Outcomes and complications of cataract surgery in patients with chronic ocular graft-versus-host-disease-a multicenter, retrospective analysis.

PURPOSE: To evaluate the outcome of phacoemulsification in patients with chronic ocular Graft-versus-host disease (oGVHD) after allogeneic hematopoietic stem cell transplantation (aHSCT). METHODS: Retrospective, observational multicenter study from 1507 oGVHD patients. From the patient files, data were collected including best-corrected visual acuity (BCVA), intraocular pressure (IOP), Schirmer's test I, tear film break-up time (TFBUT), corneal fluorescein staining score, postoperative complications, and pre- and post-operative topical therapy. RESULTS: Seventy-three patients underwent cataract surgery in 104 eyes. In n = 84 eyes, the oGVHD NIH grade was documented; 12% (n = 12) of analyzed eyes were staged oGVHD NIH grade 1, 31% (n = 32) NIH 2 and 39% (n = 41) NIH 3. The mean BCVA improved in 82% of the eyes (n = 86 eyes). BCVA significantly increased from 0.7 ± 0.5 to 0.4 ± 0.4 LogMAR after surgery independent from oGVHD severity. The mean IOP decreased from 14 ± 4 to 13 ± 4 mmHg after surgery. Visual acuity was moderately correlated to the pre-operative degree of corneal staining (Pearson p = 0.26, p = 0.002, Cohen's effect size f = 0.29). The visual acuity decreased by 0.078 LogMar units (95% CI = 0.027-0.141) with each increase of corneal staining by one grade (p = 0.05). After surgery, corneal epitheliopathy increased significantly in 42% (n = 44) of the eyes. Postoperative complications included corneal perforation (n = 6, 6%), cystoid macular edema (n = 4, 4%), and endophthalmitis (n = 1, 1%). CONCLUSION: Phacoemulsification in patients with chronic oGVHD significantly improves visual acuity, but is associated with an increased risk of complications in particular corneal epitheliopathy and corneal perforations.

Course of disease in multifocal choroiditis lacking sufficient immunosuppression: a case report.

BACKGROUND: Multifocal choroiditis with panuveitis is a rare disease. The educational merit of this case presentation results from the good documentation and the impressive ocular fundus pictures. CASE PRESENTATION: We illustrate the 3-year course of disease in a 22-year-old myopic white woman with multifocal choroiditis with panuveitis and secondary choroidal neovascularization. The activity of the disease was evaluated clinically by optical coherence tomography and fluorescein angiography. Choroidal neovascularization was treated by intravitreal bevacizumab (2.5 mg/0.1 ml). Our patient lacked systemic therapy for the first 11 months because of noncompliance. CONCLUSIONS: The case is remarkable as the delayed onset of peripheral lesions and the additional existence of high myopia made diagnosis difficult. In addition, it demonstrates that full outbreak of disease with multiple central and peripheral fundus lesions and secondary choroidal neovascularization can develop without systemic treatment.

Conjunctival fibrosis following filtering glaucoma surgery.

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.

Analysis of Varicella-Zoster Virus in Temporal Arteries Biopsy Positive and Negative for Giant Cell Arteritis.

IMPORTANCE: Giant cell arteritis (GCA) is the most common systemic vasculitis in elderly individuals. Diagnosis is confirmed by temporal artery (TA) biopsy, although biopsy results are often negative. Despite the use of corticosteroids, disease may progress. Identification of causal agents will improve outcomes. Biopsy-positive GCA is associated with TA infection by varicella-zoster virus (VZV). OBJECTIVE: To analyze VZV infection in TAs of patients with clinically suspected GCA whose TAs were histopathologically negative and in normal TAs removed post mortem from age-matched individuals. DESIGN, SETTING, AND PARTICIPANTS: A cross-sectional study for VZV antigen was performed from January 2013 to March 2015 using archived, deidentified, formalin-fixed, paraffin-embedded GCA-negative, GCA-positive, and normal TAs (50 sections/TA) collected during the past 30 years. Regions adjacent to those containing VZV were examined by hematoxylin-eosin staining. Immunohistochemistry identified inflammatory cells and cell types around nerve bundles containing VZV. A combination of 17 tertiary referral centers and private practices worldwide contributed archived TAs from individuals older than 50 years. MAIN OUTCOMES AND MEASURES: Presence and distribution of VZV antigen in TAs and histopathological changes in sections adjacent to those containing VZV were confirmed by 2 independent readers. RESULTS: Varicella-zoster virus antigen was found in 45 of 70 GCA-negative TAs (64%), compared with 11 of 49 normal TAs (22%) (relative risk [RR] = 2.86; 95% CI, 1.75-5.31; P < .001). Extension of our earlier study revealed VZV antigen in 68 of 93 GCA-positive TAs (73%), compared with 11 of 49 normal TAs (22%) (RR = 3.26; 95% CI, 2.03-5.98; P < .001). Compared with normal TAs, VZV antigen was more likely to be present in the adventitia of both GCA-negative TAs (RR = 2.43; 95% CI, 1.82-3.41; P < .001) and GCA-positive TAs (RR = 2.03; 95% CI, 1.52-2.86; P < .001). Varicella-zoster virus antigen was frequently found in perineurial cells expressing claudin-1 around nerve bundles. Of 45 GCA-negative participants whose TAs contained VZV antigen, 1 had histopathological features characteristic of GCA, and 16 (36%) showed adventitial inflammation adjacent to viral antigen; no inflammation was seen in normal TAs. CONCLUSIONS AND RELEVANCE: In patients with clinically suspected GCA, prevalence of VZV in their TAs is similar independent of whether biopsy results are negative or positive pathologically. Antiviral treatment may confer additional benefit to patients with biopsy-negative GCA treated with corticosteroids, although the optimal antiviral regimen remains to be determined.

Correlation Between Tear Film Osmolarity and the Disease Score of the International Chronic Ocular Graft-Versus-Host-Disease Consensus Group in Hematopoietic Stem Cell Transplantation Patients.

PURPOSE: To evaluate tear film osmolarity (TFO) as a diagnostic tool for detecting chronic ocular graft-versus-host disease (GVHD) in patients after hematopoietic stem cell transplantation and to assess its correlation with the new international chronic ocular GVHD score. METHODS: A group of 204 consecutive patients who underwent hematopoietic stem cell transplantation at University Hospital Wuerzburg in Germany received an ophthalmologic examination after transplantation. TFO was measured and the chronic ocular GVHD score was calculated based on the Schirmer test, corneal fluorescein staining, conjunctival injection, Ocular Surface Disease Index questionnaire, and presence of systemic GVHD. RESULTS: A total of 172 patients showed no chronic ocular GVHD. Of the remaining 32 patients using the international chronic ocular GVHD score, 21 were classified as "probably" and 11 as "definite" chronic ocular GVHD. TFO was positively correlated with the new chronic ocular GVHD score (P < 0.01, r = 0.35). TFO differed significantly between patients with no ocular GVHD (300 ± 16.5 mOsm/L) and definite ocular GVHD (337 ± 36 mOsm/L)-a receiver operating characteristic analysis showed high discrimination capability (area under the curve: 0.91 ± 0.04) and suggested a threshold level of the TFO value of 312 mOsm/L yielding a sensitivity of 91% and a specificity of 82%. CONCLUSIONS: TFO can be used for detecting chronic ocular GVHD with high sensitivity and specificity as a noninvasive objective test in addition to traditional dry eye tests. It correlates positively with the diagnostic criteria of a recently established international consensus score for diagnosing the disease.

Correlation between tear film osmolarity, dry eye disease, and rheumatoid arthritis.

PURPOSE: The aim of this study was to evaluate the reliability of tear film osmolarity as a diagnostic tool for detecting dry eye disease (DED) in patients with rheumatoid arthritis (RA). The second aim was to determine whether there is a relationship between tear film osmolarity, a commonly used RA score, and diagnostic blood tests for RA. METHODS: A group of 74 patients with RA but without any previously established diagnosis of DED at University Hospital Wurzburg was examined. A Modified Dry Eye Severity Score was calculated based on tear film osmolarity, Schirmer test, tear film break-up time, ocular surface disease index questionnaire, corneal fluorescein staining, conjunctival lissamine green staining, and meibomian gland grading score. RA assessment included disease activity score (DAS) 28-erythrocyte sedimentation rate (ESR) scoring and serum levels of C-reactive protein (CRP) and rheumatoid factor (RF). RESULTS: Tear film osmolarity showed a high coefficient of determination and correlation when regressed to Modified Dry Eye Severity Score (R = 0.727, P < 0.001). Using a cutoff point of 316 mOsmol/L, there was significant correlation between DAS28-ESR values and tear film osmolarity (P = 0.016). Spearman correlation coefficients between osmolarity and CRP or RF were not significant (CRP: r = -0.104, P = 0.353; RF: r = 0.138, P = 0.226). CONCLUSIONS: Tear film osmolarity highly correlates with a modified dry eye severity scale in patients with RA with previously unknown DED. Tear film hyperosmolarity >316 mOsmol/L is correlated significantly with a commonly used DAS of RA (DAS28-ESR) but not with serum markers such as CRP and RF. Patients with RA with a high DAS28-ESR score show higher prevalence of tear film hyperosmolarity.

Aganirsen antisense oligonucleotide eye drops inhibit keratitis-induced corneal neovascularization and reduce need for transplantation: the I-CAN study.

OBJECTIVE: Eye drops of aganirsen, an antisense oligonucleotide preventing insulin receptor substrate-1 expression, inhibited corneal neovascularization in a previous dose-finding phase II study. We aimed to confirm these results in a phase III study and investigated a potential clinical benefit on visual acuity (VA), quality of life (QoL), and need for transplantation. DESIGN: Multicenter, double-masked, randomized, placebo-controlled phase III study. PARTICIPANTS: Analysis of 69 patients with keratitis-related progressive corneal neovascularization randomized to aganirsen (34 patients) or placebo (35 patients). Patients applied aganirsen eye drops (86 µg/day/eye) or placebo twice daily for 90 days and were followed up to day 180. MAIN OUTCOME MEASURES: The primary end point was VA. Secondary end points included area of pathologic corneal neovascularization, need for transplantation, risk of graft rejection, and QoL. RESULTS: Although no significant differences in VA scores between groups were observed, aganirsen significantly reduced the relative corneal neovascularization area after 90 days by 26.20% (P = 0.014). This improvement persisted after 180 days (26.67%, P = 0.012). Aganirsen tended to lower the transplantation need in the intent-to-treat (ITT) population at day 180 (P = 0.087). In patients with viral keratitis and central neovascularization, a significant reduction in transplantation need was achieved (P = 0.048). No significant differences between groups were observed in the risk of graft rejection. However, aganirsen tended to decrease this risk in patients with traumatic/viral keratitis (P = 0.162) at day 90. The QoL analyses revealed a significant improvement with aganirsen in composite and near activity subscores (P = 0.039 and 0.026, respectively) at day 90 in the per protocol population. Ocular and treatment-related treatment-emergent adverse events (TEAEs) were reported in a lower percentage with aganirsen compared with placebo. Only 3 serious TEAEs (2 with aganirsen and 1 with placebo) were considered treatment-related. CONCLUSIONS: This first phase III study on a topical inhibitor of corneal angiogenesis showed that aganirsen eye drops significantly inhibited corneal neovascularization in patients with keratitis. The need for transplantation was significantly reduced in patients with viral keratitis and central neovascularization. Topical application of aganirsen was safe and well tolerated.

Epithelial wound healing on keratin film, amniotic membrane and polystyrene in vitro.

PURPOSE: Corneal epithelial wound healing is a major issue in ocular surface (OS) reconstruction. Aim of this study was to evaluate parameters of epithelial wound healing in vitro on transparent keratin films (KFs) derived from human hair in comparison with amniotic membrane (AM) and polystyrene. MATERIALS AND METHODS: The human corneal epithelial cell line (HCE-T) was expanded on KF, AM and commercially available 24-well polystyrene cell culture plates in vitro to compare cell proliferation, migration and attachment by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, scratch-wound healing and adhesion assay. Cells cultured on KF and AM at an air-liquid interface for 14 d were stained with hematoxylin and eosin for histology. RESULTS: The highest proliferation of HCE-T cells was observed on polystyrene at all time points (p < 0.05). At a seeding density of 5 × 10(3) cells/well, no difference in proliferation was found between AM and KF after 24 h and 72 h (p = 0.582 and p = 0.066), while higher proliferation was observed on AM compared to KF after 48 h (p = 0.005). At a seeding density of 1 × 10(4) cells/well, no difference was found between AM and KF after 24 h (p = 0.252), while higher proliferation was observed on AM compared to KF after 48 h and 72 h (p = 0.001 and p = 0.003). The significantly fastest cell migration was observed on polystyrene at all time points (p < 0.01). Cell migration was significantly higher on KF compared to AM at 48 h (p < 0.05). After 30 min, there were significantly more cells attached to AM compared to polystyrene and KF (p = 0.032 and p = 0.001). No significant difference in cell attachment was observed between KF and polystyrene (p = 0.147). Histology demonstrated that HCE-T cells cultured on KF and AM at an air-liquid interface for 14 d form a multilayered epithelium similar to normal human corneal epithelium. CONCLUSION: Transparent KFs derived from human hair support proliferation, migration, adhesion and differentiation of HCE-T cells in vitro. Therefore, it could be a promising alternative to AM for OS reconstruction.

Periocular cutaneous oncocytoma with signs of disrupted oxygen metabolism.

Oncocytomas are benign tumors most often occurring in salivary or lacrimal glands and thyroid tissue. As cutaneous oncocytoma is exceptionally rare, this tumor is uncommonly encountered by dermatopathologists. Herein, we illustrate the case of an 80-year-old man who presented with a slowly growing papule of the lower eyelid. Histopathologically, the adenomatous tumor was composed of large monomorphic cells with eosinophilic granular cytoplasm. Electron microscopy revealed abundant, enlarged and abnormally shaped mitochondria. These findings were consistent with an oncocytoma of the skin. The presented case is unique in that the thorough work-up of the tumor tissue revealed not only hyperplastic mitochondria, representing the ultrastructural correlate of the observed granular cytoplasm, but additionally disclosed functional consequences with elevated levels of reactive oxygen specimen (ROS) within the tumor. Disrupted oxygen metabolism may result from cellular aging processes and may putatively represent the underlying pathogenesis of oncocytoma.

Long-term course in type 2 idiopathic macular telangiectasia.

INTRODUCTION: To study the long-term course in patients with idiopathic macular telangiectasia and report the effect of anti VEGF and laser treatment. METHODS: A retrospective case series of 19 patients/38 eyes with symptomatic type 2 idiopathic macular telangiectasia was performed. Six eyes received intravitreal injections of bevacizumab (1-3 injections), four eyes received focal laser treatment. Follow up examinations comprised visual acuity, biomicroscopy, fluorescein angiography and assessment of macular morphology and thickness by time and spectral-domain optical coherence tomography (OCT). RESULTS: Mean follow-up time was 81 months (range 15-188 months) - the median added up to 80 months. Visual outcome at final visit varied substantially (20/200-20/20). On average visual acuity decreased 1,2 lines (range -0,5 to 6) by 3 years, 2 lines (range -0,5 to 7) by 5 years and 4,1 lines (range 0 to 12) by 10 years. Development of choroidal neovascularisation was observed in only one eye. There was no significant difference in visual acuity between eyes receiving no treatment, intravitreal bevacizumab or laser treatment after 3 and 5 years. Morphological studies by OCT revealed typical changes with retinal atrophy and intraretinal cysts. Visual acuity correlated with the eccentricity of the main manifestation-visual preservation was associated with mainly extrafoveal disease manifestation. DISCUSSION: Type 2 idiopathic macular telangiectasia is a chronic, often slowly progressing macular disease leading to retinal atrophy and visual impairment over decades. Thorough knowledge about the long term course of this disease is necessary to evaluate possible therapeutic options in the long run.

Diagnosis and treatment of ocular chronic graft-versus-host disease: report from the German-Austrian-Swiss Consensus Conference on Clinical Practice in chronic GVHD.

PURPOSE: Ocular chronic graft-versus-host disease (cGVHD) is one of the most frequent long-term complications after hematopoietic stem cell transplantation and is often associated with significant morbidity and reduced quality of life. METHODS: The German/Austrian/Swiss Consensus Conference on Clinical Practice in cGVHD aimed to summarize the currently available evidence for diagnosis and (topical) treatment and to summarize different treatment modalities of ocular cGVHD. The presented consensus was based on a review of published evidence and a survey on the current clinical practice including transplant centers from Germany, Austria, and Switzerland. RESULTS: Ocular cGVHD often affects the lacrimal glands, the conjunctiva, the lids (including meibomian glands), and the cornea but can also involve other parts of the eye such as the sclera. Up to now, there have been no pathognomonic diagnostic features identified. The main therapeutic aim in the management of ocular cGVHD is the treatment of inflammation and dryness to relieve patients' symptoms and to maintain ocular integrity and function. Therapy should be chosen in the context of the patient's overall condition, systemic immunosuppressive therapy, symptoms, ocular surface integrity, and inflammatory activity. The consensus conference proposed new grading criteria and diagnostic recommendations for general monitoring of patients with graft-versus-host-disease for use in clinical practice. CONCLUSION: The evidence levels for diagnosis and treatment of ocular cGVHD are low, and most of the treatment options are based on empirical knowledge. Topical immunosuppression, for example, with cyclosporine, represents a promising strategy to reduce inflammation and dryness in ocular cGVHD. Further clinical trials are necessary to elucidate risk factors for eye manifestation, complications, and visual loss and to evaluate staging criteria and diagnostic and therapeutic measures for ocular cGVHD.

Extracellular matrix elasticity modulates TGF-ß-induced p38 activation and myofibroblast transdifferentiation in human tenon fibroblasts.

PURPOSE: Extracellular matrix and the cytokine TGF-ß influence scar formation in an interdependent fashion. In this study, the impact of extracellular matrix elasticity on TGF-ß-induced signal transduction and myofibroblast transdifferentiation was examined. METHODS: Primary human tenon fibroblasts were seeded on collagen-coated glass coverslips (rigid environment) or collagen or polyacrylamide gels (elastic environment) of different compliance and stimulated with TGF-ß. Myofibroblast transdifferentiation was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis for the marker gene α-smooth muscle actin (SMA), and SMA incorporation into stress fibers was determined by confocal immunofluorescence microscopy. CTGF transcription was assessed by RT-qPCR. Signaling pathways were examined by Western blot using phosphospecific antibodies and by immunofluorescence microscopy. RESULTS: TGF-ß-dependent myofibroblast transdifferentiation was enhanced in a stiff environment. Increasing matrix elasticity attenuated TGF-ß-induced myofibroblast transdifferentiation and the associated CTGF expression. TGF-ß-induced p38 activation was reduced on elastic substrates. CONCLUSIONS: The results suggest that matrix elasticity influences TGF-ß-dependent activation of p38 signaling and subsequent myofibroblast transdifferentiation. Biomechanical cues represent an important determinant of scarring processes. Therefore, cellular signals elicited by mechanotransduction deserve consideration in the design of novel antifibrotic strategies.

Lovastatin inhibits TGF-beta-induced myofibroblast transdifferentiation in human tenon fibroblasts.

PURPOSE: The transdifferentiation of Tenon fibroblasts to myofibroblasts is a pivotal step in filtering bleb scarring. It is mediated by the cytokine TGF-beta, Rho-dependent contractility, and cell-matrix interactions in an interdependent fashion. HMG-CoA-reductase inhibitors (statins) have been shown to inhibit Rho-GTPase signaling; therefore, the authors studied the influence of lovastatin on TGF-beta-mediated myofibroblast transdifferentiation to assess the potential use of statins in wound healing modulation. METHODS: Human Tenon fibroblasts were grown in culture, pretreated with lovastatin, lovastatin and mevalonate, or specific inhibitors of farnesyl transferase or geranylgeranyl transferase and were stimulated with TGF-beta1. alpha-Smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF) transcription were assessed by real-time PCR. alpha-SMA protein expression and localization were studied by Western blot and confocal immunofluorescence microscopy. Cell contractility was determined in collagen gel contraction assays. Phosphorylation of the signaling proteins Smad-2/3 and p38 were detected by Western blot, and Smad-2/3 localization was determined by confocal immunofluorescence microscopy. RESULTS: Lovastatin inhibited TGF-beta-induced CTGF transcription, alpha-SMA expression and incorporation into actin stress fibers, and subsequent collagen gel contraction. These effects were reversed by mevalonate. The inhibition of geranylgeranyl transferase but not farnesyl transferase blocked TGF-beta-induced alpha-SMA expression. Lovastatin decreased TGF-beta-induced p38 activation, whereas Smad-2/3 phosphorylation and nuclear translocation were preserved. CONCLUSIONS: Lovastatin inhibits TGF-beta-induced myofibroblast transdifferentiation in human Tenon fibroblasts, most likely by interfering with Rho-signaling. Statins may, therefore, serve to inhibit scarring after filtering glaucoma surgery.

Localization of TGF-beta type II receptor and ED-A fibronectin in normal conjunctiva and failed filtering blebs.

PURPOSE: The cytokine transforming growth factor-beta (TGF-beta), and the ED-A splice variant of the extracellular matrix protein fibronectin modulate wound healing and scar formation. To further elucidate their possible role in filtering bleb scarring after glaucoma surgery in human eyes in vivo, we studied the cell type specific localization of TGF-beta receptors and the presence of ED-A fibronectin in sections of normal conjunctiva and scarred filtering blebs. METHODS: Cryosections of normal conjunctiva (four patients) and scarred filtering blebs (seven patients) were studied by double-label immunofluorescence. Antibodies against PECAM-1 and prolyl-4-hydroxylase allowed for specification of vascular endothelial cells and activated fibroblasts, respectively. TGF-beta receptor type II (TGF-beta-RII), alpha-smooth muscle actin, O-linked sialoglycoprotein, fibronectin and the ED-A fibronectin splice-variant were also detected using specific antibodies. Labeled sections were viewed with a confocal laser scanning microscope. RESULTS: Vascular endothelial cells expressed TGF-beta-RII in both normal and scarred tissue. TGF-beta-RII was sparsely detected in the fibroblasts of normal conjunctiva while it was strongly expressed in most fibroblasts of the scarred filtering blebs. Similarly, ED-A fibronectin was not detected in the extracellular matrix of normal conjunctiva but abundantly present in scarred filtering blebs. CONCLUSIONS: Filtering bleb scarring is associated with an abundant expression of TGF-beta receptors in activated fibroblasts and the deposition of the fibrogenic ED-A fibronectin splice-variant. These data support the concept of targeting TGF-beta signaling to prevent scar formation after filtering glaucoma surgery.

Substrate rigidity modulates cell matrix interactions and protein expression in human trabecular meshwork cells.

PURPOSE: Extracellular matrix (ECM) composition, tension, and rigidity modulate cell-ECM interactions and have substantial impact on cell functions. The authors studied the effects of ECM rigidity on human trabecular meshwork (HTM) cells to assess ECM rigidity as a possible pathophysiologic factor in glaucoma. METHODS: Trabecular meshwork cells derived from donor cornea rings and passaged three to seven times were plated on collagen-coated tissue culture plastic or polyacrylamide gels of different rigidity. Cell spreading and focal adhesions were assessed by immunofluorescence microscopy. Expression of focal adhesion kinase (FAK), alpha-smooth muscle actin (alpha-SMA), tubulin, alpha-B-crystallin and GAPDH, as well as phosphorylation of FAK and serum-induced activation of ERK, were studied by Western blot. The subcellular distributions of alpha-SMA and fibronectin were examined by confocal immunofluorescence microscopy. RESULTS: ECM rigidity modulated cell spreading and focal adhesion size. FAK activation and serum-induced ERK phosphorylation increased with rising substrate rigidity. Expression of alpha-SMA and recruitment of alpha-SMA to stress fibers were enhanced on rigid substrates, whereas myocilin and alpha-B-crystallin expression increased on soft substrates. The structure of fibronectin deposits differed on stiff and soft matrices. CONCLUSIONS: Extracellular matrix rigidity modulates cytoskeletal structures, protein expression patterns, signal transduction, and fibronectin deposition in HTM cells. ECM changes altering trabecular meshwork resiliency may therefore have significant effects on ocular outflow tract functions with implications in glaucoma.

Contractility as a prerequisite for TGF-beta-induced myofibroblast transdifferentiation in human tenon fibroblasts.

PURPOSE: To assess the significance of Rho-kinase-dependent contractility in TGF-beta-induced myofibroblast transdifferentiation of human tenon fibroblasts to characterize possible pharmacological targets for the inhibition of postoperative scarring after glaucoma surgery. METHODS: Human tenon fibroblasts (HTFs) were grown in culture and stimulated with TGF-beta1. The effect of TGF-beta on Rho-GTPase activity was assessed by GST-rhotekin binding domain pulldown assay and detected by Western blot analysis. Contractility was evaluated in a silicone substrate wrinkling assay and in fibroblast-populated collagen gels. The actin cytoskeleton and focal adhesions were visualized by immunofluorescence microscopy. alpha-SMA transcripts were measured by real-time RT-PCR. TGF-beta-induced Smad- and p38-activation and expression of alpha-SMA were detected by Western blot analysis. Nuclear translocation of Smad2/3 was determined by confocal immunofluorescence microscopy. The influence of Rho-dependent kinase (ROCK) and myosin light chain kinase (MLCK) were studied by using specific kinase inhibitors (Y-27632, HA-1077, H-1152, and ML-7). RESULTS: Within 10 minutes of stimulation, TGF-beta induced Rho activation that was associated with an increase in cell tension and followed by actin stress fiber enhancement. ROCK inhibitors released cell tension and averted TGF-beta-induced cytoskeletal changes, p38 activation and subsequent alpha-SMA expression, whereas Smad2-phosphorylation and nuclear translocation were preserved. MLCK inhibition also blocked alpha-SMA expression. In fibroblast-populated collagen lattices, ROCK inhibitors prevented TGF-beta-induced stress fiber assembly and contraction. CONCLUSIONS: TGF-beta induces a rapid contractile response in HTFs that precedes myofibroblast transdifferentiation. ROCK inhibitors release this contraction and block subsequent TGF-beta-induced myofibroblast transdifferentiation and may therefore serve to modulate postoperative scarring after glaucoma filtering surgery.

p38 inhibitors prevent TGF-beta-induced myofibroblast transdifferentiation in human tenon fibroblasts.

PURPOSE: The role of mitogen-activated protein kinase (MAPK) pathways in TGF-beta-induced myofibroblast transdifferentiation of human tenon fibroblasts (HTFs) was investigated to identify potential pharmacologic targets for the inhibition of scarring after glaucoma surgery. METHODS: TGF-beta-dependent activation of Smad2, p38, and Erk-1/2 was examined by Western blot analysis. TGF-beta-induced mRNA expression of collagen Ialpha1, fibronectin, and the myofibroblast transdifferentiation marker alpha smooth muscle actin (alpha-SMA) was analyzed by real-time RT-PCR. alpha-SMA protein expression and subcellular distribution were determined by Western blot analysis and immunofluorescence cytochemistry. Fibroblast contractility was assessed in three-dimensional collagen gel contraction assays, stress fiber assembly with rhodamine-phalloidin stains, and confocal microscopy. Cell proliferation was measured with an MTT assay. Specific pharmacologic kinase inhibitors were used to characterize the involvement of MAPK-dependent pathways. RESULTS: TGF-beta stimulation of HTF induced a rapid and transient activation of Smad2 and Erk, whereas p38 activation was biphasic and sustained. After 24 hours of TGF-beta stimulation, increased levels of collagen Ialpha1, fibronectin, and alpha-SMA transcripts were detected. After 3 days of stimulation, HTF displayed increased alpha-SMA protein levels, enhanced contractility, and assembly of actin stress fibers. TGF-beta also induced HTF proliferation. Specific p38 inhibitors prevented all these aspects of TGF-beta-induced myofibroblastic transdifferentiation. CONCLUSIONS: Pharmacologic inhibition of p38 abrogates TGF-beta-induced myofibroblast transdifferentiation, reduces extracellular matrix protein expression and HTF proliferation, and may therefore serve to inhibit scarring after glaucoma surgery.