transforming-growth-factor-beta and Glaucoma

Connective tissue growth factor (CTGF) is a distinct signaling molecule modulating many physiological and pathophysiological processes. This protein is upregulated in numerous fibrotic diseases that involve extracellular matrix (ECM) remodeling. It mediates the downstream effects of transforming growth factor beta (TGF-β) and is regulated via TGF-β SMAD-dependent and SMAD-independent signaling routes. Targeting CTGF instead of its upstream regulator TGF-β avoids the consequences of interfering with the pleotropic effects of TGF-β. Both CTGF and its upstream mediator, TGF-β, have been linked with the pathophysiology of glaucomatous optic neuropathy due to their involvement in the regulation of ECM homeostasis. The excessive expression of these growth factors is associated with glaucoma pathogenesis via elevation of the intraocular pressure (IOP), the most important risk factor for glaucoma. The raised in the IOP is due to dysregulation of ECM turnover resulting in excessive ECM deposition at the site of aqueous humor outflow. It is therefore believed that CTGF could be a potential therapeutic target in glaucoma therapy. This review highlights the CTGF biology and structure, its regulation and signaling, its association with the pathophysiology of glaucoma, and its potential role as a therapeutic target in glaucoma management.

Topics: Connective Tissue; Glaucoma; Humans; Intraocular Pressure; Trabecular Meshwork; Transforming Growth Factor beta

Primary open angle glaucoma (POAG), a chronic optic neuropathy, remains the leading cause of irreversible blindness worldwide. It is driven in part by the pro-fibrotic cytokine transforming growth factor beta (TGF-β) and leads to extracellular matrix remodelling at the lamina cribrosa of the optic nerve head. Despite an array of medical and surgical treatments targeting the only known modifiable risk factor, raised intraocular pressure, many patients still progress and develop significant visual field loss and eventual blindness. The search for alternative treatment strategies targeting the underlying fibrotic transformation in the optic nerve head and trabecular meshwork in glaucoma is ongoing. MicroRNAs are small non-coding RNAs known to regulate post-transcriptional gene expression. Extensive research has been undertaken to uncover the complex role of miRNAs in gene expression and miRNA dysregulation in fibrotic disease. MiR-29 is a family of miRNAs which are strongly anti-fibrotic in their effects on the TGF-β signalling pathway and the regulation of extracellular matrix production and deposition. In this review, we discuss the anti-fibrotic effects of miR-29 and the role of miR-29 in ocular pathology and in the development of glaucomatous optic neuropathy. A better understanding of the role of miR-29 in POAG may aid in developing diagnostic and therapeutic strategies in glaucoma.

Topics: Blindness; Fibrosis; Glaucoma; Glaucoma, Open-Angle; Humans; Intraocular Pressure; MicroRNAs; Optic Nerve Diseases; Transforming Growth Factor beta

Glaucoma is the leading cause of irreversible blindness worldwide and there is no effective treatment thus far. The trabecular meshwork has been identified as the major pathological area involved. Certain signaling pathways in the trabecular meshwork, including the Wnt, lysophosphatidic acid and transforming growth factor‑β pathways, have been identified as novel therapeutic targets in glaucoma treatment. Meanwhile, it has been reported that key proteins in these pathways, particularly the primary transcription regulator Yes‑associated protein (YAP) and transcriptional co‑activator with PDZ‑binding motif (TAZ), exhibit interactions with the Hippo pathway. The Hippo pathway, which was first identified in Drosophila, has drawn great focus with regard to various aspects of studies in recent years. One role of the Hippo pathway in the regulation of organ size was indicated by more recent evidence. Defining the relevant physiological function of the Hippo pathway has proven to be extremely complicated. Studies have ascribed a role for the Hippo pathway in an overwhelming number of processes, including cell proliferation, cell death and cell differentiation. Therefore, the present review aimed to unravel the roles of YAP and TAZ in the Hippo pathway and the pathogenesis of glaucoma. Furthermore, a new and creative study for the treatment of glaucoma is provided.

Topics: Animals; Glaucoma; Humans; Lysophospholipids; Models, Biological; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

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

Topics: Astrocytes; Bone Morphogenetic Proteins; Endothelins; Extracellular Matrix; Fibrosis; Glaucoma; Humans; Optic Disk; Transforming Growth Factor beta

Despite advances in surgical technique and postoperative care, fibrosis remains the major impediment to a marked reduction of intraocular pressure without the need of additional medication (complete success) following filtering glaucoma surgery. Several aspects specific to filtering surgery may contribute to enhanced fibrosis. Changes in conjunctival tissue structure and composition due to preceding treatments as well as alterations in interstitial fluid flow and content due to aqueous humor efflux may act as important drivers of fibrosis. In light of these pathophysiological considerations, current and possible future strategies to control fibrosis following filtering glaucoma surgery are discussed.

Topics: Aqueous Humor; Conjunctiva; Fibrosis; Filtering Surgery; Glaucoma; Humans; Intercellular Signaling Peptides and Proteins; Intraocular Pressure; Postoperative Complications; Signal Transduction; Trabeculectomy; Transforming Growth Factor beta; Wound Healing

In glaucoma, regardless of its etiology, retinal ganglion cells degenerate and eventually die. Although age and elevated intraocular pressure (IOP) are the main risk factors, there are still many mysteries in the pathogenesis of glaucoma. The advent of genome-wide microarray expression screening together with the availability of animal models of the disease has allowed analysis of differential gene expression in all parts of the eye in glaucoma. This review will outline the findings of recent genome-wide expression studies and discuss their commonalities and differences. A common finding was the differential regulation of genes involved in inflammation and immunity, including the complement system and the cytokines transforming growth factor β (TGFβ) and tumor necrosis factor α (TNFα). Other genes of interest have roles in the extracellular matrix, cell-matrix interactions and adhesion, the cell cycle, and the endothelin system.

Topics: Animals; Complement System Proteins; Disease Models, Animal; Gene Expression; Genome-Wide Association Study; Glaucoma; Humans; Mice; Optic Disk; Retinal Ganglion Cells; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Glaucoma is a progressive optic neuropathy frequently associated with elevated intraocular pressure, ocular vascular changes and extracellular matrix remodelling at the optic nerve head and in the trabecular meshwork. The pathogenesis is multifactorial and complex, but many recent studies have suggested that transforming growth factor-β (TGF-β) plays a major role in the process. Significantly elevated levels of TGF-β have been identified in the anterior chamber of glaucomatous eyes. TGF-β has also been shown to directly cause increased intraocular pressure. It is believed that this occurs through complex interaction with the trabecular meshwork, leading to decreased aqueous humour outflow. These processes occur through specific interactions with various proteins and signalling molecules also present in ocular tissues. By understanding the role that TGF-β plays in the pathogenesis of glaucoma, alternative therapeutic agents can be developed, which target these pathways and improve and assist in the management of disease. This review will cover previous investigative studies and discuss the current understanding of TGF-β's role in glaucoma and how it may serve as a potential therapeutic target.

Topics: Glaucoma; Humans; Signal Transduction; Transforming Growth Factor beta

Glaucoma, a leading cause of irreversible blindness, is often associated with increased resistance to aqueous outflow in trabecular tissue. Increased outflow resistance has been attributed to increased extracellular matrix (ECM) deposition in trabecular tissue. A critical balance between the synthesis and breakdown of the components of extracellular tissue is important in keeping the intraocular pressure within the normal range. Multiple mechanisms have been shown to affect ECM turnover in trabecular tissue. In this review, we examine the related literature to understand the role of TGF-beta in ECM turnover, in the development and progression of glaucoma, and in possible therapeutic strategies that can be devised by targeting the TGF-beta signaling pathways.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antihypertensive Agents; Disease Progression; Extracellular Matrix; Glaucoma; Humans; Intraocular Pressure; Signal Transduction; Trabecular Meshwork; Transforming Growth Factor beta

The molecular and physiological mechanisms that lead to the progression of glaucoma are poorly understood. Despite the fact that glaucoma afflicts millions of people worldwide, research on the disease is limited by the current animal models that do not translate well to human forms of the disease. However, recent advances in culturing and manipulating human trabecular meshwork cells may provide a means to elucidate some of the mechanisms that cause glaucoma. This review focuses on the properties of trabecular meshwork cells, from their characteristic expression profile in vivo to their responsiveness to biochemical and biophysical signals in vitro. Hopefully the study of cultured trabecular meshwork cells will provide a better understanding of glaucoma and lead to new, much needed therapies.

Topics: Cell Culture Techniques; Dexamethasone; Glaucoma; Glucocorticoids; Humans; Mechanotransduction, Cellular; Models, Biological; Oxidative Stress; Phagocytosis; Trabecular Meshwork; Transforming Growth Factor beta

Conjunctival scarring remains the major problem in filtering glaucoma surgery. Antimetabolites afford a reduction of scar formation, but considerable side effects limit their application. Here, we review the mechanisms and peculiarities of wound healing following glaucoma surgery and report on new developments in the field of wound healing modulation. The growth factor TGF-beta has a central role in wound healing and scarring. Therefore, novel concepts of wound healing modulation comprise scavenging of TGF-beta and specific inhibition of disinct downstream intracellular signalling pathways. Several compounds have entered preclinical evaluation and offer new potential to modulate scarring in future combination therapies.

Topics: Cicatrix; Conjunctival Diseases; Filtering Surgery; Glaucoma; Humans; Transforming Growth Factor beta; Wound Healing

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.

Topics: Ciliary Body; Glaucoma; Humans; Muscle Contraction; Optic Nerve; Trabecular Meshwork; Transforming Growth Factor beta; Transforming Growth Factor beta2

Development of the ocular anterior segment involves a series of inductive interactions between neural ectoderm, surface ectoderm and periocular mesenchyme. The timing of these events is well established but less is known about the molecular mechanisms involved. Various genes that participate in these processes have been identified. As the roles of more genes are determined, developmental pathways and networks will emerge. Here, we focus on recent advances made using mouse models. We summarize key morphological events in formation of anterior chamber structures, including the aqueous humor drainage structures that are involved in intraocular pressure (IOP) regulation and glaucoma. We discuss the developmental roles of genes that associate with abnormal anterior segment development and elevated IOP or glaucoma (including Bmp4, Cyp1b1, Foxc1, Foxc2, Pitx2, Lmx1b and Tyr ) and how some of these genes may fit into developmental networks.

Topics: Animals; Cornea; Disease Models, Animal; Ectoderm; Eye; Eye Proteins; Forkhead Transcription Factors; Gene Expression Regulation, Developmental; Glaucoma; Homeodomain Proteins; Humans; Intraocular Pressure; Iris; Lens, Crystalline; Mesoderm; Mice; Mice, Inbred C57BL; Models, Biological; Paired Box Transcription Factors; PAX6 Transcription Factor; Repressor Proteins; Signal Transduction; Time Factors; Transcription Factors; Transforming Growth Factor beta

Glaucoma is the major cause of irreversible blindness throughout the world. Of all of the treatments that are available at present, the most effective appears to be surgery; however, excessive conjunctival scarring can lead to surgical failure. In the last decade, the introduction of the anti-metabolites mitomycin-C and 5-fluorouracil as anti-scarring treatments have greatly improved the results of glaucoma surgery, but these agents are associated with complications that can potentially result in blindness. A possible target for a more physiological approach to anti-scarring is transforming growth factor beta. This review examines the role of transforming growth factor beta in conjunctival scarring and discusses promising new ways of modifying its activity.

Topics: Animals; Cicatrix; Conjunctival Diseases; Filtering Surgery; Glaucoma; Humans; Mice; Transforming Growth Factor beta; Wound Healing

The main problem of the surgical failure after trabeculectomy is postoperative scarring of the filtering bleb. The treatment with anti-metabolites such as 5-fluorouracil and mitomycin C to prevent scarring shows many toxic side-effects and causes patient stress. In the search for therapeutic alternatives, the focus of current research concentrates on the pathophysiological interaction between growth factors and their role in the induction of scarring. Among the growth factors, TGF-beta 2 represents an important stimulant of scarring with a key regulatory function. The TGF-beta 2 concentration in the aqueous humor of primary open angle glaucoma is significantly elevated. In vitro and in vivo studies have demonstrated promising inhibition of scarring after subconjunctival application of human monoclonal anti-TGF-beta 2 antibody, which appears to be a safe and well tolerated therapeutic approach. Currently, several prospective studies are investigating the role of anti-TGF-beta 2 antibody as an alternative to conventional long term inhibition of scarring.

Topics: Animals; Antibodies, Monoclonal; Cicatrix; Glaucoma; Humans; Postoperative Complications; Trabeculectomy; Transforming Growth Factor beta; Wound Healing

Various proteoglycans are expressed in ocular tissues. We investigated and reviewed the distribution and the potential roles of proteoglycans in cornea, trabecular meshwork, and retinal tissues.. Immunohistochemical studies were performed in rat ocular tissues. The concentration of transforming growth factor (TGF)-beta2, which regulates the expression of proteoglycans in aqueous humor from human glaucomatous eyes, was evaluated by enzyme-linked immunosorbent assay (ELISA). In retinal tissues, we examined the localization of 2 soluble nervous tissue-specific chondroitin sulfate proteoglycans, neurocan and phosphacan, by immunohistochemical analysis, then investigated the effect on the neurite outgrowth of cultivated retinal ganglion cells.. The expression of chondroitin sulfate in stroma was upregulated at early postnatal stages and reduced during development in rat eyes. In trabecular meshwork tissues, immunohistochemical studies showed the intense expression of decorin. Moreover, elevated levels of TGF-beta2 in the aqueous humor from glaucomatous patients were observed. In retinal tissues, neurocan and phosphacan were expressed mainly in nerve fiber-rich layers during rat postnatal stages. In vitro, the neurite extension from retinal ganglion cells was inhibited by neurocan and phosphacan.. Soluble extracellular proteoglycans in corneal and trabecular meshwork tissues contribute to the stromal transparency in the corneal tissues and the resistance of the aqueous humor outflow in trabecular meshwork tissues. In retinal tissues, chondroitin sulfate and heparan sulfate proteoglycans are not only secreted into the extracellular space of retinal tissues but also expressed in the membrane of the retinal cells, contributing to the neural network formation and the maintenance of the interphotoreceptor matrix.

Topics: Animals; Aqueous Humor; Cornea; Enzyme-Linked Immunosorbent Assay; Glaucoma; Immunoenzyme Techniques; Proteoglycans; Rats; Retina; Trabecular Meshwork; Transforming Growth Factor beta

The fibroblast is the central player in the wound repair and scarring processes that occur in the anterior segment of the eye. Glaucoma filtration surgery is the ultimate example of the importance of the wound healing process, as this process is the major determinant of the success of this procedure. We highlight the role of the fibroblast, and discuss some of the growth factors stimulating fibroblast proliferation, migration and extracellular matrix production in the wound environment. We also review current methods of suppressing fibroblast proliferation, the new concepts that have arisen from laboratory studies, and future directions of investigation and treatment.

Topics: Animals; Cell Division; Cell Movement; Depression, Chemical; Epidermal Growth Factor; Fibroblasts; Glaucoma; Humans; Transforming Growth Factor beta; Treatment Failure; Wound Healing

The human monoclonal antibody that neutralizes the growth factor TGFbeta(2) (CAT-152) safely and effectively inhibits in vitro and in vivo models of conjunctival scarring. This phase I/IIa clinical trial was designed to assess the safety and tolerability of CAT-152 in patients undergoing trabeculectomy.. Prospective randomized placebo-controlled clinical trial.. Twenty-four patients who were due to undergo primary trabeculectomy at Moorfields or Western Eye Hospitals in London, England, were recruited for this study and randomly assigned to treatment with either CAT-152 (100 microg in 100 microl) (n = 16) or placebo (n = 8).. The treatment regimen was a series of four 100-microl subconjunctival injections, given immediately before and after surgery, and at 1 day and 1 week postoperatively. Assessment consisted of a full ophthalmic examination with recordings of the logarithm of the minimum angle of resolution visual acuities performed at baseline and at set intervals after surgery. Any adverse events were recorded.. Logarithm of the minimum angle of resolution visual acuity, intraocular pressure, complications, and adverse events.. The results of 12 month's follow-up on all patients are documented. There were no statistically significant differences in the incidence of complications between the two groups, and no serious adverse events related to the study drug occurred. Blebs after CAT-152 antibody treatment were diffuse, noncystic, and nonavascular, unlike blebs associated with antimetabolites. The fall in intraocular pressure was greater in the CAT-152 group at 3 and 6 months (P < 0.05) and approached statistical significance at 12 months. There was a trend toward less intervention in those patients treated with CAT-152. The small number of patients included limited the power of the study (34%) to detect a difference between groups. Sixteen patients in each arm of the study would be required to obtain a power of 90% with a 5% significance level.. This is the first clinical study of CAT-152 in patients undergoing glaucoma filtration surgery. CAT-152 seems to be well tolerated, and based on these results further multicenter trials are underway.

Topics: Aged; Aged, 80 and over; Antibodies, Monoclonal; Drug Evaluation; Female; Glaucoma; Humans; Immunosuppressive Agents; Injections; Intraocular Pressure; Male; Middle Aged; Postoperative Complications; Prospective Studies; Safety; Trabeculectomy; Transforming Growth Factor beta; Transforming Growth Factor beta2; Treatment Outcome; Visual Acuity; Wound Healing

Glaucoma is a leading cause of blindness worldwide. Characteristic changes occur in the optic nerve and visual field of patients with glaucoma; optic nerve damage can be mitigated by lowering intraocular pressure. Treatment modalities include drugs and lasers; filtration surgery is necessary for patients with insufficient intraocular pressure reduction. Scar formation often contributes to glaucoma filtration surgery failure by increasing fibroblast proliferation and activation. Here, we examined the effects of ripasudil, a Rho-associated protein kinase (ROCK) inhibitor, on postoperative scar formation in human Tenon's fibroblasts.. Collagen gel contraction assays were used to compare contractility activity among ripasudil and other anti-glaucoma drugs. The effect of Ripasudil in combination with other anti-glaucoma drugs and transforming growth factor-β (TGF-β), latanoprost and timolol-induce contractions were also tested in this study. Immunofluorescence and Western blotting were used to study the expression of factors relating scarring formation.. Ripasudil inhibited contraction in collagen gel assay and reduced α-smooth muscle actin (SMA) and vimentin (scar formation-related factors) expression, which was inversely promoted by latanoprost, timolol or TGF-β. Ripasudil also inhibited contraction on TGF-β, latanoprost and timolol-induced contraction. Furthermore, we investigated the effects of ripasudil on postoperative scarring in a mouse model; ripasudil suppressed postoperative scar formation by altering the expression of α-SMA and vimentin.. These results suggest that ripasudil, ROCK inhibitor may inhibit excessive fibrosis after glaucoma filtering surgery vis inhibition the transdifferentiation of tenon fibroblast into myofibroblast and may have a potential effect as anti-scarring for glaucoma filtration surgery.

Topics: Animals; Antiglaucoma Agents; Cicatrix; Collagen; Fibroblasts; Filtering Surgery; Glaucoma; Humans; Latanoprost; Mice; Protein Kinase Inhibitors; rho-Associated Kinases; Timolol; Transforming Growth Factor beta; Vimentin

This study investigated the effect of dipeptidyl peptidase-4 inhibitors (DPP-4is) on fibrosis after glaucoma filtering surgery with clinical data and an in vitro model that used transforming growth factor-β (TGF-β) to induce human Tenon's fibroblast (HTF) fibrosis.. The medical records of 41 eyes of 35 patients with diabetes with neovascular glaucoma (NVG) who received initial trabeculectomy were retrospectively reviewed. The surgical success rate was compared between cases that received (n = 23) and did not receive (n = 18) DPP-4i treatment for diabetes. The antifibrotic effects of linagliptin (a DPP-4i) were evaluated with quantitative real-time PCR for fibrosis markers (α-smooth muscle actin, collagen Iα, and fibronectin), a scratch assay, and a collagen gel contraction assay of primary cultured HTFs treated with TGF-β1 and linagliptin. Western blotting analysis was performed to evaluate the levels of phosphorylated Smad2 and Smad3 in the presence of linagliptin.. The Kaplan-Meier curve for bleb survival was higher in patients who received DPP-4is (P = 0.017, log-rank test). The in vitro experiments demonstrated that treatment with linagliptin attenuated the elevated levels of fibrosis markers induced by TGF-β1 in HTFs. Linagliptin treatment also prevented the migration and gel contraction of HTFs. Linagliptin inhibited the phosphorylation of Smad2 and Smad3, which is the canonical pathway of TGF-β signaling.. The current study indicates the potential effect of DPP-4is for maintaining bleb function after glaucoma filtering surgery in patients with diabetes with NVG. Our results demonstrate that linagliptin attenuates fibrotic change in HTFs by inhibiting TGF-β/Smad signaling.

Topics: Cells, Cultured; Collagen; Dipeptidyl-Peptidase IV Inhibitors; Fibroblasts; Fibrosis; Glaucoma; Humans; Linagliptin; Retrospective Studies; Signal Transduction; Trabeculectomy; Transforming Growth Factor beta; Transforming Growth Factor beta1

Cells in the trabecular meshwork sense and respond to a myriad of physical forces through a process known as mechanotransduction. Whilst the effect of substratum stiffness or stretch on TM cells have been investigated in the context of transforming growth factor (TGF-β), Wnt and YAP/TAZ pathways, the role of Notch signaling, an evolutionarily conserved pathway, recently implicated in mechanotransduction, has not been investigated in trabecular meshwork (TM) cells. Here, we compare the endogenous expression of Notch pathway molecules in TM cells from glaucomatous and non-glaucomatous donors, segmental flow regions, and when subjected to cyclical strain, or grown on hydrogels of varying rigidity.. Primary TM from glaucomatous (GTM), non-glaucomatous (NTM) donors, and from segmental flow regions [high flow (HF), low flow (LF)], were utilized between passages 2-6. Cells were (i) plated on tissue culture plastic, (ii) subjected to cyclical strain (6 h and 24 h), or (iii) cultured on 3 kPa and 80 kPa hydrogels. mRNA levels of Notch receptors/ligands/effectors in the TM cells was determined by qRT-PCR. Phagocytosis was determined as a function of substratum stiffness in NTM-HF/LF cells in the presence or absence of 100 nM Dexamethasone treatment.. Innate expression of Notch pathway genes were significantly overexpressed in GTM cells with no discernible differences observed between HF/LF cells in either NTM or GTM cells cultured on plastic substrates. With 6 h of cyclical strain, a subset of Notch pathway genes presented with altered expression. Expression of Notch receptors/ligands/receptors/inhibitors progressively declined with increasing stiffness and this correlated with phagocytic ability of NTM cells. Dexamethasone treatment decreased phagocytosis regardless of stiffness or cells isolated from segmental outflow regions.. We demonstrate here that the Notch expression in cultured TM cells differ intrinsically between GTM vs NTM, and by substratum cues (cyclical strain and stiffness). Of import, the most apparent differences in gene expression were observed as a function of substratum stiffness which closely followed phagocytic ability of cells. Interestingly, on soft substrates (mimicking normal TM stiffness) Notch expression and phagocytosis was highest, while both expression and phagocytosis was significantly lower on stiffer substrates (mimicking glaucomatous stiffness) regardless of DEX treatment. Such context dependent changes suggest Notch pathway may play differing roles in disease vs homeostasis. Studies focused on understanding the mechanistic role of Notch (if any) in outflow homeostasis are thus warranted.

Topics: Aged; Aged, 80 and over; Blotting, Western; Cells, Cultured; Dexamethasone; Female; Gene Expression Regulation; Glaucoma; Glucocorticoids; Humans; Male; Mechanotransduction, Cellular; Middle Aged; Phagocytosis; Real-Time Polymerase Chain Reaction; Receptors, Notch; RNA, Messenger; Signal Transduction; Tissue Donors; Trabecular Meshwork; Transcriptional Coactivator with PDZ-Binding Motif Proteins; Transforming Growth Factor beta; Wnt Proteins; YAP-Signaling Proteins

Elevated intraocular pressure (IOP) is the main risk factor for glaucoma, which might cause the activation of astrocytes in optic nerve head. To determine the effect of mechanical stretch on the astrocytes, we investigated the changes in cell phenotype, proteins of interest and signaling pathways under biaxial stretch.. The cultured astrocytes in rat optic nerve head were stretched biaxially by 10 and 17% for 24 h, respectively. Then, we detected the morphology, proliferation and apoptosis of the stretched cells, and performed proteomics analysis. Protein expression was analyzed by Isobaric tags for relative and absolute quantification (iTRAQ) mass spectrometry. Proteins of interest and signaling pathways were screened using Gene Ontology enrichment analysis and pathway enrichment analysis, and the results were verified by western blot and the gene-chip data from Gene Expression Omnibus (GEO) database.. The results showed that rearrangement of the actin cytoskeleton in response to stimulation by mechanical stress and proliferation rate of astrocytes decreased under 10 and 17% stretch condition, while there was no significant difference on the apoptosis rate of astrocytes in both groups. In the iTRAQ quantitative experiment, there were 141 differential proteins in the 10% stretch group and 140 differential proteins in the 17% stretch group. These proteins include low-density lipoprotein receptor-related protein (LRP6), caspase recruitment domain family, member 10 (CARD10), thrombospondin 1 (THBS1) and tetraspanin (CD81). The western blot results of LRP6, THBS1 and CD81 were consistent with that of iTRAQ experiment. ANTXR2 and CARD10 were both differentially expressed in the mass spectrometry results and GEO database. We also screened out the signaling pathways associated with astrocyte activation, including Wnt/β-catenin pathway, NF-κB signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, Jak-STAT signaling pathway, ECM-receptor interaction, and transforming growth factor-β (TGF-β) signaling pathway.. Mechanical stimulation can induce changes in cell phenotype, some proteins and signaling pathways, which might be associated with astrocyte activation. These proteins and signaling pathways may help us have a better understanding on the activation of astrocytes and the role astrocyte activation played in glaucomatous optic neuropathy.

Topics: Animals; Astrocytes; beta Catenin; Glaucoma; Lipoproteins, LDL; NF-kappa B; Optic Disk; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Thrombospondin 1; Transforming Growth Factor beta; Transforming Growth Factors

Glaucoma is a neurodegenerative disease that causes irreversible blindness due to loss of retinal ganglion cells (RGCs) and their axons. We previously identified a G661R mutation of ADAMTS10 (A Disintegrin And Metalloproteinase with ThromboSpondin type 1 motif 10) as the disease-causing mutation in a beagle model of glaucoma. ADAMTS10 is a secreted matrix metalloproteinase that belongs to the ADAMTS family which is involved in extracellular matrix (ECM) turnover. Previous studies have shown that ADAMTS10 binds fibrillin microfibrils, promotes their formation, and influences their fibrillin isoform composition. Here, we established a mouse model carrying the G661R mutation of ADAMTS10 (ADAMTS10

Topics: ADAMTS Proteins; Animals; Disease Models, Animal; Fibrillins; Glaucoma; Mice; Mutation; Neurodegenerative Diseases; Optic Nerve; Optic Nerve Diseases; Retinal Ganglion Cells; Transforming Growth Factor beta

Topics: Adenosine; Animals; Biomarkers; Disease Models, Animal; Disease Susceptibility; Extracellular Matrix; Fibroblasts; Gene Expression Regulation; Glaucoma; Humans; Immunohistochemistry; Methyltransferases; Rabbits; Signal Transduction; Smad3 Protein; Tenon Capsule; Transforming Growth Factor beta; Transforming Growth Factor beta1

Elevated intraocular pressure (IOP) is a significant risk factor for vision loss due to glaucoma, which is a major cause of blindness worldwide. Glaucoma filtration surgery (GFS) is an important method to reduce IOP by guidance of aqueous humor into a newly built filtration bleb in the conjunctiva; management of the wound healing mechanism is essential for the success of GFS. Here, we investigated the roles of interleukin (IL)-6 family members during the wound healing process after GFS. At the surgical site, the expression levels of genes encoding IL-6, oncostatin M (OSM), their receptors, and collagen I were elevated at 3 h after GFS, whereas the levels of genes encoding transforming growth factor (TGF)-β, α-smooth muscle actin (SMA), type IV collagen, and fibronectin were elevated at 3 days after GFS. IL-6 trans-signaling and OSM signaling suppressed TGF-β-induced expression of α-SMA and collagen IV, as well as activation of the non-canonical TGF-β pathway, suggesting that IL-6 and OSM may aid in controlling the phase transition from inflammation to proliferation and remodeling. The suppressive effects of OSM were accompanied by STAT3 activation, such that STAT1 function was complementary to STAT3. Taken together, these observations indicated that IL-6 family members constitute early response genes after GFS, which can suppress TGF-β-induced expression of late response genes at the surgical site after GFS.

Topics: Actins; Animals; Blotting, Western; Ciliary Neurotrophic Factor; Collagen Type IV; Conjunctiva; Female; Fibroblasts; Fibrosis; Glaucoma; Humans; Interleukin-6; Leukemia Inhibitory Factor; Oncostatin M; Rabbits; Real-Time Polymerase Chain Reaction; STAT1 Transcription Factor; STAT3 Transcription Factor; Trabeculectomy; Transforming Growth Factor beta; Wound Healing

Growth factors are considered as key molecules that participating in fibrosis formation. This research aimed to clarify potential effects of p53 on regulation of transforming growth factor β (TGF-β) and fibrosis formation and investigate the associated mechanisms.. Vimentin was examined to identify human Tenon's fibroblasts (HTFs). p53-targeting small interfere RNA (siRNA) was synthesis and transfected into HTFs. Real-time PCR assay was utilized to evaluate p53 and microRNA-29b (miR-29b) expression. Immunocytochemical assay was used to observe TGF-β expression. The wound healing assay was conducted to evaluate migration of HTFs. Dual-luciferase assay was employed to identify interaction between p53 and miR-29b in HTFs.. p53 inhibited expression of TGF-β, suppressed HTFs migration and inhibited HTFs growth, by reducing miR-29b expression and interacting with miR29b gene in HTFs.

Topics: Cell Movement; Cell Proliferation; Cells, Cultured; Fibroblasts; Gene Expression Regulation; Glaucoma; Humans; Immunohistochemistry; RNA; Tenon Capsule; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Scar formation can cause the failure of glaucoma filtration surgery. We investigated the effect of AR12286, a selective Rho-associated kinase inhibitor, on myofibroblast transdifferentiation and intraocular pressure assessment in rabbit glaucoma filtration surgery models. Cell migration and collagen contraction were used to demonstrate the functionality of AR12286-modulated human conjunctival fibroblasts (HConFs). Polymerase chain reaction quantitative analysis was used to determine the effect of AR12286 on the production of collagen Type 1A1 and fibronectin 1. Cell migration and collagen contraction in HConFs were activated by TGF-β1. However, compared with the control group, rabbit models treated with AR12286 exhibited higher reduction in intraocular pressure after filtration surgery, and decreased collagen levels at the wound site in vivo. Therefore, increased α-SMA expression in HConFs induced by TGF-β1 could be inhibited by AR12286, and the production of Type 1A1 collagen and fibronectin 1 in TGF-β1-treated HConFs was inhibited by AR12286. Overall, the stimulation of HConFs by TGF-β1 was alleviated by AR12286, and this effect was mediated by the downregulation of TGF-β receptor-related SMAD signaling pathways. In vivo results indicated that AR12286 thus improves the outcome of filtration surgery as a result of its antifibrotic action in the bleb tissue because AR12286 inhibited the TGF-β receptor-related signaling pathway, suppressing several downstream reactions in myofibroblast transdifferentiation.

Topics: Animals; Cell Line; Cell Transdifferentiation; Fibrosis; Filtering Surgery; Glaucoma; Humans; Myofibroblasts; Protein Kinase Inhibitors; Rabbits; Signal Transduction; Transforming Growth Factor beta

Recessive variants in LTBP2 are associated with eye-restricted phenotypes including (a) primary congenital glaucoma and (b) microspherophakia/megalocornea and ectopia lentis with/without secondary glaucoma. Nosology of LTBP2 pathology in humans is apparently in contrast with the consolidated evidence of a wide expression of this gene in the developing embryo. Accordingly, in previously published patients with LTBP2-related eye disease, additional extraocular findings have been occasionally reported and include, among others, high-arched palate, tall stature, and variable cardiac involvement. Anyway, no emphasis was put on such systemic manifestations. Here, we report two unrelated Roma/Gypsy patients first ascertained for a multisystem disorder mainly characterized by primary congenital glaucoma, complex congenital heart defect, tall stature, long fingers, skin striae and dystrophic scarring, and resembling Marfan syndrome. Heart involvement was severe with polyvalvular heart dysplasia in one, and transposition of great arteries, thoracic arterial tortuosity, polyvalvular heart dysplasia, and neo-aortic root dilatation in the other. Both patients were homozygous for the recurrent c.895C>T[p.(R299X)] variant, typically found in individuals of Roma/Gypsy descent with an eye-restricted phenotype. Our findings point out LTBP2 as responsible of a systemic phenotype coherent with the community of syndromes related to anomalies in genes involved in the TGFβ-pathway. Among these disorders, LTBP2-related systemic disease emerges as a distinct condition with expanding prognostic implications and autosomal recessive inheritance.

Topics: Adolescent; Child; Corneal Diseases; Ectopia Lentis; Eye Diseases, Hereditary; Female; Genetic Diseases, X-Linked; Glaucoma; Heart; Heart Defects, Congenital; Homozygote; Humans; Iris; Latent TGF-beta Binding Proteins; Male; Marfan Syndrome; Phenotype; Roma; Transforming Growth Factor beta

Surgical techniques such as trabeculectomy aim to treat glaucoma by making an incision into the scleral tissue, to create an alternative drainage pathway for aqueous to flow into the sub-Tenon's/subconjunctival space. However, tissue fibrosis and wound healing occurring after the procedures can reduce the success rate. This study aims to investigate the synergistic effects of aqueous humor in combination with shear stress on the fibrosis response occurring in Tenon's capsule and conjunctival tissue (TCCT) after glaucoma surgery.. Two-dimensional (2D) and 3D in vitro TCCT models were constructed by seeding porcine Tenon's capsule + conjunctival fibroblasts in collagen gel. These were used to investigate key growth factors (singular and natural form) with shear stress, which are believed to influence tissue fibrosis after glaucoma surgery. In addition to cell proliferation assessments, a nondestructive assay to quantify neocollagen synthesis in TCCT models, in response to these factors, has been applied up to 14 days.. TCCT fibroblast proliferation increased significantly with doses of TGF-β, TNF-α, and VEGF, in comparison with the control. Furthermore, fibroblasts exposed to 50% aqueous humor had significantly increased proliferation and actin expression. Shear stress-induced mechanotransduction was also found to promote metabolic activity across experimental conditions. Neocollagen labeling cross validated the fibrosis process.. Shear stress appeared to enhance the influence of key growth factors and further promoted fibrotic response within the model. These findings offer a useful insight for further study into the wound-healing response triggered by aqueous fluid outflow after glaucoma surgery.

Topics: Actins; Animals; Aqueous Humor; Cell Proliferation; Cells, Cultured; Collagen; Conjunctiva; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Fibrosis; Glaucoma; Imaging, Three-Dimensional; Immunohistochemistry; Swine; Tenon Capsule; Trabeculectomy; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Vimentin

Loeys-Dietz syndrome (LDS), an autosomal-dominant connective tissue disorder, is characterised by systemic manifestations including arterial aneurysm and craniofacial dysmorphologies. Although ocular involvement in LDS has been reported, detailed information on those manifestations is lacking.. Retrospective chart review of patients with diagnosed LDS and comparison with age-matched control patients.. Mean age was 37.8±14.6 years (patients with LDS) and 38.4±13.5 years (controls). Patients with LDS less frequently had iris transillumination, cataract and glaucoma compared with controls. Scleral and retinal vascular abnormalities were not found in any of the LDS eyes. Ectopia lentis was found in one patient with LDS. The eyes of patients with LDS tended to be more myopic (spherical equivalent, -2.47±2.70 dioptres (dpt) vs -1.30±2.96dpt (controls); P=0.08) and longer (24.6±1.7mm vs 24.1±1.5mm (controls); P=0.10). Central corneal thickness was significantly reduced in LDS eyes (521±48µm vs 542±37µm (controls); P=0.02). Corneal curvature (43.06±1.90dpt (LDS) versus 43.00±1.37dpt (controls); P=0.72) and interpupillary distance (65.0±6.0mm (LDS) vs 64.3±4.8mm (controls); P=0.66) did not differ significantly between both groups. Visual acuity was similar between both groups (0.03±0.09logarithm of the minimum angle of resolution (logMAR) for LDS eyes and 0.05±0.17logMAR for control eyes, P=0.47).. Ocular features of LDS include decreased central corneal thickness and mild myopia. Ectopia lentis may be slightly more common than in controls but appears less common than in Marfan syndrome. Hypertelorism, scleral and retinal vascular abnormalities were not features of LDS.

Topics: Adolescent; Adult; Aged; Biometry; Cataract; Corneal Diseases; Ectopia Lentis; Female; Glaucoma; Humans; Iris Diseases; Loeys-Dietz Syndrome; Male; Middle Aged; Myopia; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Retrospective Studies; Smad3 Protein; Transforming Growth Factor beta; Visual Acuity; Young Adult

We aimed at discussing the biological function of lncRNA GAS5 (Growth Arrest Specific 5) on the proliferative, apoptotic and differentiative abilities of retinal ganglion cells.. GAS5 expression was knocked down by transfection with siRNA targeting GAS5 (siGAS5) in retinal ganglion cells. After transfection, qRT-PCR was utilized to evaluate the mRNA level of GAS5 expression. The protein levels of smad2 and smad3 were detected by Western blot. Proliferative ability was accessed by Cell Counting Kit-8 (CCK-8) assay, and apoptosis and cell cycle changes were tested by flow cytometry. Also, the differentiative ability of RGC-5 cells was evaluated. TGF-β was exogenously administered to test its function on regulation of GAS5.. Knockdown of GAS5 promoted the cell proliferation and differentiation, but negatively regulated cell apoptosis, and had no effect on cell cycle. Exogenous administration of TGF-β could decrease the expression level of GAS5 in a dose-dependent manner. Meanwhile, lowly expressed GAS5 could improve the phosphorylation of smad2 and smad3.. In our study, we found that down-regulation of lncRNA GAS5 may maintain retinal ganglion cell survival in glaucoma through the activation of TGF-β pathway to promote cell proliferation and differentiation.

Topics: Apoptosis; Cell Differentiation; Cell Line; Cell Proliferation; Down-Regulation; Gene Knockdown Techniques; Glaucoma; Humans; Retinal Ganglion Cells; RNA, Long Noncoding; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

To investigate the effect of tacrolimus on the proliferation of fibroblasts after glaucoma surgery.. Biopsy was applied in this study. Under aseptic conditions, tissues were collected from rabbits, cut into small pieces and cultured. Morphology of fibroblasts was observed under a microscope. Features of fibroblasts were identified via immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Western blotting and RT-PCR were performed to detect the expressions of related proteins after treatment. Flow cytometry and cell counting kit-8 (CCK-8) assay were employed to examine the proliferation of human Tenon's capsule fibroblasts (HTFs) after tacrolimus treatment.. Tacrolimus decreased the levels of survivin and α-smooth muscle actin (α-SMA) after transforming growth factor-β (TGF-β) treatment. Besides, it inhibited proliferation and induced apoptosis of HTFs.. Tacrolimus reduces proliferation and promotes apoptosis of HTFs by inhibiting the expression of survivin, which may be a strategy for treating hypertrophic scar after glaucoma surgery.

Topics: Actins; Animals; Apoptosis; Cell Proliferation; Cells, Cultured; Cicatrix; Fibroblasts; Glaucoma; Humans; In Vitro Techniques; Rabbits; Survivin; Tacrolimus; Tenon Capsule; Transforming Growth Factor beta

To evaluate the effect of treatment with monocyte chemoattractant protein-1 receptor inhibitor (MCP-Ri) to maintain bleb survival and prevent fibrosis in an experimental model of glaucoma filtration surgery (GFS).. GFS was performed on one eye of C57/Bl6 mice (n = 36) that was treated with MCP-Ri, mitomycin-C (MMC), or vehicle at the time of surgery. Real-time polymerase chain reaction was used to evaluate conjunctival expression of monocyte chemoattractant protein-1 (MCP-1), TGFB1, TGFB2, collagen 1a1 (Col1a1), sparc (Sparc), and fibronectin at 2 and 7 days following surgery. Anterior segment slit-lamp examination, optical coherence tomography, and confocal microscopy were performed in vivo at day 14. Eyes were processed for immunohistochemical staining of F4/80, a monocyte-macrophage marker, at day 2. In vitro experiments were also performed to compare the effect of MMC, MCP-Ri, and vehicle on the viability of mouse Tenon's fibroblasts.. Treatment with MCP-Ri results in a greater reduction in the percentage of F4/80-positive cells in conjunctival blebs and lesser MCP-1 gene expression following experimental GFS than MMC or control. Both MMC and MCP-Ri reduced Col1a1 and Sparc expression, but not fibronectin. TGFB1 decreased with MCP-Ri but not MMC; MMC but not MCP-Ri reduced TGFB2. MMC and MCP-Ri treatment resulted in the preservation of bleb height at day 14, as compared to control. MCP-Ri was less toxic to mouse Tenon's fibroblasts in comparison with MMC.. Targeting MCP-1 results in prolonged bleb survival following experimental GFS with less cellular toxicity as compared to MMC. MCP inhibition could provide a safer alternative to conventional antifibrotic adjunctive treatments in GFS.

Topics: Animals; Antifibrinolytic Agents; Cell Survival; Chemokine CCL2; Collagen; Conjunctiva; Disease Models, Animal; Enzyme Inhibitors; Fibroblasts; Fibronectins; Fibrosis; Filtering Surgery; Glaucoma; Glaucoma Drainage Implants; Mice; Mice, Inbred C57BL; Mitomycin; Osteonectin; Receptors, CCR2; Transforming Growth Factor beta

Fibrosis and a hypoxic environment are associated with the trabecular meshwork (TM) region in the blinding disease glaucoma. Hypoxia has been shown to alter DNA methylation, an epigenetic mechanism involved in regulating gene expression such as the pro-fibrotic transforming growth factor (TGF) β1 and the anti-fibrotic Ras protein activator like 1 (RASAL1). The purpose of this study was to compare DNA methylation levels, and the expression of TGFβ1 and RASAL1 in primary human normal (NTM) with glaucomatous (GTM) cells and in NTM cells under hypoxic conditions.. Global DNA methylation was assessed by ELISA in cultured age-matched NTM and GTM cells. qPCR was conducted for TGFβ1, collagen 1α1 (COL1A1), and RASAL1 expression. Western immunoblotting was used to determine protein expression. For hypoxia experiments, NTM cells were cultured in a 1%O2, 5%CO2 and 37°C environment. NTM and GTM cells were treated with TGFβ1 (10ng/ml) and the methylation inhibitor 5-azacytidine (5-aza) (0.5μM) respectively to determine their effects on DNA Methyltransferase 1 (DNMT1) and RASAL1 expression.. We found increased DNA methylation, increased TGFβ1 expression and decreased RASAL1 expression in GTM cells compared to NTM cells. Similar results were obtained in NTM cells under hypoxic conditions. TGFβ1 treatment increased DNMT1 and COL1A1, and decreased RASAL1 expression in NTM cells. 5-aza treatment decreased DNMT1, TGFβ1 and COL1A1 expression, and increased RASAL1 expression in GTM cells.. TGFβ1 and RASAL1 expression, global DNA methylation, and expression of associated methylation enzymes were altered between NTM and GTM cells. We found that hypoxia in NTM cells induced similar results to the GTM cells. Furthermore, DNA methylation, TGFβ1 and RASAL1 appear to have an interacting relationship that may play a role in driving pro-fibrotic disease progression in the glaucomatous TM.

Topics: Aged; Aged, 80 and over; Azacitidine; Cells, Cultured; DNA Methylation; Epigenesis, Genetic; Extracellular Matrix Proteins; Female; Gene Expression; Glaucoma; GTPase-Activating Proteins; Humans; Hypoxia; Infant, Newborn; Male; Trabecular Meshwork; Transforming Growth Factor beta

Retinal ganglion cells (RGCs) are commonly experienced optic nerve diseases including glaucoma-induced injury that results in decrease of cell survival. However, the underlying mechanism remains to be elaborated. This present study was to focus on the miR-187 and Transforming growth factor-β (TGF-β) signal and investigated their roles in RGCs apoptosis and proliferation.. RGC-5 retinal ganglion cell line was chose in present study and subjected to miR-187 mimic or inhibitor transfection. Cell apoptosis was evaluated using flow cytometry-based Annexin V-PI assay. Cell proliferation was examined using CCK-8. Protein levels of Smad2/3/7 were determined using western blotting.. miR-187 negatively regulated cell survival via inhibiting cell apoptosis and promoting cell proliferation. We observed that alteration expression of miR-187 is closely related to phosphorylation levels of Smad2 and Smad3. This correlation is associated with down-regulation of Smad7 induced by miR-187 via targeting Smad7 3'-UTR. From result of co-transfection of Smad7-plasmid and miR-187 mimic or siSmad7 and miR-187 inhibitor, we concluded that cell proliferation and apoptosis was mediated by miR-187/Smad7 axis.. In summary, cell internal signal transduction, miR-187 regulating Smad7 expression, plays a vital role in retinal ganglion cell survival.

Topics: 3' Untranslated Regions; Animals; Apoptosis; Binding Sites; Cell Line; Cell Proliferation; Dose-Response Relationship, Drug; Glaucoma; MicroRNAs; Rats, Sprague-Dawley; Retinal Ganglion Cells; RNA Interference; Signal Transduction; Smad7 Protein; Transfection; Transforming Growth Factor beta; Up-Regulation

Excess scarring of the conjunctiva after glaucoma filtration surgery is a major cause of failure. Transforming growth factor (TGF)-β is critically involved in post-operative scarring. Lithium inhibits TGF-β-induced gene protein expression in corneal fibroblasts and inhibits TGF-β-induced epithelial mesenchymal transition. Here, we investigated the effects of LiCl on TGF-β1-mediated signaling pathways and on myofibroblast transdifferentiation of human Tenon's capsule fibroblasts (HTFs). LiCl treatment reduced expression of TGF-β1-induced α-SMA expression in HTFs. LiCl also decreased Akt phosphorylation induced by TGF-β1. TGF-β1-induced α-SMA expression was significantly decreased by LY294002 and Akt siRNA indicating that these changes are mediated by the PI3K/Akt pathway. Thus, LiCl induces the suppression of transdifferentiation stimulated by TGF-β1 by the regulation of PI3K/Akt signaling in HTFs.

Topics: Cell Differentiation; Cicatrix; Fibroblasts; Glaucoma; Humans; Lithium Chloride; Myofibroblasts; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Tenon Capsule; Transforming Growth Factor beta

The purpose of this work was to look into the effects of infliximab on wound healing in experimental glaucoma filtration surgery and to compare the antifibrotic effects of this agent to that of mitomycin-C (MMC).. Twenty-eight male New Zealand White rabbits were randomly assigned to four groups, each including seven rabbits: control group, sham group, MMC group, and infliximab group. The rabbits in the control group were not operated on and did not receive any treatment. The rabbits in the sham group underwent trabeculectomy and had one drop of saline instilled four times a day for 14 days. The rabbits in the MMC treatment group underwent trabeculectomy, and a sponge soaked in 0.4 mg/mL MMC was applied intraoperatively to the scleral surgical site for three minutes. The rabbits in the infliximab treatment group underwent trabeculectomy and one drop of 10 mg/mL infliximab was instilled four times a day for 14 days after surgery. On day 14 of the experiment, the operated and control eyes were enucleated and histologically and immunohistochemically analyzed.. The mean fibroblast and mononuclear cell (MNC) numbers and the mean immunostaining intensities of transforming growth factor-β (TGF-β), fibroblast growth factor-β (FGF-β), and platelet-derived growth factor (PDGF) in the sham group were higher than those of the control group (P<0.01). The mean fibroblast and MNC numbers and the mean immunostaining intensities of TGF-β, FGF-β, and PDGF in the MMC and infliximab groups were statistically significantly lower than those of the sham group (P<0.01). The mean fibroblast and MNC numbers and the mean TGF-β, FGF-β, and PDGF immunostaining intensities of the MMC and infliximab groups were similar (P>0.05).. Our study suggests that topical infliximab effectively suppresses the subconjunctival wound healing response after experimental glaucoma filtration surgery, reducing the MNC and fibroblast numbers and immunostaining intensities of TGF-β, FGF-β, and PDGF.

Topics: Animals; Antibodies, Monoclonal; Fibroblast Growth Factors; Filtering Surgery; Glaucoma; Infliximab; Male; Mitomycin; Platelet-Derived Growth Factor; Rabbits; Trabeculectomy; Transforming Growth Factor beta; Wound Healing

The chloride channel protein 2 (CLC‑2) is important in maintaining the volume of trabecular meshwork cells by adjusting the outflow of aqueous solutions and maintaining the fluid balance. However, little is known concerning the functions of CLC‑2 in the cytoskeleton, specifically in human trabecular meshwork (HTM) cells. In the present study, two CLC-2 specific siRNAs (siRNA1 and siRNA2) that target CLC-2 mRNA were designed. The siRNAs were transfected into the HTM cells and the results showed that siRNA1 in particular decreased the expression of CLC‑2 by ~45%. Furthermore, an siRNA1‑mediated CLC-2 knockdown significantly reconstructed the actin cytoskeleton and formed cross‑linked actin networks. In addition, the downregulation of the expression of CLC‑2 was associated with increased TGF‑β and Smad2 activities in the HTM cells following 24 h of transfection. In conclusion, these results suggest that CLC‑2 knockdown promotes trabecular meshwork cytoskeletal disorders and may activate the TGF‑β/Smad signaling pathway. Thus, CLC‑2 may be a promising and potential novel therapeutic strategy for combating primary open‑angle glaucoma.

Topics: Actins; beta Catenin; Cells, Cultured; Chloride Channels; CLC-2 Chloride Channels; Cytoskeleton; Gene Expression; Gene Knockdown Techniques; Glaucoma; Humans; RNA, Small Interfering; Signal Transduction; Smad Proteins; Stress Fibers; Trabecular Meshwork; Transforming Growth Factor beta; Vinculin

The purposes of this study were to investigate the effects of topically administrated Tacrolimus and Octreotide on modulation of postoperative scarring in experimental glaucoma filtration surgery and to compare the antifibrotic properties of these agents with mitomycin-C (MMC).. A total of 28 New Zealand rabbits weighing 2.5-3 kg were randomly divided into a surgical control (SC) group and three experimental groups. Standard filtration surgeries were performed on the right eyes of all the rabbits. The rabbits in the SC group received only vehicle after the surgeries, whereas the rabbits in the three experimental groups were treated either with 0.4 mg/mL MMC during the surgery (MMC group) or with 0.3 mg/mL Tacrolimus drop four times a day (TT group) or with 10 µg/mL Octreotide drop three times a day (OT group) for 14 days. The animals were killed on day 14, eyes were enucleated and histologically and immunohistochemically analyzed.. In SC group mean fibroblast, mononuclear cell number and fibroblast growth factor-β (FGF-β), transforming growth factor-β (TGF-β) immunostaining intensity was higher than all treatment groups. In OT group mean fibroblast number was lesser than MMC (p < 0.01) and TT (p < 0.05) group. In TT group mean fibroblast number was lesser than MMC group (p < 0.05). Mean mononuclear cell number was similar between MMC, OT and TT groups (p > 0.05). In MMC, OT and TT groups mean TGF-β and FGF-β immunostaining intensity was similar (p > 0.05).. Topically administration of Tacrolimus and Octreotide effectively reduced the subconjuntival scarring response 2 weeks after experimental glaucoma filtration surgery.

Topics: Administration, Topical; Alkylating Agents; Animals; Cicatrix; Conjunctival Diseases; Disease Models, Animal; Fibroblast Growth Factor 2; Fibroblasts; Fibrosis; Filtering Surgery; Glaucoma; Immunoenzyme Techniques; Immunosuppressive Agents; Leukocyte Count; Mitomycin; Octreotide; Ophthalmic Solutions; Postoperative Complications; Rabbits; Tacrolimus; Transforming Growth Factor beta

To investigate the effects of gelatin hydrogel (GH) containing a chymase inhibitor (CI) on intraocular pressure (IOP) and conjunctival scarring in a canine model of glaucoma surgery.. Glaucoma surgery models were made in beagles. As the first experiment, GH was implanted. IOP was measured for 4 weeks, followed by histologic evaluation. As the second experiment, GH containing a CI or GH alone was implanted. IOP and bleb features were evaluated for 12 weeks. The densities of proliferative cell nuclear antigen (PCNA)-positive cells, fibroblasts, and mast cells were quantified. The mRNA levels of transforming growth factor-β (TGF-β) and chymase were determined by real-time PCR.. In the first experiment, IOP was significantly lower in the eyes treated with GH than that in the control eyes. The conjunctival area normalized by the scleral area was reduced in the treated eyes. In the second experiment, IOP reduction was maintained for 12 weeks in the eyes treated with GH containing a CI, but not in the eyes treated with GH alone. In addition, the bleb score was larger, whereas the adhesion score and densities of PCNA-positive cells, fibroblasts, mast cells, and chymase-positive cells were lower in the eyes treated with GH containing a CI. The mRNA levels of TGF-β and chymase were significantly decreased in the eyes treated with GH containing a CI.. Implanting GH alone maintained IOP reduction, whereas GH containing a CI enhanced the IOP-reducing effect by suppressing cell proliferation. This drug delivery system might be useful for maintaining filtering blebs for a longer duration after glaucoma surgery.

Topics: Animals; Chymases; Cicatrix; Conjunctival Diseases; Disease Models, Animal; Dogs; Drug Combinations; Drug Delivery Systems; Fibroblasts; Fibrosis; Gelatin; Glaucoma; Hydrogels; Immunoenzyme Techniques; Intraocular Pressure; Mast Cells; Oligopeptides; Polyglutamic Acid; Proliferating Cell Nuclear Antigen; Real-Time Polymerase Chain Reaction; RNA, Messenger; Tonometry, Ocular; Trabeculectomy; Transforming Growth Factor beta

African-Americans experience an excessive prevalence of a number of apparently disparate disorders that all appear to be, at least in part, mediated by the over-expression or activation of transforming growth factor-beta (TGF-beta) signaling pathways, and that certain genotypes including the codon 10 polymorphism occur more commonly among African-Americans and appears to predispose to these disorders. These disorders, fibrosing in nature, include hypertension, focal glomerulosclerosis, diabetic nephropathy, end stage renal disease, sarcoidosis, uterine leiomyoma, keloids, myocardial fibrosis, and glaucoma. The specific polymorphism for TGF-beta, codon 10, has been implicated in glomerulosclerosis and diabetic nephropathy as well as cardiac transplant rejection. Although TGF-beta over-expression is not the sole factor in these disorders, it is suggested that by designing future clinical studies that consider genomic differences in TGF-beta expression, a more complete understanding of these clinical disorders will be possible. A more thorough understanding of the genetic basis of disease will like promote improved therapeutic regimens and may help reduce the disparate health outcomes for African-Americans as well as improve treatment of individuals of various and diverse ethnic backgrounds.

Topics: Black People; Female; Glaucoma; Heart Transplantation; Humans; Keloid; Leiomyoma; Male; Sarcoidosis; Transforming Growth Factor beta

Oxidative stress and TGFbeta-induced disturbance of cells and tissues are implicated in initiation and progression of pathophysiology of cells/tissues. Using primary human trabecular meshwork (TM) cells from normal and glaucomatous subjects, this study demonstrated that peroxiredoxin (PRDX) 6, an antioxidant, offsets the deleterious effects of oxidative stress on TM cells by optimizing ROS and TGFbeta levels. An analysis of glaucomatous TM cells revealed a reduced expression of PRDX6 mRNA and protein. Biochemical assays disclosed enhanced levels of ROS, as well as high levels of TGFbetas and these cells expressed elevated extracellular matrix (ECM) and Tsp1 proteins with reduced MMP2; conditions implicated in the pathophysiology of glaucoma. Non-glaucomatous TM cells exposed to TGFbetas/ROS showed similar features as in glaucomatous cells. The abnormalities induced were reversed by delivery of PRDX6. The data provide evidence that oxidative stress-induced abnormality in TM may be related to reduced PRDX6 expression and provide a foundation for antioxidant-based therapeutics for treating glaucoma.

Topics: Actins; Cells, Cultured; Cellular Senescence; DNA Damage; Dose-Response Relationship, Drug; Extracellular Matrix Proteins; Fibronectins; Glaucoma; GTP-Binding Proteins; Humans; Hydrogen Peroxide; Matrix Metalloproteinase 2; Oxidants; Oxidative Stress; Peroxiredoxin VI; Phenotype; Plasminogen Activator Inhibitor 1; Protein Glutamine gamma Glutamyltransferase 2; Reactive Oxygen Species; Recombinant Fusion Proteins; RNA, Messenger; Thrombospondin 1; Trabecular Meshwork; Transduction, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Transglutaminases; Tropomyosin

Topics: Aqueous Humor; Dinoprostone; Enzyme-Linked Immunosorbent Assay; Filtering Surgery; Glaucoma; Humans; Transforming Growth Factor beta

In glaucoma, the optic nerve head (ONH) is the likely site of initial injury and elevated intraocular pressure (IOP) is the best-known risk factor. This study determines global gene expression changes in the pressure-injured ONH.. Unilateral sustained IOP elevation (glaucoma, n = 46) or optic nerve transection (n = 10) was produced in rats. ONHs were removed, and the retrobulbar optic nerves were graded for degeneration. Gene expression in the glaucomatous ONH with extensive injury was compared with that in the fellow ONH (n = 6/group), by using cDNA microarrays. Data from 12 arrays were normalized, significant differences in gene expression determined, and significantly affected gene classes identified. For the remaining ONH, grouped by experimental condition and degree of injury, quantitative reverse transcriptase-PCR (qPCR) and ANOVA were used to compare selected message levels.. Microarray analysis identified more than 2000 significantly regulated genes. For 225 of these genes, the changes were greater than twofold. The most significantly affected gene classes were cell proliferation, immune response, lysosome, cytoskeleton, extracellular matrix, and ribosome. A 2.7-fold increase in ONH cellularity confirmed glaucoma model cell proliferation. By qPCR, increases in levels of periostin, collagen VI, and transforming growth factor beta1 were linearly correlated to the degree of IOP-induced injury. For cyclinD1, fibulin 2, tenascin C, TIMP1, and aquaporin-4, correlations were significantly nonlinear, displaying maximum change with focal injury.. In the ONH, pressure-induced injury results in cell proliferation and dramatically altered gene expression. For specific genes, expression levels were most altered by focal injury, suggesting that further array studies may identify initial, and potentially injurious, altered processes.

Topics: Animals; Cell Proliferation; Disease Models, Animal; Extracellular Matrix; Gene Expression Profiling; Gene Expression Regulation; Genes, MHC Class II; Glaucoma; Immunoenzyme Techniques; Intraocular Pressure; Lipids; Lysosomes; Microglia; Oligonucleotide Array Sequence Analysis; Optic Disk; Optic Nerve Diseases; Polymerase Chain Reaction; Rats; Rats, Inbred BN; Ribosomal Proteins; RNA, Messenger; Transforming Growth Factor beta; Up-Regulation

The aim of the present study was to evaluate the effect of latanoprost monotherapy on the aqueous humour concentrations of TGF-beta1, MMP-2, TIMP-2, MMP-9 and gelatinolytic activity in patients treated for exfoliative glaucoma (XFG). Aqueous samples from 50 XFG patients treated with latanoprost and 50 age-matched XFG patients treated with timolol were collected during phacoemulsification cataract surgery. The concentrations of TGF-beta1, MMP-2, TIMP-2, MMP-9 and gelatinase activity were determined by commercial immunoassays. The mean active TGF-beta1 concentration in the aqueous was significantly lower in XFG patients treated with latanoprost compared with those treated with timolol (3.1 +/- 0.65 vs 13.4 +/- 1.5 pg ml(-1)); (P = 0.0014). The mean total MMP-2 concentration was lower in latanoprost treated patients (31.75 +/- 3.8 vs 81.5 +/- 7.2 ng ml(-1)); (P < 0.0001). The TIMP-2 concentration was also lower in XFG-latanoprost treated patients (73.8 +/- 6.81 vs 101.28 +/- 7.29 ng ml(-1)); (P = 0.0096). Latanoprost monotherapy has a marked effect on the aqueous concentration of TGF-beta1, MMP-2 and TIMP-2 in XFG patients. A better understanding of its effect on the pathobiology of the disease may lead to its earlier use in the disease process to prevent progression from XFS to XFG.

Topics: Adrenergic beta-Antagonists; Aged; Antihypertensive Agents; Aqueous Humor; Case-Control Studies; Gelatinases; Glaucoma; Humans; Latanoprost; Prostaglandins F, Synthetic; Timolol; Transforming Growth Factor beta; Transforming Growth Factor beta1

The purpose of this study was to design microspheres combining sustained delivery and enhanced intracellular penetration for ocular administration of antisense oligonucleotides. Nanosized complexes of antisense TGF-beta2 phosphorothioate oligonucleotides (PS-ODN) with polyethylenimine (PEI), and naked PS-ODN were encapsulated into poly(lactide-co-glycolide) microspheres prepared by the double-emulsion solvent evaporation method. The PS-ODN was introduced either naked or complexed in the inner aqueous phase of the first emulsion. We observed a marked influence of microsphere composition on porosity, size distribution and PS-ODN encapsulation efficiency. Mainly, the presence of PEI induced the formation of large pores observed onto microsphere surface. Introduction of NaCl in the outer aqueous phase increased the encapsulation efficiency and reduced microsphere porosity. In vitro release kinetic of PS-ODN was also investigated. Clearly, the higher the porosity, the faster was the release and the higher was the burst effect. Using an analytical solution of Fick's second law of diffusion, it was shown that the early phase of PS-ODN and PS-ODN-PEI complex release was primarily controlled by pure diffusion, irrespectively of the type of microsphere. Finally, microspheres containing antisense TGF-beta2 nanosized complexes were shown, after subconjunctival administration to rabbit, to significantly increase intracellular penetration of ODN in conjunctival cells and subsequently to improve bleb survival in a rabbit experimental model of filtering surgery. These results open up interesting prospective for the local controlled delivery of genetic material into the eye.

Topics: Animals; Conjunctiva; Delayed-Action Preparations; Female; Glaucoma; Nanostructures; Oligonucleotides, Antisense; Polyethyleneimine; Rabbits; Thionucleotides; Transforming Growth Factor beta; Transforming Growth Factor beta2

To investigate the effect of IOP on retinal ganglion cell (RGC) apoptosis and correlate the effects with IOP-induced changes in extracellular matrix (ECM) in the retina and optic nerve head (ONH) in glaucomatous rat eyes.. Thirty-seven Dark Agouti rats had elevated IOP induced in the left eye by hypertonic saline episcleral vein injections. Eyes were examined at 3 months histologically for RGC apoptosis and expression of specific ECM components.. RGC apoptosis was significantly related to IOP exposure (integral DeltaIOP P <0.001; peak IOP P <0.01). In the RGC layer, elevated IOP correlated positively to a significant increase in MMP-9 activity (P <0.001), tissue inhibitor of matrix metalloproteinase (TIMP-1) (P <0.05), and collagen I (P <0.01), and negatively correlated to deposition of laminin (P <0.05) and TGF-beta2 (P <0.05). There was a significant correlation between MMP-9 activity and both RGC apoptosis (P <0.001) and loss of laminin (P <0.01). IOP exposure was also associated with increased deposition of TGF-beta2 and collagen I at the ONH (P <0.01).. The results demonstrated that RGC apoptosis in glaucoma correlates strongly with elevated IOP and is significantly associated with IOP-induced changes in specific ECM components in the RGC layer. The study shows for the first time a link between MMP-9, laminin degradation, RGC apoptosis, and IOP exposure in glaucoma. The findings suggest that abnormal ECM remodeling in the glaucomatous retina may relate to RGC death and support the notion that the retina is a primary site of injury in glaucoma.

Topics: Animals; Apoptosis; Collagen Type I; Extracellular Matrix Proteins; Glaucoma; Intraocular Pressure; Male; Matrix Metalloproteinase 9; Optic Disk; Rats; Rats, Inbred Strains; Retina; Retinal Ganglion Cells; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Transforming Growth Factor beta2

To analyze the effect of perioperative decorin in an experimental setting of glaucoma filtration surgery.. Glaucoma filtration surgery, similar to that performed in clinical practice, was performed on 35 chinchilla rabbits (ChBB:CH). The animals received a unilateral subconjunctival injection of decorin (40-100 microg) or the vehicle alone before surgery and at different time intervals thereafter. Antifibrotic efficacy was established by clinical response and histologic examination. The animals were killed on day 14, and the eyes processed for histology.. Both the vehicle and the decorin solution were well tolerated. No adverse effects such as inflammation or blurring of the optical media were observed. Conjunctival scarification occurred within 1 week in the control groups but was suppressed in the experimental groups. The intraocular pressure correlated with the fibrotic process and reached normal levels within 7 days after surgery in control animals, but remained significantly (P <0.001) reduced in the experimental groups. Histologic examination of the surgical area 14 days after surgery disclosed massive fibrosis in the control animals, but little deposition of extracellular matrix in the experimental groups.. The data of this pilot study suggest that perioperative subconjunctival decorin applications significantly affect conjunctival scarring and surgical outcome of glaucoma filtration treatments in rabbits.

Topics: Animals; Cicatrix; Conjunctiva; Decorin; Disease Models, Animal; Extracellular Matrix Proteins; Female; Fibrosis; Filtering Surgery; Glaucoma; Injections; Intraocular Pressure; Perioperative Care; Pilot Projects; Proteoglycans; Rabbits; Transforming Growth Factor beta; Wound Healing

Connective tissue growth factor (CTGF) appears to play a significant role in mediating fibrosis in several tissues. To gain further understanding of the role of CTGF in the scar formation that occurs after glaucoma filtering surgery (GFS), experiments were performed in a rabbit model.. . Three experiments were performed: (1) CTGF and transforming growth factor (TGF)-beta expression were measured quantitatively after GFS, using ELISA. (2) After GFS conjunctival bleb tissues were immunostained for the presence of CTGF and TGF-beta. (3) Exogenous CTGF was injected into mitomycin-C (MMC)-treated filtering blebs and the scaring response compared to TGF-beta and physiological saline-injected blebs.. CTGF and TGF-beta were expressed maximally by day 5 after surgery and were both shown to be present in the bleb tissues after GFS. The addition of exogenous CTGF and TGF-beta increased the rate of failure of GFS blebs.. These data support the hypothesis that CTGF plays an important role in scarring and wound contracture after GFS. Inhibition of CTGF synthesis or its action may help prevent bleb failure and improve long-term GFS outcomes.

Topics: Animals; Conjunctiva; Connective Tissue Growth Factor; Enzyme-Linked Immunosorbent Assay; Filtering Surgery; Glaucoma; Immediate-Early Proteins; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Male; Models, Animal; Rabbits; Trabecular Meshwork; Transforming Growth Factor beta

Physiological pressure inside the eye is maintained by a resistance mechanism provided by the trabecular meshwork tissue. In most cases, prolonged, elevated pressure leads to an eye pathology characterized by retinal ganglion cell (RGC) degeneration, optic nerve damage, and non-remedial blindness. We are investigating the regulation of trabecular meshwork genes in response to elevated pressure. Using perfused organ cultures from postmortem human donors, we have previously demonstrated the presence of a homeostatic mechanism at 2-4 days of pressure insult (Borrás et al. 2002, Invest Ophthalmol Vis Sci 43:33-40). Here, we sought to identify trabecular meshwork genes whose expression was altered during this homeostatic period. By macroarray hybridization, we compared the expression profiles of high-pressure (HP) and normal-pressure (NP) treated eyes from the same individual (n = 3 pairs). Our results identified 40 upregulated and 14 downregulated genes. The highest proportion of upregulated genes encoded proteins involved in signal transduction (32%). Among the potentially relevant genes, PIP 5K1C, VIP, tropomodulin, and MMP2 encoded mediators known to influence outflow resistance. Others encoded functions which are new for the trabecular meshwork, but which are intrinsic to unrelated tissues. These new mechanisms appear as they could be of benefit for trabecular meshwork function. Matrix Gla protein (MGP), perlecan, osteomodulin, and osteoblast-specific factor are essential in cartilage and bone physiology whereas spectrin and ICAM4 are specific for blood cells and crucial in maintaining their shape and adhesion. In addition, MGP transcripts were stimulated by extracellular calcium and downregulated by TGF-beta1. We propose that MGP might be an important player in the adaptive homeostatic mechanism by contributing to maintain a softer trabecular meshwork tissue and facilitate aqueous humor outflow.

Topics: Aged; Calcium; Chromosomes; Down-Regulation; Gene Expression Profiling; Gene Expression Regulation; Gene Library; Glaucoma; Homeostasis; Humans; Intraocular Pressure; Trabecular Meshwork; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation; Vision, Ocular

The scarring response is an important factor in many diseases throughout the body. In addition, it is a major problem in influencing results of surgery. In the eye, for example, post-operative scarring can determine the outcome of surgery. This is particularly the case in the blinding disease glaucoma, where several anti-scarring regimens are currently used to improve glaucoma surgery results, but are of limited use clinically because of severe complications. We have recently identified transforming growth factor-beta (TGF-beta) as a target for post-operative anti-scarring therapy in glaucoma, and now report the first study of novel second-generation antisense phosphorothioate oligonucleotides against TGF-beta in vivo. Single applications of a TGF-beta OGN at the time of surgery in two different animal models closely related to the surgical procedure performed in glaucoma patients, significantly reduced post-operative scarring (P<0.05) and improved surgical outcome. Our findings suggest that TGF-beta antisense oligonucleotides have potential as a new therapy for reducing post-surgical scarring. Its long-lasting effects after only a single administration at the time of surgery make it particularly attractive clinically. Furthermore, although we have shown this agent to be useful in the eye, it could have widespread applications anywhere in the body where the wound-healing response requires modulation.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cicatrix; Cornea; Female; Filtering Surgery; Genetic Therapy; Glaucoma; Injections; Mice; Mice, Inbred BALB C; Models, Animal; Molecular Sequence Data; Oligonucleotides, Antisense; Protein Isoforms; Protein Serine-Threonine Kinases; Rabbits; Random Allocation; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Sequence Analysis, DNA; Transforming Growth Factor beta; Wound Healing

Topics: Animals; Antibodies, Monoclonal; Cardiomyopathies; Cicatrix; Clinical Trials, Phase III as Topic; Diastole; Fibrosis; Glaucoma; Humans; Oligonucleotides, Antisense; Rats; Transforming Growth Factor beta; Transforming Growth Factor beta2; Treatment Outcome

Postoperative subconjunctival wound healing remains the commonest cause of late bleb failure after glaucoma filtration surgery. This study was undertaken to investigate whether the human monoclonal antibody that neutralizes transforming growth factor-beta2 (CAT-152; lerdelimumab) could be used as a postoperative agent to prevent scarring after glaucoma surgery and compared it with 5-fluorouracil (5-FU), to benchmark its potential clinical benefit.. In a randomized, controlled, masked-observer study, after modified glaucoma surgery, 48 rabbits were randomly allocated to receive a postoperative course of seven subconjunctival injections of CAT-152 (1 mg/mL), 5-FU (50 mg/mL), or no treatment. Bleb characteristics, the presence of subconjunctival drainage, and local reaction to treatment were assessed. Animals were killed on days 10, 21, and 30. Immunohistochemistry, histologic staining and electron microscopy were performed to demonstrate the mechanism of CAT-152-mediated effects on the extracellular matrix.. CAT-152 significantly improved surgical outcome (log rank test, P < 0.001) and reduced subconjunctival collagen deposition (P < 0.01) compared with 5-FU and control. Median bleb survival was increased in the CAT-152 group (23.5 days) compared with the 5-FU (20 days) and control (16 days) treatment groups. CAT-152 treatment improved bleb morphology (P < 0.05) and was well tolerated. 5-FU prolonged the duration of corneal epitheliopathy (P < 0.01).. Postoperative administration of CAT-152 significantly improved surgical outcome, reduced subconjunctival scarring, and minimized the risk of corneal side effects compared with the anti-scarring agent 5-FU. These findings suggest that CAT-152 may offer therapeutic benefit as a postoperative agent to prevent subconjunctival scarring after glaucoma filtration surgery.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Cicatrix; Conjunctival Diseases; Fibrosis; Filtering Surgery; Glaucoma; Injections; Postoperative Complications; Rabbits; Transforming Growth Factor beta; Transforming Growth Factor beta2; Wound Healing

To study the expression of beta-Fibroblastic Growth Factor (bFGF) and Transforming Growth Factor beta 1 (TGF beta 1) in filtering area following sclerectomy and the interaction between growth factors and interferon alpha-2b (IFN alpha-2b).. Immunohistochemical technique were applied to test the expression bFGF and TGF beta 1 in frozen sections of filtration area following sclerectomy on postoperative day three, five, seven and 14 in white rabbits, respectively. 16 eyes of eight rabbits in all were randomly and equally divided into two groups. The experimental eyes had subconjunctivally been administered IFN alpha-2b 5 x 10(5) IU 0.2 ml each time at filtering bleb following operation whereas the control eyes hadn't taken.. In control group, positive cell and substance expressing bFGF and TGF beta 1 on postoperative day three were revealed in the filtration area, on postoperative day five and seven, they were gradually increased. On the postoperative day 14, all significant cells and substance markly decreased. In experimental group only small amount of positive cell and bFGF or TGF beta 1-expressing substance were sparely distributed in the filtration area.. The filtration area after glaucoma filtering surgery was "bathed" in growth factors such as bFGF and TGF beta 1 which well-known play an important role in stimulating wound healing response, IFN alpha-2b had an inhibitory effect on bFGF or TGF beta 1 expression in wound environment.

Topics: Animals; Female; Fibroblast Growth Factor 2; Filtering Surgery; Glaucoma; Interferon alpha-2; Interferon-alpha; Male; Rabbits; Random Allocation; Recombinant Proteins; Sclera; Sclerostomy; Transforming Growth Factor beta; Transforming Growth Factor beta1; Wound Healing

To investigate the distribution and potential participation of microglia, the resident defense cells of the central nervous system, in the optic nerve head (ONH) in glaucoma, histological paraffin sections of optic nerves from normal and glaucoma patients with mild to advanced nerve damage were studied using double labeling immunohistofluorescence. A monoclonal antibody for HLA-DR, indicating activated microglia, was colocalized with antibodies for functional proteins. In normal ONHs, microglia do not contain TGF-beta2, COX-2, or TNF-alpha and are not positive for PCNA; however, in glaucomatous ONHs, microglia contain abundant TGF-beta2, TNF-alpha, and PCNA. In glaucomatous eyes, a few microglia are usually positive for COX-2. In normal ONHs, there are rarely microglia containing TGF-beta1, NOS-2, TSP, TIMP-2, and CD68, but, in glaucomatous tissue, a few microglia are positive from the prelaminar to the postlaminar regions. MMP-1, MMP-2, MMP-3, and MMP-14 are constitutively present in the perivascular microglia in normal ONHs and appear to be more abundant in glaucomatous tissue. COX-1, TNF-R1, TIMP-1, and c-fms are constitutively present in normal tissues and appear to be increased in microglia in the glaucomatous ONHs. HSP27 is not present in microglia. In glaucomatous ONHs, microglia become activated and phagocytic and produce cytokines, mediators, and enzymes that can alter the extracellular matrix. Our findings suggest that activated microglia may participate in stabilizing the tissue early in the disease process, but, as the severity of the glaucomatous damage increases, the activities of microglia may have detrimental consequences for the pathological course of glaucomatous optic neuropathy.

Topics: Aged; Aged, 80 and over; Antigens, CD; Cyclooxygenase 1; Cyclooxygenase 2; Glaucoma; Humans; Immunohistochemistry; Isoenzymes; Matrix Metalloproteinases; Membrane Proteins; Microglia; Middle Aged; Mitosis; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Optic Disk; Phagocytosis; Prostaglandin-Endoperoxide Synthases; Receptor Protein-Tyrosine Kinases; Receptors, Cell Surface; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Thrombospondins; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Tumor Necrosis Factor-alpha

Remodeling of the extracellular matrix occurs in the lamina cribrosa in progressed glaucomatous optic nerve damage including disc cupping. We examined immunohistochemical changes in the transforming growth factor (TGF)-beta and platelet derived growth factor (PDGF) in the optic nerve heads in experimentally induced glaucoma.. We used 3 cynomolgus and 2 Japanese monkey eyes. Glaucoma was induced by repeated argon laser photocoagulation of the chamber angle. Eyes were enucleated after disc cupping had formed 3 to 5 months after treatment. The optic nerve head was examined for expression of TGF beta 1, beta 2, and beta 3, and PDGF A and B in frozen sections and by the biotin-ExtrAvidin-Alkali Phosphatase method.. Normal monkey eyes showed TGF beta 1, beta 2, and beta 3, and PDGF A, and B in the optic nerve head including the nerve fibers, glial cells, and vascular cells. Glaucomatous eyes showed stronger expression of TGF beta 1 and beta 2 in the glial cells around the lamina cribrosa. The staining intensities for TGF beta 3, PDGF A, and PDGF B were the same as in normal eyes.. Eyes with experimental glaucoma showed higher expressions of TGF beta 1 and beta 2 around the lamina cribrosa. This finding may show upregulation of extracellular matrix production as related to remodeling of the lamina cribrosa in glaucoma.

Topics: Animals; Glaucoma; Immunohistochemistry; Macaca; Macaca fascicularis; Optic Disk; Platelet-Derived Growth Factor; Transforming Growth Factor beta

Currently available anti-scarring regimens for glaucoma filtration surgery have potentially blinding complications and thus the need for alternative and safer agents. The effects of a new antibody to transforming growth factor (TGF)-beta2 on in vitro and in vivo conjunctival scarring and after glaucoma filtration surgery were investigated.. The activity of a novel recombinant monoclonal neutralizing antibody (mAb) to human TGF-2 (rhAnti-TGF-beta2 mAb) was studied in conjunctival fibroblast-mediated proliferation, migration, and collagen contraction. Its safety in subconjunctival administration was assessed in vivo, and, in a rabbit model of glaucoma filtration surgery, its effects on conjunctival scarring and filtration surgery outcome were investigated.. The rhAnti-TGF-beta2 mAb effectively inhibited TGF-beta2-mediated conjunctival scarring activity in vitro, at 50% inhibitory concentrations (IC50) of less than 1 nM. It significantly improved glaucoma filtration surgery outcome in an animal model of aggressive conjunctival scarring compared with control (P = 0.0291) and was clinically safe, nontoxic, and well tolerated after subconjunctival administration.. Subconjunctival rhAnti-TGF-beta2 mAb treatment significantly affects surgical outcome and effectively reduces conjunctival scarring both in vitro and in vivo. It appears safe for subconjunctival administration and when compared with mitomycin-C treatment histologically, much less destructive to local tissue. rhAnti-TGF-beta2 mAb may have potential as a new anti-scarring agent for use in glaucoma filtration surgery.

Topics: Animals; Antibodies, Monoclonal; Cell Division; Cell Movement; Cicatrix; Collagen; Conjunctival Diseases; Connective Tissue; Female; Fibroblasts; Filtering Surgery; Glaucoma; Humans; Rabbits; Recombinant Proteins; Safety; Transforming Growth Factor beta

Topics: ATP-Binding Cassette Transporters; Chromosome Mapping; Corneal Dystrophies, Hereditary; Cytoskeletal Proteins; Extracellular Matrix Proteins; Eye Proteins; Glaucoma; Glycoproteins; Humans; Macular Degeneration; Mutation; Neoplasm Proteins; Photoreceptor Cells; Trabecular Meshwork; Transforming Growth Factor beta

To investigate whether transforming growth factor-beta 2 (TGF-beta 2), a strong immunosuppressive factor normally present in aqueous humor, is involved in the inflammatory process of clinical uveitis.. Mature TGF-beta 2 levels were determined in aqueous humor samples of 9 patients with Fuchs' heterochromic cyclitis, aqueous humor samples of 21 patients with other uveitis entities, and vitreous fluid samples of 19 patients with uveitis by using a commercially available sandwich ELISA: Total TGF-beta 2 levels in ocular fluids were measured after heat activation. Aqueous humor samples from patients with cataract and glaucoma and vitreous fluid samples from eye bank eyes were tested as controls. Albumin levels, determined by radial immunodiffusion, were used as a measure of the disruption of the blood aqueous barrier.. Significantly lower mature TGF-beta 2 levels were detected in aqueous humor samples of patients with uveitis, compared to the two control groups without intraocular inflammation. Samples of patients with uveitis without detectable mature TGF-beta 2 did contain latent TGF-beta 2 levels (504 to 6024 pg/ml). In aqueous humor, there was a significant negative correlation between mature TGF-beta 2 and albumin levels. No mature TGF-beta could be detected in vitreous fluid. Total TGF-beta 2 levels in vitreous fluid were significantly lower in samples from patients with uveitis than in samples from eye bank eyes.. These results indicate that the mature TGF-beta 2 levels in aqueous humor and the total TGF-beta 2 levels in vitreous fluid are reduced during ocular inflammation. In aqueous humor, this might be caused by binding of mature TGF-beta to serum proteins, for instance, alpha 2-macroglobulin, or by a disturbance in the activation process of latent TGF-beta 2.

Topics: alpha-Macroglobulins; Aqueous Humor; Cataract; Enzyme-Linked Immunosorbent Assay; Glaucoma; Glucocorticoids; Humans; Serum Albumin; Transforming Growth Factor beta; Uveitis; Vitreous Body