Günther Schlunck

Differential expression of angioregulatory factors in normal and CNV-derived human retinal pigment epithelium

BackgroundChoroidal neovascularization (CNV) causes loss of vision in age-related macular degeneration (AMD). In CNV, choroidal capillaries penetrate Bruch’s membrane and the retinal pigment epithelium (RPE). Angiogenic factors produced by RPE cells are suspected as major contributors to CNV development. We therefore studied the differential expression of angioregulatory factors in normal and CNV-derived RPE.MethodsCultures of normal (ARPE-19) and CNV-derived RPE (CNV-RPE) were compared by quantitative PCR. Differential expression was verified on the protein level by immunohistochemistry in tissue samples.ResultsThe angioregulatory factors VEGF-A, VEGF-B, VEGF-C, Angiopoietin-1 (Ang-1) and Angiopoietin-2, Semaphorin-3A, PEDF, HIF-1, FGF-2, and the receptors VEGF-R2, Neuropilin-1 and Neuropilin-2 were detected in both, ARPE-19 and CNV-RPE. Transcription of PEDF, FGF-2, Neuropilin-2, Ang-1 and Ang-2 was significantly upregulated in CNV-RPE. EphA7, VEGF-R1 and leptin were transcribed exclusively in CNV-RPE and Eph-A7 and VEGF-R1 proteins were present exclusively in CNV specimens.ConclusionsA set of common factors controlling angiogenesis was detected in both, ARPE-19 cells and CNV-RPE cells. Surprisingly, PEDF and other factors inhibiting angiogenesis are strongly upregulated in CNV-RPE; thus, at least in later stages, the RPE has a potential to control angiogenesis in age-related macular degeneration.

Elasticity-dependent modulation of TGF-β responses in human trabecular meshwork cells.

PURPOSE To gain further insight into a possible role of biomechanical cues in glaucoma, the authors assessed the influence of extracellular matrix (ECM) elasticity on TGF-β2-induced changes in trabecular meshwork (TM) cells. METHODS Human TM cells derived from donor cornea rings were plated on rigid collagen-coated tissue culture plastic or polyacrylamide gels of different elasticity and treated with vehicle or TGF-β2. Activation of Smad-2/3, ERK, and AKT signaling and expression of α-SMA and PAI-1 proteins were assessed by Western blot analysis. Subcellular localization of α-SMA was determined by confocal immunofluorescence microscopy. Transcription of collagen-I, -IV, and -VI, α-SMA, PAI-1, fibronectin, fibrillin-1, cochlin, and MGP-1 was studied by RT-qPCR. The contribution of non-Smad signaling pathways to TGF-β-induced α-SMA and PAI-1 expression was assessed using the small molecule inhibitors U0126 and LY294002. RESULTS TGF-β2-induced activation of Smad-2/3, ERK, and AKT signaling as well as expression of collagen-1, α-SMA, fibulin, and MGP-1 were attenuated with increasing elasticity. In contrast, TGF-β2-induced collagen-6, fibronectin, PAI-1, and cochlin expression were enhanced on elastic substrates. The MEK-inhibitor U0126 blocked TGF-β-induced PAI-1 expression, whereas α-SMA expression was enhanced. PI3K inhibition with LY294002 reduced α-SMA expression. CONCLUSIONS ECM elasticity modulates TGF-β-induced signaling and protein expression in human TM cells. Non-Smad signaling contributes to TGF-β-induced alterations. Increasing ECM elasticity in vitro promotes protein expression patterns reminiscent of patterns reported in primary open-angle glaucoma. Therefore, changes in TM elasticity and mechanical load may have a significant role in primary open-angle glaucoma.

Trabeculectomy versus canaloplasty (TVC study) in the treatment of patients with open-angle glaucoma: a prospective randomized clinical trial.

To compare the outcomes of canaloplasty and trabeculectomy in open‐angle glaucoma.

Ultra-soft PDMS-based magnetoactive elastomers as dynamic cell culture substrata.

Mechanical cues such as extracellular matrix stiffness and movement have a major impact on cell differentiation and function. To replicate these biological features in vitro, soft substrata with tunable elasticity and the possibility for controlled surface translocation are desirable. Here we report on the use of ultra-soft (Young’s modulus <100 kPa) PDMS-based magnetoactive elastomers (MAE) as suitable cell culture substrata. Soft non-viscous PDMS (<18 kPa) is produced using a modified extended crosslinker. MAEs are generated by embedding magnetic microparticles into a soft PDMS matrix. Both substrata yield an elasticity-dependent (14 vs. 100 kPa) modulation of α-smooth muscle actin expression in primary human fibroblasts. To allow for static or dynamic control of MAE material properties, we devise low magnetic field (≈40 mT) stimulation systems compatible with cell-culture environments. Magnetic field-instigated stiffening (14 to 200 kPa) of soft MAE enhances the spreading of primary human fibroblasts and decreases PAX-7 transcription in human mesenchymal stem cells. Pulsatile MAE movements are generated using oscillating magnetic fields and are well tolerated by adherent human fibroblasts. This MAE system provides spatial and temporal control of substratum material characteristics and permits novel designs when used as dynamic cell culture substrata or cell culture-coated actuator in tissue engineering applications or biomedical devices.

Are there filtering blebs after canaloplasty

PurposeAim of the study was to assess the development of filtering blebs after canaloplasty. MethodsTwenty eyes of 20 consecutive patients receiving canaloplasty were included. All eyes were examined clinically (slit lamp), and by anterior segment optical coherence tomography and high-frequency ultrasound biomicroscopy to detect filtering blebs. Preoperative and postoperative intraocular pressure (IOP) and medications were recorded. No antimetabolites were used at any time. Two success criteria were defined to assess a possible correlation of bleb formation and success: (1) IOP ⩽21 mm Hg and minimum 20% IOP reduction without medication and (2) IOP <18 mm Hg without medication. ResultsNo filtering blebs were detected clinically. One patient had a filtering bleb-like structure as detected by anterior segment optical coherence tomography and ultrasound biomicroscopy. Mean IOP decreased significantly from 22.15±9.5 mm Hg preoperatively to 13.3±9.9 mm Hg at last follow-up (at 245±120.0 d). The number of medications was reduced significantly from 3.15±1.2 preoperatively to 0.55±0.94 postoperatively. Complete success rate was 65% for both success criteria. ConclusionsFiltering blebs occur rarely after canaloplasty. In canaloplasty, IOP reduction seems to be independent of subconjunctival aqueous drainage, thus, avoiding the problems of conjunctival scarring.

Effect of bradykinin on the cytosolic free calcium activity and phosphoinositol turnover in human glomerular epithelial cells

The effect of bradykinin (BK) on the intracellular free calcium activity [Ca2+]i and phosphoinositide (PI) turnover was investigated in human glomerular epithelial cells (GEC) in culture. Human GEC exhibited a baseline [Ca2+]i of 114 +/- 3 nmol (n = 81). BK (ED50 10(-9) mol/l) caused a rapid and transient increase in [Ca2+]i, which could also be observed in the absence of extracellular calcium. The effect of BK (10(-8) mol/l) on the [Ca2+]i was inhibited by the BK2 antagonist Hoe 140 (IC50 10(-8) mol/l). BK also induced PI turnover in a time- and dose-dependent manner. A transient increase in (1,4,5)-inositol-triphosphate (InsP3) formation from 1,445 +/- 119 to 4,629 +/- 323 cpm occurred after 5 s. Stimulation of protein kinase C (PKC) by short-term preincubation (15 min) of human GEC with phorbol-12-myristate-13-acetate (PMA) induced a dose-dependent inhibition of the BK-stimulated (10(-7) mol/l) inositol-phosphate formation. Downregulation of PKC by preincubation of human GEC with PMA (24 h, 10(-6) mol/l) or inhibition of PKC by pretreatment with staurosporin (1 h, 10(-6) mol/l) resulted in a slight but significant augmentation of the BK-induced InsP3 stimulation. The data indicate that BK induces stimulation of [Ca2+]i and PI turnover via a BK2 receptor in human GEC. PKC might exert a negative feedback function for the BK-induced PI turnover.

MicroRNA Profiling in Aqueous Humor of Individual Human Eyes by Next-Generation Sequencing

PURPOSE Extracellular microRNAs (miRNAs) in aqueous humor were suggested to have a role in transcellular signaling and may serve as disease biomarkers. The authors adopted next-generation sequencing (NGS) techniques to further characterize the miRNA profile in single samples of 60 to 80 μL human aqueous humor. METHODS Samples were obtained at the outset of cataract surgery in nine independent, otherwise healthy eyes. Four samples were used to extract RNA and generate sequencing libraries, followed by an adapter-driven amplification step, electrophoretic size selection, sequencing, and data analysis. Five samples were used for quantitative PCR (qPCR) validation of NGS results. Published NGS data on circulating miRNAs in blood were analyzed in comparison. RESULTS One hundred fifty-eight miRNAs were consistently detected by NGS in all four samples; an additional 59 miRNAs were present in at least three samples. The aqueous humor miRNA profile shows some overlap with published NGS-derived inventories of circulating miRNAs in blood plasma with high prevalence of human miR-451a, -21, and -16. In contrast to blood, miR-184, -4448, -30a, -29a, -29c, -19a, -30d, -205, -24, -22, and -3074 were detected among the 20 most prevalent miRNAs in aqueous humor. Relative expression patterns of miR-451a, -202, and -144 suggested by NGS were confirmed by qPCR. CONCLUSIONS Our data illustrate the feasibility of miRNA analysis by NGS in small individual aqueous humor samples. Intraocular cells as well as blood plasma contribute to the extracellular aqueous humor miRNome. The data suggest possible roles of miRNA in intraocular cell adhesion and signaling by TGF-β and Wnt, which are important in intraocular pressure regulation and glaucoma.

Increased Expression of Angiogenic and Inflammatory Proteins in the Vitreous of Patients with Ischemic Central Retinal Vein Occlusion

Background Central retinal vein occlusion (CRVO) is a common disease characterized by a disrupted retinal blood supply and a high risk of subsequent vision loss due to retinal edema and neovascular disease. This study was designed to assess the concentrations of selected signaling proteins in the vitreous and blood of patients with ischemic CRVO. Methods Vitreous and blood samples were collected from patients undergoing surgery for ischemic CRVO (radial optic neurotomy (RON), n = 13), epiretinal gliosis or macular hole (control group, n = 13). Concentrations of 40 different proteins were determined by an ELISA-type antibody microarray. Results Expression of proteins enriched in the vitreous (CCL2, IGFBP2, MMP10, HGF, TNFRSF11B (OPG)) was localized by immunohistochemistry in eyes of patients with severe ischemic CRVO followed by secondary glaucoma. Vitreal expression levels were higher in CRVO patients than in the control group (CRVO / control; p < 0.05) for ADIPOQ (13.6), ANGPT2 (20.5), CCL2 (MCP1) (3.2), HGF (4.7), IFNG (13.9), IGFBP1 (14.7), IGFBP2 (1.8), IGFBP3 (4.1), IGFBP4 (1.7), IL6 (10.8), LEP (3.4), MMP3 (4.3), MMP9 (3.6), MMP10 (5.4), PPBP (CXCL7 or NAP2) (11.8), TIMP4 (3.8), and VEGFA (85.3). In CRVO patients, vitreal levels of CCL2 (4.2), HGF (23.3), IGFBP2 (1.23), MMP10 (2.47), TNFRSF11B (2.96), and VEGFA (29.2) were higher than the blood levels (vitreous / blood, p < 0.05). Expression of CCL2, IGFBP2, MMP10, HGF, and TNFRSF11B was preferentially localized to the retina and the retinal pigment epithelium (RPE). Conclusion Proteins related to hypoxia, angiogenesis, and inflammation were significantly elevated in the vitreous of CRVO patients. Moreover, some markers known to indicate atherosclerosis may be related to a basic vascular disease underlying RVO. This would imply that local therapeutic targeting might not be sufficient for a long term therapy in a systemic disease but hypothetically reduce local changes as an initial therapeutic approach.

TGF-β2-Induced Invadosomes in Human Trabecular Meshwork Cells

Primary open-angle glaucoma (POAG) is a leading cause of blindness due to chronic degeneration of retinal ganglion cells and their optic nerve axons. It is associated with disturbed regulation of intraocular pressure, elevated intraocular levels of TGF-β2, aberrant extracellular matrix (ECM) deposition and increased outflow resistance in the trabecular meshwork (TM). The mechanisms underlying these changes are not fully understood. Cell-matrix interactions have a decisive role in TM maintenance and it has been suggested that TGF-β-induced inhibition of matrix metalloproteases may drive aberrant ECM deposition in POAG. Invadopodia and podosomes (invadosomes) are distinct sites of cell-matrix interaction and localized matrix-metalloprotease (MMP) activity. Here, we report on the effects of TGF-β2 on invadosomes in human trabecular meshwork cells. Human TM (HTM) cells were derived from donor tissue and pretreated with vehicle or TGF-β2 (2 ng/ml) for 3d. Invadosomes were studied in ECM degradation assays, protein expression and MMP-2 activity were assessed by western blot and zymography and ECM protein transcription was detected by RT-qPCR. HTM cells spontaneously formed podosomes and invadopodia as detected by colocalization of Grb2 or Nck1 to sites of gelatinolysis. Pretreatment with TGF-β2 enhanced invadosomal proteolysis and zymographic MMP-2 activity as well as MMP-2, TIMP-2 and PAI-1 levels in HTM cell culture supernatants. Rho-kinase inhibition by H1152 blocked the effects of TGF-β2. Concomitant transcription of fibronectin and collagens-1, -4 and -6 was increased by TGF-β2 and fibrillar fibronectin deposits were observed in areas of invadosomal ECM remodelling. In contrast to a current hypothesis, our data indicate that TGF-β2 induces an active ECM remodelling process in TM cells, characterized by concurrent increases in localized ECM digestion and ECM expression, rather than a mere buildup of material due to a lack of degradation. Invadosomal cell adhesion and signaling may thus have a role in POAG pathophysiology.

Long-term results of Erbium YAG-laser-assisted deep sclerectomy

PurposeTo evaluate the long-term results of Erbium YAG-laser-assisted deep sclerectomy (DS). In this procedure, the delicate dissection of a deep corneoscleral lamella is greatly simplified by using the Erbium YAG-laser.MethodsData of 14 consecutive patients (10 male, four female, age 67.7±10.4 years), who underwent surgery from 1999 to 2000 were analysed retrospectively. The procedure was begun as a standard DS. The deep corneoscleral lamella was dissected with a pulsed Erbium YAG-laser (energy: 40–100 mJ, frequency: 5–10 Hz). Schlemms canal was unroofed and the lamella thinned until aqueous percolated continuously through the membrane.ResultsThe mean follow-up time was 50.4±6.8 months. The mean preoperative intraocular pressure (IOP) was 37.7±10.5 mmHg. The mean postoperative IOP was 16.1±3.9 mmHg at 1 month, 15.1±4.3 mmHg at 3 months, 16.4±4.5 mmHg at 12 months, and 17.6±8.7 mmHg at 50.5 months. The complete success rates (IOP⩽21 mmHg+IOP reduction ⩾20% without glaucoma medication) were 83.3% at 3 months and 50% at 12 and 50.5 months. Rates for qualified success (IOP⩽21 mmHg+IOP reduction ⩾20% with glaucoma medication) were 91.7% at 3 months, 92.9% at 12 months, and 78.6% at 50.5 months. The number of glaucoma medications was reduced from 3.07±0.92 preoperatively to 1.14±1.41 at 50.5 months. A single case of anterior-chamber penetration, requiring iridectomy, was the only intraoperative complication.ConclusionsErbium YAG-laser-assisted DS has the advantage of a greatly simplified dissection, while offering a successful long-term IOP control comparable to conventional DS.