagen I-coated flexible-bottom culture plates, rinsed with Dulbecco's phosphate-buffered saline, and left for 24 hours in serum-free media. They were subjected to 3% or 12% cyclic equiaxial stretch for 2 or 24 hours using a commercial strain-unit system. Control cells were serum-deprived and incubated without stretch for 24 hours. Nano liquid chromatography-mass spectrometry analysis with isobaric tags for relative and absolute quantitation labeling was used to determine protein regulation. In all, 526 proteins were discovered at a 95% confidence limit. Analysis of associated pathways and functional annotation indicated that the LC cells reacted in vitro to mechanical strain by activating pathways involved in protein synthesis, cellular movement, cell-to-cell signaling, and inflammation. These pathways indicated consistent major protein hubs across all stretch/time conditions involving transforming growth factor-β1 (TGFβ1), tumor necrosis factor (TNF), caspase-3 (CASP3), and tumor protein-p53 (p53). Among proteins of particular interest, also found in multiple stretch/time conditions, were bcl-2-associated athanogene 5 (BAG5), nucleolar protein 66 (NO66), and eukaryotic translation initiation factor 5A (eIF-5A). Pathway analysis identified major protein hubs (TGFβ1, TNF, CASP3, p53) and pathways all previously implicated in cellular activation and in the pathogenesis of glaucomatous optic neuropathy. Several specific proteins of interest (BAG5, NO66, eIF-5A) were identified for future investigation as to their role in ONH glial activation.
Article
Full-text available
The aim of this study was to investigate the expression level of several biomarkers in the in the aqueous humor of 14 patients with primary open angle glaucoma who underwent glaucoma surgery, and 11 nonglaucomatous normals who underwent cataract extraction surgery. The aqueous humor proteome of 25 patients was analyzed using an antibody microarray. Fourteen patients with uncontrolled intraocular pressure-despite profound therapeutic interventions-who underwent filtering procedures and 11 control subjects who underwent surgery for senile cataracts were included in the present study. Protein expression was evaluated using Cy3/Cy5 labeling, column purification, and hybridization on antibody-spotted glass microarrays. Fluorescent signals were detected by fluorescence laser scanning. The levels of 13 proteins were significantly increased in the aqueous humor of glaucomatous patients compared with expression levels in healthy controls. One of the 13 proteins (ELAM 1) was involved in inflammation. Two of these proteins (apolipoprotein B and E) were involved in the delivery of cholesterol to cells. Five of the 13 proteins (myotrophin, myoblast determination protein 1, myogenin, vasodilator-stimulated phosphoprotein, and ankyrin-2) were involved in muscle cell differentiation and function. Three proteins (heat shock 60 kilodaltons (kDa) and 90 kDa proteins, and ubiquitin fusion degradation 1-like) were involved in stress response and the removal of damaged proteins; and two proteins (phospholipase C β and γ) were involved in signal transduction and neural development. The expressions of these proteins in the aqueous humor of glaucomatous patients reflect the damage occurring in anterior chamber endothelia, mainly including the trabecular meshwork, which is the main structure of this ocular segment injured by glaucoma.
Article
Purpose: We have previously demonstrated elevated levels of connective tissue growth factor (CTGF/CCN2) in the aqueous humor (AqH) of pseudoexfoliation glaucoma (PXFG) patients when compared with cataract controls. Furthermore, there is a significant trabecular meshwork (TM) and lamina cribrosa (LC) fibrotic phenotype associated with glaucoma, possibly driven by CTGF. The purpose of this study was to investigate the potential of anti-CTGF immunotherapy in glaucoma. Methods: Primary TM and LC cells were cultured from human donors with (GTM/GLC) and without (NTM/NLC) primary open angle glaucoma (POAG). Aqueous humor samples from PXFG, POAG, and control cataract patients were applied to N/GTM and N/GLC cells in the presence or absence of a therapeutic, humanized monoclonal anti-CTGF antibody FG-3019 (10 μg/mL). Hydrogen peroxide (H2O2) was also used as a stimulus. Expression of fibrotic genes (fibronectin-1, fibrillin-1, CTGF, collagen type I α1, and α-smooth muscle actin) was assessed by q-PCR. Protein expression of collagen 1A1 and α-smooth muscle actin was examined in N/G TM cells by SDS-PAGE. The modulatory effect of FG-3019 (10 μg/mL) and IgG (10 μg/mL) were also assessed. Results: Treatment of cells with AqH from PXFG and POAG patients and H2O2 induced a significant (P < 0.05) increase in expression of profibrotic genes, which was significantly reduced by pretreatment with FG-3019 (P < 0.05). FG-3019 also reduced expression of α-smooth muscle actin and collagen 1A1 protein expression in N/GTM cells. Conclusions: FG-3019 is effective in blocking extracellular matrix production in TM and LC cells, thus supporting a role for the use of anti-CTGF immunotherapy in the treatment of glaucoma.
Article
In glaucoma treatment, beside the traditional reduction of intraocular pressure, additional therapeutic strategies have come into consideration. Therefore pleiotropic effects of medications, defined as positively acting effects independent of the main mechanism of action, represent a new research sub-field in medical therapy and play an increasingly important role in internal medicine. Using the example of local beta-blockers, alpha-2-agonists, carbonic anhydrase inhibitors and prostaglandin analogues, their pleiotropic spectra will be shown and discussed. Georg Thieme Verlag KG Stuttgart · New York.
Article
Full-text available
Oxidative stress plays a key role in the pathophysiology of glaucoma. This study was designed to assess ethyl pyruvate (EP) as a novel antioxidative agent in cultured human trabecular meshwork (hTM) cells. Primary hTM cells were cultured on collagen matrices. Tolerance to EP was assessed at various concentrations using fluorescent vital dyes (live/dead) and metabolic (1-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. After the candidate doses were identified, cells received either preincubation with EP before hydrogen peroxide stressing or pre- and coincubation with EP before and during stressing. Live/dead and metabolic activity assays were used to quantify oxidative damage. Cultured hTM cells were well tolerant of EP concentrations at or below 10 mM while higher doses showed significant levels of cytotoxicity. In the peroxide stress assays, samples that received pre- and cotreatment with all concentrations of EP showed significantly increased cell survival and maintenance of metabolic activity. However, samples that received only pretreatment did not show a significant increase in survival rates and lost nearly all metabolic activity after peroxide-induced stressing. This work suggests that EP is a potent antioxidant that is well tolerated by hTM cells; however, EP's potential as a therapeutic agent for glaucoma is limited by its inability to enhance endogenous antioxidant capacity. A continuous drug delivery system may be needed to realize the full therapeutic potential of EP for treatment of glaucoma.
Article
Full-text available
Purpose: Loss or dysfunction of trabecular meshwork (TM) cells has been associated with the development of pathologically elevated IOP, and it is conceivable that replacement of damaged TM cells could restore function to the TM. We propose that the use of TM-like cells derived from induced pluripotent stem cells (iPSCs) created from a patient's own dermal fibroblasts offers the best solution to this challenge. Here we demonstrate that mouse iPSCs can be induced to differentiate into TM-like cells suitable for autologous transplantation. Methods: Directed induction of stem cell differentiation was achieved through coculture of mouse iPSCs with human TM cells for up to 21 days. The resultant TM-like cells (iPSC-TM) were characterized morphologically, immunohistochemically, and functionally. Results: The iPSC-TM cells closely resembled cultured human TM cells morphologically and began to express many markers of TM cells while ceasing to express pluripotency markers such as Nanog, Oct4, and Sox2. Functionally, these cells developed the ability to phagocytose particles. Finally, exposure to dexamethasone or phorbol 12-myristate acetate caused a distinct increase in the production and secretion of myocilin and matrix metalloproteinase-3, respectively, behavior characteristic of TM cells. Conclusions: Our data demonstrate that iPSCs can be induced to assume a phenotype that resembles native TM cells in many important aspects. Not only do these cells represent a valuable research tool, but transplantation into glaucomatous eyes with elevated IOP may also restore function to the TM, resulting in re-establishment of IOP.
Article
Structural changes in the course of glaucoma disease affect the trabecular meshwork and ciliary body in addition to the optic disc as the primary site of glaucoma damage. Latest results from experimental studies, animal models and measurements in human eyes are presented and discussed. The presenting scenario is complex with age, biochemical and mechanical stress factors leading to subsequent, irreversible tissue change in the trabecular meshwork and cribriform plate of the optic nerve, resulting in neuronal tissue loss. Knowledge of these changes will be the key for future glaucoma therapies.
Article
Glaucoma is one of the world's leading causes of blindness. Astragalus membranaceus is a well-known traditional Chinese herbal medicine widely used for a long time. The aim of our study is to evaluate the activity of lowering intraocular pressure through the use of Astragalus membranaceus extract (AME) in an experimental glaucoma model. The rats used in the study were divided into six groups: one sham group, two positive control groups with topical brimonidine instillation and oral acetazolamide therapy, and three groups treated with AME (low, medium and high dosage). The antioxidant activity of AME was accessible by MDA and GPx levels. The ability to lower intraocular pressure (IOP) signified the efficiency of treating glaucoma. The results revealed that AME may decrease the MDA production and restore the GPx level in the periocular blood. This extremely beneficial effect may be by the same as that of brimonidine. Furthermore, AME also showed the ability to significantly lower IOP as is the case with brimonidine and acetazolamide. AME is a relatively safe Chinese herbal medicine with no observed side effects such as body-weight loss, or pathological change. In conclusion, the extract of Astragalus membranaceus is beneficial in treating glaucoma during the development of progression of this disease due to its significant IOP and antioxidant activities.
Article
Der uveosklerale Abflussweg erfolgt über den Ziliarmuskel zu den suprachoroidalen Räumen hin zu dem orbitalen Blut- und Lymphsystem. Die treibende Kraft für den uveoskleralen Abfluss ist der onkotische Druck in der Aderhaut. Der CyPass (Alcon Pharma GmbH, Freiburg) ist derzeit der einzige verfügbare Stent für die ab interno suprachoroidale minimalinvasive Glaukomchirurgie. Der CyPass erreicht eine maximale Drucksenkung auf 16–17 mm Hg im 1‑ bis 2‑Jahres-Verlauf und ist für frühe bis moderate Glaukomstadien beim primären Offenwinkelglaukom zugelassen. Es ist eine einfach anzuwendende minimalinvasive glaukomchirurgische Stentoperation und hat wenig intraoperative sowie Kurzzeit- und Langzeitkomplikationen. Das postoperative Management ist unkompliziert, und die postoperativen Belastungen für die Patienten sind gering, was zu einer beschleunigten Rekonvaleszenz führt. Für eine gute Erfolgsquote ist allerdings die richtige Auswahl der Patienten entscheidend. Langzeitstudien müssen noch zeigen, wie lange die drucksenkende Wirkung anhält.
Preprint
Full-text available
Background: Hyperglycemia, which can lead to apoptosis, hypertrophy, and fibrosis, induces hyperinflammation through inflammasome activation. A major cause of diabetic vascular complications is increased oxidative stress due to hyperglycemia. In order to elucidate the potential dual roles and regulatory signal transduction of TGF-β1 and TGF-β2 in human trabecular meshwork cells (HTMCs) in vitro, we established an oxidative cell model in HTMCs using 5.5, 25, 50, and 100 mM D-glucose-supplemented media and characterized the TGF-β-related oxidative stress pathway. Methods: Further analysis was conducted to investigate oxidative damage and protein alterations in the trabecular meshwork caused by the signal transduction. This was done through a series of qualitative cell function studies, such as cell viability/apoptosis analysis, intracellular reactive oxygen species (ROS) detection, analysis of calcium release concentration, immunoblot analysis to detect the related protein expression alteration, and analysis of cell fibrosis to study the effect of different severities of hyperglycemia. We also illustrated the role of TGF-β1 and TGF-β2 in oxidative stress-induced injury by shRNA-mediated knockdown or stimulation with recombinant human TGF-β1 protein (rhTGF-β1). Results: Results from the protein expression analysis showed that p-JNK, p-p38, p-AKT, TGF-β1, TGF-β2, and related SMAD family members were upregulated in HTMCs under hyperglycemia. In the cell functional assays, HTMCs treated with rhTGFβ-1 (1 ng/mL) and under hyperglycemic conditions showed higher proliferation rates and lower ROS and calcium levels than those with higher concentration or without rhTGFβ-1 treated. Furthermore, TGF-β1 and TGF-β2 were found to be associated with the activation of fibrosis-response proteins, α-SMA, collagen I, and laminin, which may lead to HTMC damage. Conclusions: To summarize, mechanistic analyses in HTMCs showed that hyperglycemia-induced oxidative stress activated TGF-β1 along with its associated pathway: at low concentrations, TGF-β1 protects cells from antioxidation, whereas at high concentrations, it accumulates in the extracellular matrix, causing further HTMC dysfunction.
Article
The implication of oxidative stress associated with increased oxidant production in mammalian and human cells characterized by the release of free radicals, resulting in cellular degeneration, is involved in many ocular diseases, such as age-related macular degeneration, retinopathy of prematurity, retinal light damage, primary open-angle glaucoma (POAG), and cataract. Cataract is the leading cause of blindness, accounting for 50% of blindness worldwide. Glaucoma, the leading cause of irreversible blindness, is considered as a progressive optic neuropathy often caused by elevated intraocular pressure (IOP) consequent to abnormally high resistance to aqueous humor (AH) drainage via the trabecular meshwork (TM) and Schlemm's canal. Morphological and biochemical analyses of the TM of patients with POAG revealed the loss of cells, increased accumulation of extracellular matrix proteins (ECM), changes in the cytoskeleton, cellular senescence, and the process of subclinical inflammation. The TM is the target tissue of glaucoma in the anterior chamber, and the development and progression of glaucoma are accompanied by the accumulation of oxidative damage in this tissue. The separate studies were conducted to comparatively evaluate the sensitivity to oxidative stress and lipid peroxidation (LPO) of anterior chamber tissues including TM. Accumulation of the primary, secondary, and end products of LPO (diene and triene conjugates, Schiff's bases) was noted in the studied extracts. Significant differences in the levels of all mentioned LPO products in comparison with the control were observed. The data may be considered as an evidence of LPO participation in the destruction of the trabecule and Schlemm's canal in POAG. Treatment of TM cells with oxidative stress induced POAG-typical changes such as ECM accumulation, cell death, disarrangement of the cytoskeleton, advanced senescence, and the release of inflammatory markers. By pretreatment with antioxidants, prostaglandin analogs, beta-blockers, or local carbonic anhydrase inhibitors, these effects were markedly reduced. Oxidative stress can induce characteristic glaucomatous TM changes, and these oxidative stress-induced TM changes can be minimized by the use of antioxidants and IOP-lowering substances. It is tempting to speculate that the prevention of oxidative stress exposure to the TM may help to reduce the progression of POAG. The author's laboratory has developed and patented the dual combination therapy with N-acetylcarnosine lubricant eye drops and oral formulation of nonhydrolyzed carnosine in ripe cataracts and POAG. The specific regimen for the treatment in each stage of age-related ophthalmic disease has been taken up. In the treatment of POAG, this dual therapy can be combined with conventional antiglaucoma therapy with beta-blocking and/or adrenergic agonist medicines providing the significant IOP-lowering effect and significant increase in outflow facility. The developed therapy is a prominent management care of the glaucomatous neurodegeneration.
Article
Full-text available
The isolation and characterization of 25 strains of human diploid fibroblasts derived from fetuses are described. Routine tissue culture techniques were employed. Other than maintenance of the diploid karyotype, ten other criteria serve to distinguish these strains from heteroploid cell lines. These include retention of sex chromatin, histotypical differentiation, inadaptability to suspended culture, non-malignant characteristics in vivo, finite limit of cultivation, similar virus spectrum to primary tissue, similar cell morphology to primary tissue, increased acid production compared to cell lines, retention of Coxsackie A9 receptor substance, and ease with which strains can be developed.Survival of cell strains at − 70 °C with retention of all characteristics insures an almost unlimited supply of any strain regardless of the fact that they degenerate after about 50 subcultivations and one year in culture. A consideration of the cause of the eventual degeneration of these strains leads to the hypothesis that non-cumulative external factors are excluded and that the phenomenon is attributable to intrinsic factors which are expressed as senescence at the cellular level.With these characteristics and their extremely broad virus spectrum, the use of diploid human cell strains for human virus vaccine production is suggested. In view of these observations a number of terms used by cell culturists are redefined.
Article
Full-text available
The trabecular meshwork (TM) of glaucomatous eyes is characterized by cell loss, increased accumulation of extracellular matrix (ECM), and cellular senescence. One factor increasingly discussed in the pathogenesis of primary open-angle glaucoma (POAG) is oxidative stress. The goal of this study was to determine whether oxidative stress is able to trigger these typical glaucomatous changes in vitro and whether these oxidative stress-induced TM changes can be reduced by the application of prostaglandin analogues. Cultured human TM cells were exposed to 200 to 800 microM hydrogen peroxide (H(2)O(2)) for 1 hour. Cell loss was detected by cell-viability assay. Levels of fibronectin and MMP-2 mRNA were determined by real-time PCR analysis. Senescence-associated beta-galactosidase (SA-beta-Gal) activity was investigated by histochemical staining. The effects of prostaglandin analogues and benzalkonium chloride (BAC) on these glaucoma typical TM changes were investigated by preincubation of nonstressed or H(2)O(2)-treated cells with 1:100 diluted commercial solutions of bimatoprost, travoprost, and latanoprost or their corresponding BAC concentrations. H(2)O(2) induced cell death and fibronectin mRNA expression, but decreased the amount of MMP-2 mRNA. H(2)O(2) increased SA-beta-Gal activity. Additional pretreatment with BAC further increased the typical glaucomatous TM changes in vitro. These effects were reduced by preincubation with prostaglandin analogues in H(2)O(2)-treated and, to a lesser extent, in nonstressed cells. No reduction occurred in the presence of prostaglandin F receptor antagonists in H(2)O(2)-treated cells. Oxidative stress is able to induce characteristic glaucomatous TM changes in vitro, and these oxidative stress-induced TM changes can be minimized by the use of prostaglandin analogues. Thus, prevention of oxidative stress exposure to the TM may help to reduce the progression of POAG.
Article
Full-text available
Primary open-angle glaucoma (POAG) is a highly prevalent cause of irreversible blindness which associates cupping of the optic disc and alteration of the visual field, elevation of intraocular pressure being a major risk factor. Provided diagnosis is made at an early stage, treatments are available to prevent visual impairment. A locus, GLC1A, has been mapped on chromosome 1q23-q25 in several families affected with juvenile-onset POAG (JOAG) and also in some families affected with juvenile and middle-age onset POAG. Recently, three mutations of the TIGR (Trabecular meshwork-Induced Glucocorticoid Response) gene were shown to be responsible for the disease in several American families and in unrelated POAG patients. We now describe five new mutations in eight French families. All mutations known to date appear to concentrate in the evolutionarily conserved C-terminal domain of TIGR which bears homology to frog olfactomedin, an extracellular matrix glycoprotein of the olfactory epithelium, to rat and human neuronal olfactomedin-related proteins and to F11C3.2, a protein from Caenorhabditis elegans . Moreover, this conserved domain of TIGR is encoded by a single exon to which mutation screening could be limited. Surprisingly, the TIGR message, which is abundantly transcribed in the trabecular meshwork and also in the ciliary body and the sclera, is not expressed in the optic nerve whose degeneration is, however, the primary lesion of POAG.
Article
Full-text available
A glaucoma locus, GLC1A, was identified previously on chromosome 1q. A gene within this locus (encoding the protein myocilin) subsequently was shown to harbor mutations in 2-4% of primary open angle glaucoma patients. A total of 1703 patients was screened from five different populations representing three racial groups. There were 1284 patients from primarily Caucasian populations in Iowa (727), Australia (390) and Canada (167). A group of 312 African American patients was from New York City and 107 Asian patients from Japan. Overall, 61 different myocilin sequence variations were identified. Of the 61 variations, 21 were judged to be probable disease-causing mutations. The number of probands found to harbor such mutations in each population was: Iowa 31/727 (4.3%), African Americans from New York City 8/312 (2.6%), Japan 3/107 (2.8%), Canada 5/167 (3.0%), Australia 11/390 (2.8%) and overall 58/1703 (3. 4%). Overall, 16 (76%) of 21 mutations were found in only one population. The most common mutation observed, Gln368Stop, was found in 27/1703 (1.6%) glaucoma probands and was found at least once in all groups except the Japanese. Studies of genetic markers flanking the myocilin gene suggest that most cases of the Gln368Stop mutations are descended from a common founder. Although the specific mutations found in each of the five populations were different, the overall frequency of myocilin mutations was similar ( approximately 2-4%) in all populations, suggesting that the increased rate of glaucoma in African Americans is not due to a higher prevalence of myocilin mutations.
Article
Full-text available
The glaucomas are a group of optic neuropathies comprising the leading cause of irreversible blindness worldwide. Elevated intraocular pressure due to a reduction in normal aqueous outflow is a major causal risk factor. We found that endothelial leukocyte adhesion molecule-1 (ELAM-1), the earliest marker for the atherosclerotic plaque in the vasculature, was consistently present on trabecular meshwork (TM) cells in the outflow pathways of eyes with glaucomas of diverse etiology. We determined expression of ELAM-1 to be controlled by activation of an interleukin-1 (IL-1) autocrine feedback loop through transcription factor NF-kappaB, and activity of this signaling pathway was shown to protect TM cells against oxidative stress. These findings characterize a protective stress response specific to the eye's aqueous outflow pathways and provide the first known diagnostic indicator of glaucomatous TM cells. They further indicate that common mechanisms contribute to the pathophysiology of the glaucomas and vascular diseases.
Article
Full-text available
The purpose was to study whether any differences exist in the optic nerve head (ONH) and peripapillary retinal blood flow between the two eyes of patients with unilateral exfoliation glaucoma or ocular hypertension (OHT) with exfoliation syndrome. This cross-sectional study included 50 patients. All had exfoliation syndrome with glaucoma or OHT in one eye, and these eyes comprised the study group. The fellow eyes, all normotensive, comprised the control group. Blood flow was measured with scanning laser Doppler flowmetry in the lamina cribrosa region, in the rim area, and on the peripapillary retina. Multiple linear regression analyses were used to identify any associations between different factors and differences in flow. Flow in the rim area was significantly higher in the study eyes than in the control eyes, with a mean difference of 172 arbitrary units (P = 0.001). The difference of 40 units in the laminar area was of borderline significance (P = 0.065) and no significant difference was found in the peripapillary retina (P = 0.530). In the study eyes, blood flow of the ONH lamina and rim area decreased with increasing glaucomatous damage, and treatment with topical timolol was associated with reduced blood flow in the lamina cribrosa and rim area. Perfusion pressure was associated only with flow in the peripapillary area (P = 0.021). Advanced glaucomatous damage was associated with reduced flow both in the lamina cribrosa and the rim area but not in the peripapillary retina. Treatment with topical timolol was associated with decreased flow in the ONH.
Article
Full-text available
Telomere shortening limits the number of cell divisions of primary human cells and might affect the regenerative capacity of organ systems during aging and chronic disease. To test whether the telomere hypothesis applies to human cirrhosis, the telomere length was monitored in cirrhosis induced by a broad variety of different etiologies. Telomeres were significantly shorter in cirrhosis compared with noncirrhotic samples independent of the primary etiology and independent of the age of the patients. Quantitative fluorescence in situ hybridization showed that telomere shortening was restricted to hepatocytes whereas lymphocytes and stellate cells in areas of fibrosis had significantly longer telomere reserves. Hepatocyte-specific telomere shortening correlated with senescence-associated beta-galactosidase staining in 84% of the cirrhosis samples, specifically in hepatocytes, but not in stellate cells or lymphocytes. Hepatocyte telomere shortening and senescence correlated with progression of fibrosis in cirrhosis samples. This study demonstrates for the first time that cell type-specific telomere shortening and senescence are linked to progression of human cirrhosis. These findings give a novel explanation for the pathophysiology of cirrhosis, indicating that fibrotic scarring at the cirrhosis stage is a consequence of hepatocyte telomere shortening and senescence. The data imply that future therapies aiming to restore regenerative capacity during aging and chronic diseases will have to ensure efficient targeting of specific cell types within the affected organs.
Article
Full-text available
No information is available on the interaction between cigarette smoke, the most important man-made carcinogen, and light, the most widespread natural carcinogen. In order to clarify this issue, SKH-1 hairless mice were exposed to environmental smoke and/or to the light emitted by sunlight-simulating halogen quartz bulbs. After 28 days, intermediate biomarkers were evaluated in skin, respiratory tract, bone marrow and peripheral blood. The results showed that, individually, the light produced extensive alterations not only in the skin but even at a systemic level, as shown by formation of bulky DNA adducts in both lung and bone marrow and induction of cytogenetic damage in bone marrow and peripheral blood erythrocytes. Smoke damaged the respiratory tract and produced significant alterations in the skin as well as an evident cytogenetic damage in both bone marrow and peripheral blood. Interestingly, as compared with exposure to smoke only, alternate daily cycles of exposure to both light and smoke significantly increased malondialdehyde concentrations and DNA adduct levels in lung and the frequency of micronuclei in pulmonary alveolar macrophages. The oral administration of sulindac, a non-steroidal anti-inflammatory drug, attenuated several biomarker alterations due to the combined exposure of mice to light and smoke. In conclusion, the light induces a systemic genotoxic damage, which is presumably due to the UV-mediated formation in the skin of long-lived derivatives, such as aldehydes. This damage may mechanistically be involved in light-related hematopoietic malignancies. In addition, the light displayed an insofar unsuspected synergism with smoke in the induction of DNA damage in the respiratory tract.
Article
Full-text available
The linear and nonlinear viscoelastic response of networks of cross-linked and bundled cytoskeletal filaments demonstrates remarkable scaling with both frequency and applied prestress, which helps elucidate the origins of the viscoelasticity. The frequency dependence of the shear modulus reflects the underlying single-filament relaxation dynamics for 0.1-10 rad/sec. Moreover, the nonlinear strain stiffening of such networks exhibits a universal form as a function of prestress; this is quantitatively explained by the full force-extension relation of single semiflexible filaments.
Article
Full-text available
Current evidence suggests that vascular insufficiencies in the optic nerve head play an important part in the pathogenesis of glaucomatous optic neuropathy. Trabeculectomy is the most common operative procedure for the treatment of medically uncontrolled glaucoma. This study was conducted to investigate whether trabeculectomy may improve ocular haemodynamics. 30 patients with primary open angle glaucoma about to undergo trabeculectomy were included in the study. Patients were evaluated before surgery and at 2 and 10 weeks after trabeculectomy. Optic nerve head blood flow (OnhBF) was assessed with scanning laser Doppler flowmetry. Fundus pulsation amplitude (FPA) measurements were obtained with laser interferometry. Because of the decrease in intraocular pressure there was a significant increase in ocular perfusion pressure (OPP) following trabeculectomy (18.5% (SD 12.0%) and 19.0% (17.1%) at 2 and 10 weeks postoperatively; p <0.001). A significant increase in OnhBF was observed after trabeculectomy (11.6% (16.4%) and 16.2% (20.2%) for each postoperative visit, respectively; p <0.001). FPA was also significantly higher compared with baseline values (17.2% (17.3%) and 17.4% (16.3%), respectively; p <0.001). A significant association between the increase in OPP and the increase in OnhBF and FPA was observed 10 weeks after surgery (r = 0.47; p = 0.009, and r = 0.50; p = 0.005, respectively). The results of this study suggest that trabeculectomy improves ocular blood flow in patients with chronic open angle glaucoma.
Article
Full-text available
This review describes the three mammalian glutathione transferase (GST) families, namely cytosolic, mitochondrial, and microsomal GST, the latter now designated MAPEG. Besides detoxifying electrophilic xenobiotics, such as chemical carcinogens, environmental pollutants, and antitumor agents, these transferases inactivate endogenous alpha,beta-unsaturated aldehydes, quinones, epoxides, and hydroperoxides formed as secondary metabolites during oxidative stress. These enzymes are also intimately involved in the biosynthesis of leukotrienes, prostaglandins, testosterone, and progesterone, as well as the degradation of tyrosine. Among their substrates, GSTs conjugate the signaling molecules 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) and 4-hydroxynonenal with glutathione, and consequently they antagonize expression of genes trans-activated by the peroxisome proliferator-activated receptor gamma (PPARgamma) and nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). Through metabolism of 15d-PGJ2, GST may enhance gene expression driven by nuclear factor-kappaB (NF-kappaB). Cytosolic human GST exhibit genetic polymorphisms and this variation can increase susceptibility to carcinogenesis and inflammatory disease. Polymorphisms in human MAPEG are associated with alterations in lung function and increased risk of myocardial infarction and stroke. Targeted disruption of murine genes has demonstrated that cytosolic GST isoenzymes are broadly cytoprotective, whereas MAPEG proteins have proinflammatory activities. Furthermore, knockout of mouse GSTA4 and GSTZ1 leads to overexpression of transferases in the Alpha, Mu, and Pi classes, an observation suggesting they are part of an adaptive mechanism that responds to endogenous chemical cues such as 4-hydroxynonenal and tyrosine degradation products. Consistent with this hypothesis, the promoters of cytosolic GST and MAPEG genes contain antioxidant response elements through which they are transcriptionally activated during exposure to Michael reaction acceptors and oxidative stress.
Article
Full-text available
To evaluate the intensity of oxidative molecular damage and its clinical correlations: visual field damage, intraocular pressure, age, and disease duration. DNA was extracted from human trabecular meshwork specimens collected from 17 glaucoma-affected patients using standard filtration surgery. Twenty-one specimens from healthy eyes collected for cornea transplants serve as controls. Oxidative DNA damage was evaluated by determining 8-hydroxy-2'-deoxyguanosine levels. All patients underwent a Humphrey 30-2 visual field examination and diurnal tonometry before surgery. The mean +/- SD DNA oxidative damage was 8.51 +/- 5.44 and 1.75 +/- 1.80 8-hydroxy-2'-deoxyguanosine molecules/10(5) normal nucleotides in patients with glaucoma and controls, respectively. A statistically significant correlation was found among human trabecular meshwork DNA oxidative damage, visual field damage, and intraocular pressure. No other statistically significant correlations were found. Oxidative stress may represent an important pathogenetic step in primary open-angle glaucoma because it could induce human trabecular meshwork degeneration, favoring an intraocular pressure increase, thus priming the glaucoma pathogenetic cascade.
Article
Full-text available
Genome-wide analyses of yeast provide insight into cellular responses to reactive oxygen species (ROS). Many deletion mutants are sensitive to at least one ROS, but no one oxidant is representative of 'oxidative stress' despite the widespread use of a single compound such as H(2)O(2). This has major implications for studies of pathological situations. Cells have a range of mechanisms for maintaining resistance that involves either induction or repression of many genes and extensive remodeling of the transcriptome. Cells have constitutive defense systems that are largely unique to each oxidant, but overlapping, inducible repair systems. The pattern of the transcriptional response to a particular ROS depends on its concentration, and 'classical' antioxidant systems that are induced by high concentrations of ROS can be repressed when cells adapt to low concentrations of ROS.
Article
The Hayflick limit—senescence of proliferative cell types—is a fundamental feature of proliferative cells in vitro. Various human proliferative cell types exposed in vitro to many types of subcytotoxic stresses undergo stress-induced premature senescence (SIPS) (also called stress-induced premature senescence-like phenotype, according to the definition of senescence). The known mechanisms of appearance the main features of SIPS are reviewed: senescent-like morphology, growth arrest, senescence-related changes in gene expression, telomere shortening. Long before telomere-shortening induces senescence, other factors such as culture conditions or lack of ‘feeder cells’ can trigger either SIPS or prolonged reversible G0 phase of the cell cycle. In vivo, ‘proliferative’ cell types of aged individuals are likely to compose a mosaic made of cells irreversibly growth arrested or not. The higher level of stress to which these cells have been exposed throughout their life span, the higher proportion of the cells of this mosaic will be in SIPS rather than in telomere-shortening dependent senescence. All cell types undergoing SIPS in vivo, most notably the ones in stressful conditions, are likely to participate in the tissular changes observed along ageing. For instance, human diploid fibroblasts (HDFs) exposed in vivo and in vitro to pro-inflammatory cytokines display biomarkers of senescence and might participate in the degradation of the extracellular matrix observed in ageing.
Article
The molecular mechanisms contributing to the progressive malfunction of the trabecular meshwork (TM)/Schlemm's canal (SC) conventional outflow pathway during aging and in Primary Open Angle Glaucoma (POAG) are still poorly understood. Progressive accumulation of damaged and cross-linked proteins is a hallmark of aging tissues and has been proposed to play a major role in the tissue abnormalities associated with organismal aging and many age-related diseases. Such progressive accumulation of damaged proteins with age is believed to result from both, increased oxidative stress that results in faster rates of protein damage, as well as from a functional decline in the cellular proteolytic machinery that eliminates misfolded and damaged proteins. Here, we review the reported data that supports the occurrence of oxidative damage and the alterations in the intracellular proteolytic systems in the TM in aging and POAG. Finally, we discuss how the functional decline of the cellular proteolytic machinery in the TM might lead to the observed physiologic alterations of the outflow pathway in glaucoma.
Article
We previously demonstrated that ultraviolet (UV) light (254 nm) induced the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in DNA via a singlet oxygen mechanism. In the present paper, we provide novel findings that DNA structure and base composition significantly affect the yield of 8-OHdG by UV radiation. Unlike ionizing radiation that induces 8-OHdG both in free 2'-deoxyguanosine (dG) and in DNA, UV light induced 8-OHdG formation in intact DNA and polydG.dC, but not in dG. When thermally denatured DNA was irradiated with UV light, the yield of 8-OHdG was reduced by more than 80% compared to intact DNA. Oxygenation of the denatured DNA solution did not restore the yield of UV-induced 8-OHdG. Irradiation of DNA with different AT/GC ratios showed that the yield of UV-induced 8-OHdG varied in proportion to the AT content, suggesting that AT base pairs in DNA enhance generation of the oxidizing species and subsequent oxidation of dG. The natural antioxidants genistein, estradiol, protocatechuic acid (PCA), and oleanolic acid (OA) were investigated for their inhibition of UV-induced 8-OHdG. Genistein and estradiol, that intercalate into DNA as shown by a computer modeling, significantly quenched UV-induced 8-OHdG, whereas PCA and OA did not fit into DNA and exhibited weak or no effect. These results suggest that the intercalation of genistein and estradiol into DNA may alter the DNA structural integrity, interrupt the production of oxidizing species, and subsequently reduce the formation of 8-OHdG by UV radiation.
Article
Para-inflammation is a tissue adaptive response to noxious stress or malfunction and has characteristics that are intermediate between basal and inflammatory states (Medzhitov, 2008). The physiological purpose of para-inflammation is to restore tissue functionality and homeostasis. Para-inflammation may become chronic or turn into inflammation if tissue stress or malfunction persists for a sustained period. Chronic para-inflammation contributes to the initiation and progression of many human diseases including obesity, type 2 diabetes, atherosclerosis, and age-related neurodegenerative diseases. Evidence from our studies and the studies of some others suggests that para-inflammation also exists in the aging retina in physiological conditions and might contribute to age-related retinal pathologies. The purpose of this review is to introduce the notion of “para-inflammation” as a state between frank, overt destructive inflammation and the non-inflammatory removal of dead or dying cells by apoptosis, to the retinal community.
Article
Oxidative stress plays an important role in pathogenesis of glaucoma. The purpose of this study is to investigate the novel effect of antiglaucoma drugs on the expression of antioxidant peroxiredoxins of trabecular meshwork (TM) cells. The expression of the peroxiredoxin family was investigated using immortalized TM cell lines. Cells were treated with antiglaucoma drugs and analyzed for the expression of peroxiredoxin, and cellular sensitivity to oxidative stress. Furthermore, the effect of antiglaucoma drugs on the molecular regulation of the expression of peroxiredoxin was examined using a reporter assay and siRNA strategy. Glaucomatous TM cells highly express peroxiredoxin 2 when compared with normal TM cells. Nipradilol and timolol, but not latanoprost, induce the expression of peroxiredoxin 2 through the activation of the Foxo3a transcription factor. TM cells showed reduced sensitivity to H(2)O(2) when cells were treated with either nipradilol or timolol, but not with latanoprost. In addition, both Foxo3a and PRDX2 expression were enhanced by drug-induced signal transduction through its receptor. These results indicate that both nipradilol and timolol possess a novel mechanism of action and function as potent protective agents against oxidative stress.
Article
Early age-related macular degeneration (AMD) is characterized by thickening of Bruch's membrane due to the accumulation of extracellular matrix (ECM). This finding could be related to hypoxia of the retinal pigment epithelium (RPE). In the present study, we investigated the effects of hypoxia and reoxygenation on the expression of connective tissue growth factor (CTGF), plasminogen activator inhibitor-1 (PAI-1), collagen type IV (Col IV) and fibronectin (Fn) in cultured human RPE cells. Cultured human RPE cells were kept for 12-36h under hypoxic conditions (1% O(2)). Reoxygenation was conducted for 24h. Hypoxia-mediated CTGF and PAI-1 expression were analyzed by using immunohistochemistry, Northern and Western blot analysis. Actinomycin D was added to examine whether hypoxia induces the transcription of CTGF and PAI-1 mRNA. Furthermore, cells were transfected with siRNA against hypoxia-inducible factor-1alpha (HIF-1alpha) and kept under hypoxic conditions. The effects of antioxidants on hypoxia/reoxygenation-mediated CTGF and PAI-1 expression were tested by real-time PCR analysis. Production of Col IV and Fn were investigated by real-time PCR and Western blot analysis. Both hypoxia and hypoxia/reoxygenation increased the expression of CTGF, PAI-1, Col IV and Fn. Actinomycin D prevented the new transcription of CTGF and PAI-1 mRNA by hypoxia. Using siRNA against HIF-1alpha, the hypoxia-mediated increase of CTGF and PAI-1 was inhibited. Antioxidants attenuated the reoxygenation-mediated increase of CTGF and PAI-1. The process of hypoxia/reoxygenation in the RPE may lead to an increase of ECM in the RPE and thus may contribute to the accumulation of ECM in Bruch's membrane.
Article
The global society is aging at an increasing rate, with a continually larger proportion of the population consisting of those over the age of 65. Age-related vascular changes have been demonstrated in ocular tissue, and the incidence and prevalence of diseases such as macular degeneration, glaucoma and vascular occlusive diseases increase significantly with age. This article reviews the current body of literature examining age-associated ocular vascular changes, and summarizes the aggregate findings. We discuss the potential role of the aging vasculature in the etiology of age-associated ocular disease, focusing on glaucoma. Our working hypothesis is that although advancing age is a physiological phenomenon, there are stepwise hemodynamic and vascular changes that occur, predisposing the eye and other tissue beds to pathological conditions. Advancing age does not independently give rise to disease, but does generate increasingly vulnerable vascular beds that are susceptible to further insults. These results compel a need for further investigation of age-related changes in ocular physiology and pathophysiology.
Article
Degenerative ocular diseases are widespread in the population and represent a major cause of reversible and irreversible blindness. Scientific evidences have been accumulating supporting the role of genotoxic damage and gene environment interactions in the pathogenesis of these diseases mainly including glaucoma, age-related macular degeneration, and cataract. Glaucoma, in its degenerative form, is characterized by the degeneration of the trabecular meshwork, the tissue of the anterior chamber of the eye devoted to aqueous-humour outflow. Such a degenerative process results in intra-ocular pressure increase and progressive damage of optic nerve head. Oxidative stress and DNA damage play an important role in inducing the degeneration of these well differentiated target tissues in which DNA damage results in a progressive cell loss. Macular degeneration is a common age-related disease affecting the central regions of the retina inducing progressive accumulation of oxidized lipoproteins and neovascularization. Environmental genotoxic risk factors include diet, light, and cigarette smoke paralleled by individual susceptibility as determined by adverse genetic assets. Cataract is a progressive opacity of the crystalline lens resulting from molecular damages induced by various risk factors including UV-containing light. This disease has been related to a failure in antioxidant defences. Experimental study provides evidence that cataract patients possess higher basal level of DNA damage, as evaluated by Comet test, in lymphocytes than controls. This finding is paralleled by the higher susceptibility to oxidative stress observed in the same patients. These novel experimental data further support the role of DNA damage as a main factor contributing to cataract onset. In conclusion, the examined degenerative ocular diseases recognise environmental risk factors often displaying genotoxic attitudes. Whenever these factors target individuals who are susceptible due their genetic assets the results is the onset of a specific eye disease depending on the affected ocular tissue.
Article
Elevated intraocular pressure (IOP) constitutes the best characterized risk for primary open-angle glaucoma (POAG). Elevated IOP is believed to result from an increase in aqueous humor outflow resistance at the level of the trabecular meshwork (TM)/Schlemm's canal. Malfunction of the TM in POAG is associated with the expression of markers for inflammation, cellular senescence, oxidative damage, and decreased cellularity. Current POAG treatments rely on lowering IOP, but there is no therapeutic approach available to delay the loss of function of the TM in POAG patients. We evaluated the effects of chronic administration of the dietary supplement resveratrol on the expression of markers for inflammation, oxidative damage, and cellular senescence in primary TM cells subjected to chronic oxidative stress (40% O2). Resveratrol treatment effectively prevented increased production of intracellular reactive oxygen species (iROS) and inflammatory markers (IL1alpha, IL6, IL8, and ELAM-1), and reduced expression of the senescence markers sa-beta-gal, lipofuscin, and accumulation of carbonylated proteins. Furthermore, resveratrol exerted antiapoptotic effects that were not associated with a decrease in cell proliferation. These results suggest that resveratrol could potentially have a role in preventing the TM tissue abnormalities observed in POAG.
Article
The content of the fibronectin, an extracellular glycoprotein in the drainage outflow system of human eyes was determined by the indirect immunoperoxidase staining technique. The degree of fibronectin accumulation in ocular tissues was evaluated by quantitative morphometric analysis. It was shown that the fibronectin level was elevated in the ocular drainage outflow system of humans in ageing and was rapidly increased at different stages of primary open-angle glaucoma development. Increased deposit of fibronectin in trabecular tissues, mainly, in the inner wall of Schlemm's canal and juxtacanalicular, or cribriform part of trabecular meshwork, was demonstrated along with ageing and glaucoma disease progression. A hypothesis underlying the glaucomatous process as an adhesive impairment was proposed.
Article
The trabecular meshwork of 5 rhesus monkeys (Macaca mulatta), 4 saimiris and 8 rats was studied by electron microscopy, and the optic nerve heads were examined by light microscopy as well. The eyes were enucleated at various intervals (15 min to 80 days) after injection of 0.5–1.2 C. Hb units of alphachymotrypsin in the posterior chamber. The intraocular pressure was determined by electromanometry or the Schiötz tonometer. Electron microscopy revealed fine fibrillar material (zonular fragments) in the intertrabecular spaces, which had the same appearance as the remnants of the zonular fibers in the region of the ciliary body. These fibrils, which were about 100 Å thick and up to 0.5 μm long, showed no regular periodicity. The distribution of these zonular fragments varied in different sectors of the eyes. In some sectors, zonular fragments were so numerous that the entire filtering area of the inner wall of Schlemm's canal was blocked. In rats, distinct pinocytotic processes could be observed within the endothelium of the inner wall, through which a part of the zonular material might be transported into Schlemm's canal and the adjacent outflow channels.
Article
There is evidence that H2O2 present in aqueous humor arises from ascorbic acid which is also present in this fluid, but the extent to which peroxide is derived from ascorbic acid is not known. We have measured the concentrations of H2O2 and ascorbic acid normally present in the aqueous humor of various species and also under conditions in which the level of ascorbic acid in the fluid was experimentally altered. In aqueous humor of rabbit and guinea pig the concentration of ascorbic acid was 10 times higher than that present in aqueous of rat and frog. Similarly, the concentration of H2O2 was four to 10 times higher in rabbit and guinea pig aqueous compared to that in rat and frog. Consistent with the higher concentration of ascorbic acid in posterior compared to anterior aqueous humor in the rabbit, the concentration of H2O2 was also significantly higher in the posterior aqueous. When ascorbic acid in rabbit aqueous humor was elevated by intraperitoneal administration of the compound, there was a significant increase in the level of H2O2 in both anterior and posterior aqueous humor. Moreover, when the level of ascorbic acid was lowered experimentally by placing guinea pigs on an ascorbic acid deficient diet, a 10-fold decrease in the level of both ascorbic acid and H2O2 was observed in the aqueous humor. Upon returning the animals to a normal diet, the concentrations of both compounds returned to control values. The direct correlation between the concentrations of ascorbic acid and H2O2 in aqueous humor suggests that ascorbic acid is the primary source of H2O2 in this fluid.
Article
This experiment was conducted to investigate the possible association between an increased frequency of glutathione-S-transferase (GST)1 gene deletion and the presence of cataracts in elderly patients. Genomic DNA was isolated from blood samples obtained from 138 elderly patients who had undergone cataract surgery, and from 62 random blood donors. All subjects lived in the same geographic area (Ibaraki Prefecture, Japan). The DNA sequences among three different exon ranges (exons 3-5, exons 4-5 and exons 5-6) of the GST1 gene were amplified by the polymerase chain reaction (PCR) technique to determine if GST1 gene deletion occurred. Cataract patients had a significantly higher frequency of GST1 gene deletion than random controls did (P < 0.001, odds ratio = 2.91, 1.56-5.44; 95% of confidence interval). Mean age of cataract patients lacking GST1 gene was significantly younger (n = 101, mean age = 70.4, s.d. = 10.2) than that of patients possessing the GST1 gene (n = 37, mean age = 75.0, s.d. = 8.7) (P < 0.02). These results show that the deletion of the GST1 gene may be one of determinants of genetic susceptibility to cataractgenic agents.
Article
A role of the oxygen radical generating system hypoxanthine-xanthine oxidase in hypoxia-reoxygenation injury was proposed 15 years ago. In recent years, however, new understanding of hypoxia-reoxygenation injury has been achieved and the significance of other oxygen radical generating systems has been acknowledged too. The hypothesis that an oxygen radical disease exists in preterm infants has recently been strengthened; an important observation is that preterm infants have lower activities of erythrocyte Cu/Zn superoxide dismutase compared to term babies. New actions of oxygen radicals have also been emphasized, and recently it has been demonstrated that the degree of protein oxidation of the lung of newborn infants is associated with chronic lung injury. The new insight into the interaction of oxygen radicals with other systems as excitatory amino acids and the NO system also increases the possibility to understand and hence prevent oxygen radical injury in the preterm infant as well as in adults exposed to an increased load of oxygen radicals.
Article
Mitochondria are cellular organelles where the generation of reactive oxygen species may be high. They are, however, effectively protected by their high capacities of antioxidative systems, as enzymes and either water or lipid soluble low molecular weight antioxidants. These antioxidative defence systems can be effectively regenerated after or during an oxidative stress as long as the mitochondria are in an energized state. Energization of mitochondria mainly depends on the availability of suitable respiratory substrates which can provide hydrogen for the reduction of either the glutathione- or α-tocopherol-system, since GSH is regenerated by glutathione reductase with the substrate NADPH and the α-tocopheroxyl-radical likely by reduced coenzyme Q. It was shown that mitochondria do not undergo damages as long as they can keep a high energy state. The delicate balance between prooxidative/antioxidative activities can be shifted towards oxidation, if experimentally prooxidants were added. After exhaustion of the antioxidative defence systems damages of mitochondrial functions become expressed followed by membrane injuries along with the oxidation and degradation of mitochondrial lipids and proteins leading finally to the total degradation of the mitochondria. Extramitochondrial antioxidants may assist the mitochondrial antioxidative defence systems in a complex way, whereby particularly ascorbic acid can act both as prooxidant and as antioxidant. (Mol Cell Biochem 174: 199–205, 1997)
Article
The mitochondrial electron transport system consumes more than 85% of all oxygen used by the cells, and up to 5% of the oxygen consumed by mitochondria is converted to superoxide, hydrogen peroxide, and other reactive oxygen species (ROS) under normal physiologic conditions. Disruption of mitochondrial ultrastructure is one of the earliest pathologic events during vitamin E depletion. The present studies were undertaken to test whether a direct link exists between vitamin E and the production of hydrogen peroxide in the mitochondria. In the first experiment, mice were fed a vitamin E-deficient or-sufficient diet for 15 weeks, after which the mitochondria from liver and skeletal muscle were isolated to determine the rates of hydrogen peroxide production. Deprivation of vitamin E resulted in an approximately 5-fold increase of mitochondrial hydrogen peroxide production in skeletal muscle and a 1-fold increase in liver when compared with the vitamin E-supplemented group. To determine whether vitamin E can dose-dependently influence the production of hydrogen peroxide, four groups of male and female rats were fed diets containing 0, 20, 200, or 2000 lU/kg vitamin E for 90 d. Results showed that dietary vitamin E dose-dependently attenuated hydrogen peroxide production in mitochondria isolated from liver and skeletal muscle of male and female rats. Female rats, however, were more profoundly affected by dietary vitamin E than male rats in the suppression of mitochondrial hydrogen peroxide production in both organs studied. These results showed that vitamin E can directly regulate hydrogen peroxide production in mitochondria and suggest that the overproduction of mitochondrial ROS is the first event leading to the tissue damage observed in vitamin E-deficiency syndromes. Data further suggested that by regulating mitochondrial production of ROS, vitamin E modulates the expression and activation of signal transduction pathways and other redox-sensitive biologic modifiers, and thereby delays or prevents degenerative tissue changes.
Article
To test the hypothesis that the aging mammalian heart and brain might have increased vulnerability to acute stress, DNA fragmentation was studied after hypoxia-reoygenation in young adult (6 months) and old (22-24 months) F344 rats. Heart and brain tissue were examined at the following time points: 30, 60, or 90 min of hypoxia (H, 5% O2, 95% N2) plus 2 h of reoxygenation (R, room air, 21% O2). With increasing duration of hypoxia preceding the reoxygenation, the extent of DNA fragmentation (in situ terminal dUTP nick end labeling, TUNEL, positive cells) was progressively higher in both age groups, greater in the old compared to that of the young adult rat. The levels of the anti-apoptotic proteins bcl-2 and bcl-xL, were similar in young and old at baseline and tended to increase in both age groups after hypoxia/reoxygenation. The pro-apoptotic protein, bax, was higher at baseline in the old; it rose after hypoxia/reoxygenation in the young adult heart and brain, but was unchanged in the old heart and was decreased in the old brain. The ratios of bcl-2/bax and of bcl-xL/bax were higher in the old heart and brain compared to that in the young adult after hypoxia/reoxygenation. Thus, compared to that of the young adult, the heart and brain of the old rat have lower thresholds and are more vulnerable to injury induced by hypoxia/reoxygenation, despite rapid and heightened expression of the anti-apoptotic proteins bcl-2 and bcl-xl. This could be due partly to the age-associated increase in the basal expression of the pro-apoptotic protein bax, as well as possibly other factors.
Article
Cellular stress can initiate prostaglandin (PG) biosynthesis which, through changes in gene expression, can modulate cellular functions, including cell growth. PGA(2), a metabolite of PGE(2), induces the expression of stress response genes, including gadd153 and hsp70, in HeLa cells and human diploid fibroblasts. PGs, gadd153, and hsp70 expression are also influenced by the cellular redox status. Polyphenolic glutathione conjugates retain the ability to redox cycle, with the concomitant generation of reactive oxygen species. One such conjugate, 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ), is a potent nephrotoxic and nephrocarcinogenic metabolite of the nephrocarcinogen, hydroquinone. We therefore investigated the effects of TGHQ on PGE(2) synthesis and gene expression in a renal proximal tubular epithelial cell line (LLC-PK(1)). TGHQ (200 microM, 2 h) increases PGE(2) synthesis (2-3-fold) in LLC-PK(1) cells with only minor (5%) reductions in cell viability. This response is toxicant-specific, since another proximal tubular toxicant, S-(1, 2-dichlorovinyl)-L-cysteine (DCVC), stimulates PGE(2) synthesis only after massive (68%) reductions in cell viability. Consistent with the ability of TGHQ to generate an oxidative stress, both deferoxamine mesylate and catalase protect LLC-PK(1) cells from TGHQ-mediated cytotoxicity. Only catalase, however, completely blocks TGHQ-mediated PGE(2) synthesis, implying a major role for hydrogen peroxide in this response. TGHQ induces the early (60 min) expression of gadd153 and hsp70. However, while inhibition of cyclooxygenase with aspirin prevents TGHQ-induced PGE(2) synthesis, it does not affect TGHQ-mediated induction of gadd153 or hsp70 expression. In contrast, a stable PGE(2) analogue, 11-deoxy-16, 16-dimethyl-PGE(2) (DDM-PGE(2)), which protects LLC-PK(1) cells against TGHQ-mediated cytotoxicity, modestly elevates the levels of gadd153 and hsp70 expression. In addition, catalase and, to a lesser extent, deferoxamine mesylate block TGHQ-induced gene expression. Therefore, although TGHQ-induced generation of reactive oxygen species is required for PGE(2) synthesis and stress gene expression, acute TGHQ-mediated increases in gadd153 and hsp70 mRNA levels are independent of PGE(2) synthesis.
Article
We tested the long-term effects of sublethal oxidative stresses on replicative senescence. WI-38 human diploid fibroblasts (HDFs) at early cumulative population doublings (CPDs) were exposed to five stresses with 30 microM tert-butylhydroperoxide (t-BHP). After at least 2 d of recovery, the cells developed biomarkers of replicative senescence: loss of replicative potential, increase in senescence-associated beta-galactosidase activity, overexpression of p21(Waf-1/SDI-1/Cip1), and inability to hyperphosphorylate pRb. The level of mRNAs overexpressed in senescent WI-38 or IMR-90 HDFs increased after five stresses with 30 microM t-BHP or a single stress under 450 microM H(2)O(2). These corresponding genes include fibronectin, osteonectin, alpha1(I)-procollagen, apolipoprotein J, SM22, SS9, and GTP-alpha binding protein. The common 4977 bp mitochondrial DNA deletion was detected in WI-38 HDFs at late CPDs and at early CPDs after t-BHP stresses. In conclusion, sublethal oxidative stresses lead HDFs to a state close to replicative senescence.
Article
Replicative senescence of human diploid fibroblasts (HDFs) or melanocytes is caused by the exhaustion of their proliferative potential. Stress-induced premature senescence (SIPS) occurs after many different sublethal stresses including H(2)O(2), hyperoxia, or tert-butylhydroperoxide. Cells in replicative senescence share common features with cells in SIPS: morphology, senescence-associated beta-galactosidase activity, cell cycle regulation, gene expression and telomere shortening. Telomere shortening is attributed to the accumulation of DNA single-strand breaks induced by oxidative damage. SIPS could be a mechanism of accumulation of senescent-like cells in vivo. Melanocytes exposed to sublethal doses of UVB undergo SIPS. Melanocytes from dark- and light- skinned populations display differences in their cell cycle regulation. Delayed SIPS occurs in melanocytes from light-skinned populations since a reduced association of p16(Ink-4a) with CDK4 and reduced phosphorylation of the retinoblastoma protein are observed. The role of reactive oxygen species in melanocyte SIPS is unclear. Both replicative senescence and SIPS are dependent on two major pathways. One is triggered by DNA damage, telomere damage and/or shortening and involves the activation of the p53 and p21(waf-1) proteins. The second pathway results in the accumulation of p16(Ink-4a) with the MAP kinase signalling pathway as possible intermediate. These data corroborate the thermodynamical theory of ageing, according to which the exposure of cells to sublethal stresses of various natures can trigger SIPS, with possible modulations of this process by bioenergetics.
Article
Kidney aging has been recognized as a chronic process of compromised renal function and structural changes in the tubulointerstitium and glomerulus. Cell senescence is associated with alterations in cell structure and function, including expression of cytokines and structural and regulatory components of extracellular matrix proteins. In this investigation, we tested the hypothesis that senescent renal cells may accumulate in vivo with advancing age. We also evaluated the expression of transforming growth factor (TGF)-beta1 and p21WAF1/CIP1 in aging kidneys. Sprague-Dawley rats at the ages of 3, 12, and 24 months were used for this study. Renal tissues were processed for morphometric and senescence analysis. Expression of TGF-beta1 and p21WAF1/CIP1 was evaluated by Northern or Western blot analysis and immunohistochemistry. Substantial tubulointerstitial injury occurred at the age of 12 months, but significant glomerular structure alteration was observed at the age of 24 months. Tubular cells developed senescence, which was detected by beta-galactosidase staining. This staining increased in frequency and intensity with age. Renal cortices showed a significant increase in the mRNA expression for TGF-beta1 and protein level for p21WAF1/CIP1. The enhanced expression of TGF-beta1 and p21WAF1/CIP1 was localized in the tubulointersititial cells. These data suggest that tubular cells undergo senescence and express increased TGF-beta1 and p21WAF1/CIP1 with advancing age. These age-related cellular and molecular alterations may play an important role in the initiation and/or progression of tubulointerstitial fibrosis and glomerulosclerosis in aging.
Article
The number of protein-bound carbonyl groups is an established marker of protein oxidation. Recent evidence indicates a significant increase in actin carbonyl content in both Alzheimer's disease brains and ischemic hearts. The enhancement of actin carbonylation, causing the disruption of the actin cytoskeleton and the loss of the barrier function, has also been found in human colonic cells after exposure to hypochlorous acid (HOCl). Here, the effects of oxidation induced by HOCl on purified actin are presented. Results show that HOCl causes a rapidly increasing yield of carbonyl groups. However, when carbonylation becomes evident, some Cys and Met residues have been already oxidized. Covalent intermolecular cross-linking as well as some noncovalent aggregation of carbonylated actin have been found. The covalent cross-linking, unaffected by reducing and denaturing agents, parallels an increase in dityrosine fluorescence. Moreover, HOCl-mediated oxidation induces the progressive disruption of actin filaments and the inhibition of F-actin formation. The molar ratios of HOCl to actin that lead to inhibition of actin polymerization seem to have effect only on cysteines and methionines. The process that involves oxidation of amino acid side chains with formation of a carbonyl group would occur at an extent of oxidative insult higher than that causing the oxidation of some critical amino acid residues. Therefore, the increase in actin content of carbonyl groups found in vivo would indicate drastic oxidative modification leading to drastic functional impairments.
Article
Preservatives are an important component of ophthalmic preparations, providing antimicrobial activity in the bottle and preventing decomposition of active drug. Often underrecognized, however, are the significant cytotoxic effects of preservatives associated with long-term therapy and especially use of multiple preserved drugs. The most common preservatives in ophthalmic preparations for glaucoma and surface eye disease-benzalkonium chloride (BAK), chlorobutanol, sodium perborate, and stabilized oxychloro complex (SOC)-were reviewed. Compared with other preservatives, SOC caused the least amount of damage to rabbit corneal epithelial cells. BAK has demonstrated cytotoxic effects in cell culture, as well as in animal and human studies. Physicians should consider treatment with new-generation preparations containing low-risk preservatives such as SOC, especially in patients receiving multiple ophthalmic medications.
Article
Among the consequences resulting from the exposure of endothelial cells (ECs) to ischemia/reperfusion is angiogenesis, involving degradation of vascular basement membrane and extracellular matrix. Matrix metalloproteinase (MMP)-2, a member of the MMP family, partakes in this process. MMP-2, secreted as a proenzyme, undergoes activation through interaction with membrane type (MT)1-MMP and the endogenous tissue inhibitor of MMPs (TIMP)-2. Although hypoxia and reoxygenation (H/R) are major constituents of ischemia/reperfusion processes, their direct effects on endothelial MMP-2 have been scarcely investigated. This study examined the in vitro effects of H/R on human macrovascular ECs (EAhy 926). The level of MMP-2 mRNA (Northern blot) and protein (zymography, ELISA) and the mRNA of its activator (MT1-MMP) and inhibitor (TIMP-2) were analyzed. Short (6-hour) hypoxia inhibited the mRNA expression of MMP-2, MT1-MMP, and TIMP-2, culminating in reduced latent and active MMP-2 protein. Prolonged (24-hour) hypoxia further suppressed MT1-MMP and TIMP-2 mRNA, whereas it enhanced MMP-2 mRNA and enzyme secretion (after 48-hour hypoxia). Reoxygenation did not influence the inhibited TIMP-2 but upregulated MMP-2 and MT1-MMP mRNA expression, leading to enhanced secretion of active MMP-2 protein. These results demonstrate H/R-mediated modulation of EC MMP-2 at both transcriptional and posttranscriptional levels. Prolonged hypoxia of ECs appears to enhance MMP-2 production and secretion, whereas reoxygenation further increases its level. These H/R-mediated effects on MMPs have the potential of enabling EC migration and possible angiogenesis.
Article
Prostaglandin E(2) (PGE(2)), a product of the cyclooxygenation of arachidonic acid released from membrane phospholipids, plays a critical role in inflammatory neurodegenerative conditions. Despite its classic role as a proinflammatory molecule, exogenous PGE(2) was suggested to have protective roles against neuronal death, although the exact protective mechanisms of PGE(2) are not yet defined. Thus, the aim of this study was to examine the effect of exogenous PGE(2) on inflammatory neurotoxicity. Lipopolysaccharide (LPS) induced neuronal toxicity, which was associated with terminal transferase dUTP nick end labeling (TUNEL)-positive neuronal death with increased caspase-3 activity. In neuron-glial coculture, LPS markedly induced inducible nitric oxide synthase/nitric oxide (iNOS/NO) release from microglial cells, but not from neurons; however, LPS-induced oxidative stress such as reactive oxygen species (ROS), measured with 2,7-dichlorofluorescein diacetate oxidation, was increased in neurons, but not in microglial cells. Exogenous PGE(2) (1 microg/ml) rescued the neurons, reducing iNOS/NO release from microglial cells and ROS formation from neurons. PGE(2) has been known to increase intracelluar cyclic adenosine monophosphate (cAMP) levels. In this study, we found that intracellular cAMP elevating agents, forskolin, and cAMP analogue, dbcAMP and 8-Br-cAMP, also prevented LPS-induced neuronal death. Thus, these results indicate that exogenous PGE(2) protects against LPS-induced neuronal apoptotic cell death through the intracellular cAMP system, and is associated with the modulation of NO from microglial cells and ROS production from neurons.
Article
Genetic factors have long been implicated in the pathophysiology of primary open-angle glaucoma (POAG). Recently, myocilin, a gene of unknown function, was associated with both juvenile open-angle glaucoma (JOAG) and POAG. Forty-three different myocilin mutations have been reported in open-angle glaucoma patients, and several large studies have suggested that as a group these mutations are associated with 3-4% of POAG in patient populations worldwide. Support for the pathogenicity of the individual myocilin mutations has been obtained from in vitro assays, statistical methods, and conservation of gene sequence arguments. Several of these myocilin mutations were observed in multiple patients allowing the identification of mutation-specific glaucoma phenotypes (maximum intraocular pressure and age at diagnosis). Associations between myocilin and other forms of open-angle glaucoma have been explored. At present there is no evidence to link myocilin mutations and steroid-induced ocular hypertension or normal-tension glaucoma. Clinical vignettes of POAG patients from four generations of a family harboring the TYR437HIS myocilin mutation are presented, highlighting the benefits of elucidating the genetics of glaucoma.
Article
Little is known about the molecular mechanisms responsible for the development of glaucoma, the leading cause of irreversible blindness worldwide. Some investigators have hypothesized that oxidative damage may be involved. We evaluated oxidative deoxyribonucleic acid (DNA) damage, in terms of 8-hydroxy-2'-deoxyguanosine (8-OH-dG), in the eyes of glaucoma patients. Levels of 8-OH-dG were measured in the trabecular meshwork region from 42 patients with glaucoma and 45 controls of similar age and sex. Genotypes of glutathione S-transferase isoenzymes (GSTM1 and GSTT1) were assessed by polymerase chain reaction in the same DNA samples. Levels of 8-OH-dG were significantly higher in glaucoma patients than in controls. Oxidative DNA damage in patients with glaucoma correlated significantly with intraocular pressure; in patients with primary open-angle glaucoma, it also correlated with visual field defects. GSTT1 was similar in the two groups, and had no effect on 8-OH-dG levels. Conversely, 8-OH-dG levels were significantly higher in GSTM1-null than in GSTM1-positive subjects. The GSTM1-null genotype was significantly more common in patients with primary open-angle glaucoma than in controls. Oxidative DNA damage is significantly increased in the trabecular meshwork of glaucoma patients. GSTM1 gene deletion, which has been associated with an increased risk of cancer at various sites and molecular lesions in atherosclerosis, predisposes to more severe oxidative DNA damage in glaucoma patients. These findings may contribute to understanding the pathogenesis of glaucoma and may be useful in the prevention and treatment of this disease.
Article
The mechanisms of trabecular cell loss in glaucoma patients are poorly understood. In order to determine whether drug-induced apoptosis could be one of the mechanisms by which trabecular cells die in glaucoma, we evaluated the effect of benzalkonium-preserved (BAC+) or preservative-free (BAC-) antiglaucoma medications on apoptotic marker expression by cultured human trabecular meshwork (HTM) cells. Normal and glaucomatous trabecular cell lines were treated for 15 min with antiglaucoma drugs (1/100 and 1/10 dilutions): timolol BAC+ or BAC-, betaxolol BAC+ or BAC-, latanoprost BAC+ or pure BAC. Apo2.7 expression, annexin V binding and DNA content were evaluated by flow cytometry and confocal microscopy. Results obtained in the two cell lines were similar for all tested drugs and criteria. In a 1/100 dilution, unpreserved beta-blockers had no apoptotic effect, preserved beta-blockers and latanoprost significantly increased Apo2.7 expression only, while BAC significantly increased all three apoptotic markers. When tested in a 1/10 dilution, all drugs except unpreserved timolol triggered a 2- to 3.5-fold increase in apoptotic features, whereas up to 95% of the cells underwent apoptosis upon treatment with BAC (representing a 9-fold increase over the background level). At concentrations higher than those supposed to be found in the aqueous humor after instillation (1/100 dilution), unpreserved beta-blocker exhibited no proapoptotic activity on HTM cells in vitro. Benzalkonium-containing beta-blockers and prostaglandin analogue triggered mild expression of one out of three apoptotic markers, while the pro-apoptotic effect observed with BAC appeared to be largely hindered by active compounds in the preserved eyedrops.
Article
Glaucoma is a progressive blinding disease characterized by gradual loss of vision due to optic neuropathy and retinal ganglion cell death. Increased intraocular pressure is a common feature of glaucoma that is thought to arise from an increased resistance to outflow of aqueous humor through the trabecular meshwork. Mutations of the myocilin gene are one cause of autosomal dominant juvenile- and adult-onset primary open angle glaucoma, but the mechanism by which mutant myocilins cause disease is poorly understood. We have found that disease-causing myocilin mutants are misfolded, are highly aggregation-prone and accumulate in large aggregates in the endoplasmic reticulum (ER) of human embryonic kidney cells and differentiated primary human trabecular meshwork (HTM) cells. In HTM cells, Pro370Leu mutant myocilin is not secreted under normal culture conditions and prolonged expression results in abnormal cell morphology and cell killing. Culturing HTM cells at 30 degrees C, a condition known to facilitate protein folding, promotes secretion of mutant myocilin, normalizes cell morphology and reverses cell lethality. Our results indicate that myocilin-associated glaucoma is an ER storage disease and suggest a progression of events in which chronic expression of misfolded, non-secreted myocilin leads to HTM cell death, trabecular meshwork dysfunction and, ultimately, a dominant glaucoma phenotype. The beneficial effects of facilitating folding and secretion of mutant myocilin suggest a new type of treatment for this form of glaucoma.
Article
To review and synthesize information concerning the pathogenesis of age-related macular degeneration (AMD). Review of the English-language literature. Five concepts relevant to the cell biology of AMD are as follows: (1) AMD involves aging changes plus additional pathological changes (ie, AMD is not just an aging change); (2) in aging and AMD, oxidative stress causes retinal pigment epithelial (RPE) and, possibly, choriocapillaris injury; (3) in AMD (and perhaps in aging), RPE and, possibly, choriocapillaris injury results in a chronic inflammatory response within the Bruch membrane and the choroid; (4) in AMD, RPE and, possibly, choriocapillaris injury and inflammation lead to formation of an abnormal extracellular matrix (ECM), which causes altered diffusion of nutrients to the retina and RPE, possibly precipitating further RPE and retinal damage; and (5) the abnormal ECM results in altered RPE-choriocapillaris behavior leading ultimately to atrophy of the retina, RPE, and choriocapillaris and/or choroidal new vessel growth. In this sequence of events, both the environment and multiple genes can alter a patient's susceptibility to AMD. Implicit in this characterization of AMD pathogenesis is the concept that there is linear progression from one stage of the disease to the next. This assumption may be incorrect, and different biochemical pathways leading to geographic atrophy and/or choroidal new vessels may operate simultaneously. Better knowledge of AMD cell biology will lead to better treatments for AMD at all stages of the disease. Many unanswered questions regarding AMD pathogenesis remain. Multiple animal models and in vitro models of specific aspects of AMD are needed to make rapid progress in developing effective therapies for different stages of the disease.
Article
To review current knowledge of key pathogenetic pathways in age-related macular disease (AMD). Experimental evidence and clinical observations are reviewed. A number of common downstream pathophysiologic pathways appear to be relevant in AMD manifestations irrespective of primary heterogeneous etiologies. These include sequelae of oxidative damage, retinal pigment epithelium (RPE) cell dysfunction with accumulation of lipofuscin and impairment of lysosomal functions, deposition of subsequently incompletely degraded material at the basal RPE cell side and alterations in Bruch's membrane extracellular matrix, immunologic responses to extracellular material (drusen) with subsequent growth of drusen, induction of choroidal neovascularization as a result of imbalance between anti-angiogenetic and proangiogenetic factors as well as cell death (geographic atrophy) without prior neovascular events. Understanding is expanding regarding the sequence of events that lead to early and late lesions in AMD. Therapeutic approaches that focus on the molecular mechanisms are more likely to succeed than currently available treatment options as exemplified by the management of choroidal neovascularisations.
Article
Experimentally induced ischemia of the endothelial cells surrounding the aqueous drainage sites led to a rise in intraocular pressure (IOP) in monkey and pigeon eyes. Clinical conditions associated with a rise in IOP in human eyes, e.g. peripheral retinal detachments, subluxated lenses, and occlusion of the internal carotid artery, can result in ischemia of the endothelial cells lining Schlemm's canal. These findings led to the hypothesis that ischemia of the endothelial cells of Schlemm's canal induces hypertension in human eyes. Thus, the ischemia hypothesis should be considered in discussions of the etiology of age-related open-angle glaucoma.
Article
Separation of clathrin-coated pits from the plasma membrane, a key event during endocytosis, is thought to be driven by dynamin and the actin cytoskeleton. However, the mechanism for the actin-mediated endocytosis remains elusive. RNA interference-mediated suppression of cortactin, an F-actin binding protein that promotes Arp2/3 complex-mediated actin polymerization, effectively blocked transferrin uptake. Depletion of cortactin in brain cytosol inhibited formation of clathrin-coated vesicles by 70% as analyzed in a cell-free system. Interestingly, the interaction between cortactin and dynamin 2 in cells was dependent on actin polymerization and was attenuated upon cell exposure to cytochalasin D as analyzed by immunofluorescence and immunoprecipitation. Moreover, a cortactin mutant deficient in Arp2/3 binding colocalized less efficiently with dynamin 2 and inhibited the uptake of transferrin. The effect of actin polymerization on the interaction between cortactin and the dynamin proline-rich domain (PRD) was further evaluated under a condition for actin polymerization in vitro. Cortactin binds to the dynamin PRD with an equilibrium dissociation constant of 81 nM in the presence of the Arp2/3 complex and actin, and 617 nM in the absence of actin polymerization. Taken together, these data demonstrate that Arp2/3-mediated actin polymerization regulates the accessibility of cortactin to dynamin 2 and imply a novel mechanism by which cortactin and dynamin drive the fission of clathrin-coated pits in an actin polymerization dependent manner.
Article
Glaucoma, one of the leading causes of blindness in the world, is characterized by progressive visual field loss and distinctive excavation of the optic nerve head. Although elevated intraocular pressure is the major risk factor, there is increasing evidence that the pathogenesis of glaucoma is also linked to altered ocular blood flow. This review summarizes the recent publications relevant to blood flow in glaucoma. Several studies indicate that a perfusion instability, rather than a steady reduction of ocular blood flow, might contribute to glaucomatous optic neuropathy. The main cause of the instability is a disturbed autoregulation in the context of a general vascular dysregulation. The underlying mechanism of such a vascular dysregulation is not known. A dysfunction of both the autonomic nervous system and vascular endothelial cells is discussed. The mechanical and vascular theories are not mutually exclusive; on the contrary, a vascular dysregulation increases the susceptibility to intraocular pressure. Therapeutically, therefore, both an intraocular pressure reduction and an improvement of the ocular blood flow might be considered.
Article
Hematopoietic cells are often exposed to transient hypoxia and reoxygenation as they develop and migrate. Given that bone marrow (BM) failure occurred in patients with Fanconi anemia (FA), we reason that hypoxia-then-reoxygenation represents a physiologically relevant stress for FA hematopoietic progenitor/stem cells. Here we show that expansion of Fancc-/- BM cells enriched for progenitor and stem cells was significantly decreased after 2 continuous cycles of hyperoxic-hypoxic-hyperoxic treatments compared with wild-type (WT) BM cells. This inhibition was attributable to a marked decrease of lineage-depleted (Lin-) ScaI- c-kit+ cells and more primitive Lin- ScaI+ c-kit+ cells in Fancc-/- BM cells following reoxygenation. Evaluation of the cell-cycle profile of long-term BM culture (LTBMC) revealed that a vast majority (70.6%) of reoxygenated Fancc-/- LTBMC cells was residing in the G0 and G1 phases compared with 55.8% in WT LTBMC cells. Fancc-/- LTBMC cells stained intensely for SA-beta-galactosidase activity, a biomarker for senescence; this was associated with increased expression of senescence-associated proteins p53 and p21(WAF1/CIP1). Taken together, these results suggest that reoxygenation induces premature senescence in Fancc-/- BM hematopoietic cells by signaling through p53, up-regulating p21, and causing senescent cell-cycle arrest. Thus, reoxygenation-induced premature senescence may be a novel mechanism underlying hematopoietic cell depletion and BM failure in FA.
Article
This review summarizes the Ernst H. Bárány Prize Lecture given at the XVI. meeting 2004 of the International Society of Eye Research in Sydney, Australia. The article describes the author's early studies starting with the determination of the site of aqueous humour outflow resistance and its regulation through ciliary muscle contraction, which were performed in collaborations with Bárány. It continues with the qualitative and quantitative evaluation of the trabecular meshwork (TM) changes seen in different kinds of glaucoma diseases. A comparison of correlations between meshwork pathology, IOP, and axon loss in the optic nerve between eyes with pseudoexfoliation glaucoma (PEXG) and primary open angle glaucoma (POAG) indicates that in the secondary glaucoma (PEXG) optic neuropathy is mainly induced by an increase in IOP. In eyes with POAG, the correlations point towards a more complex pathogenesis of the disease. Common factors might be involved in both the TM and the optic nerve changes. In vitro studies performed in cell cultures of human TM cells and optic nerve astrocytes as well as organ culture studies of the anterior eye segment indicate that TGFbeta2 might be one of the factors involved in the development of POAG. The paper is primarily focused on studies performed by the author and complete reference to other previous or contemporary studies is therefore not given as the purpose is not to present a comprehensive review article.