GDF-15: a novel serum marker for metastases in uveal melanoma patients.

BACKGROUND: About 50% of patients with uveal melanoma (UM) develop metastases during the course of their disease. We analyzed serum levels of Growth Differentiation Factor-15 (GDF-15), with the aim of identifying patients with early metastases. METHODS: GDF-15 concentration was measured using an enzyme-linked immunosorbent assay (ELISA) in serum samples from 188 UM patients (170 patients without metastases; 18 patients with clinically detectable metastases) and 18 healthy control individuals. Data were analyzed with respect to differences between patients with and without clinically detectable UM metastases. GDF-15 serum levels were further analyzed with regard to significant patient and tumor characteristics as revealed by histology and multiplex ligation-dependent probe amplification (MLPA) to determine chromosome 3 copy number. GDF-15 expression in UM was investigated by immunohistochemistry. RESULTS: Patients with clinically detectable metastases had significantly higher GDF-15 serum levels compared to those without clinically detectable metastases as well as to healthy individuals (ANOVA; p < 0.001). GDF-15 concentrations in UM patients with overt clinically detectable metastases were significantly higher than those in UM patients with a second malignancy in remission but without clinically detected UM metastases (ANOVA; p < 0.001). No association between serum concentration of GDF-15 and clinical, pathological, and genetic features was observed. GDF-15 protein was only expressed in a minority of UM cells in most tumors. CONCLUSIONS: Our data suggest that GDF-15 can be used as a serum marker for the diagnosis of metastases in UM patients. Further data collection and analysis are necessary to evaluate a possible prognostic role of GDF-15 in predicting early metastases.

The cross-linked biopolymer hyaluronic acid as an artificial vitreous substitute.

PURPOSE: Biopolymers are promising substances in the development of a new vitreous substitute to overcome the drawbacks associated with current hydrophobic tamponade materials. METHODS: Different hydrogels were assembled by cross-linking hyaluronic acid either with adipic dihydrazide (ADH) by carboxylation with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI) after hydrazation or by photocrosslinking with UV-light and N-vinyl-pyrrolidinone. The refractive index and rheologic properties of the obtained gels were investigated. To quantify the degradation of the hydrogels over time, free hyaluronic acid was measured photometrically by means of the degradation product uronic acid. For biocompatibility testing, the hydrogels were applied on top of cultured retinal pigment epithelial (RPE) cells and analyzed by the cell viability, MTT, and alamar blue viability cytotoxicity assays and flow cytometry, with Annexin V-FITC and propidium iodide co-staining. The in vivo biocompatibility of the hydrogels was tested in vitrectomized rabbit eyes for up to 6 weeks. RESULTS: The synthesized hydrogels were all clear and transparent and had a refractive index similar to human vitreous. The rheologic measurements suggested sufficient viscosity and elasticity for intraocular use. Quantification of the degradation products revealed only a small decay of the gels over 1 month. However, the ADH cross-linked hydrogels induced mild cytotoxicity in the RPE cells. The UV cross-linked hydrogels showed no toxicity or induction of apoptosis. In vivo the UV cross-linked biogels remained in place for 6 weeks, and electrophysiology and histology showed excellent tissue biocompatibility. CONCLUSIONS: Biopolymers based on UV cross-linked hyaluronic acid may be promising vitreous substitutes.

Toxicity testing of the VEGF inhibitors bevacizumab, ranibizumab and pegaptanib in rats both with and without prior retinal ganglion cell damage.

PURPOSE: To evaluate the effects of intravitreally introduced vascular endothelial growth factor (VEGF) inhibitors in rat eyes with healthy retinal ganglion cells (RGC) and into others with N-methyl-D-aspartate (NMDA)-induced RGC damage. METHODS: Bevacizumab, ranibizumab and pegaptanib were intravitreally injected each at two different concentrations. Respective vehicles of the three substances served as controls. In a different group, additionally a rat anti-VEGF antibody was injected after NMDA treatment. Retrogradely labelled RGC were counted on retinal wholemounts 1 week or 2 months after intravitreal introduction of the VEGF inhibitors. Electron microscopy (EM) was performed on normal rat eyes 2 months after introduction of the VEGF inhibitors. RESULTS: RGC counts in healthy rat eyes were essentially unchanged from those of the control animals after the administration of both low and high concentrations of bevacizumab, ranibizumab or pegaptanib. Compared to the other two substances, however, high doses of pegaptanib and its respective vehicle significantly decreased RGC after 1 week and led to a marked increase of mitochondrial swelling in EM. In eyes with NMDA-induced RGC damage, no changes of RGC numbers were detected after rat anti-VEGF antibody or bevacizumab, ranibizumab and pegaptanib at both tested concentrations. CONCLUSIONS: Even at higher doses, bevacizumab and ranibizumab showed no toxic effects on RGC in vivo in either untreated rats or in the NMDA-induced RGC damage model. Also a rat anti-VEGF antibody showed no adverse effects after NMDA. Anti-VEGF therapy therefore appears safe even for eyes with additional excitotoxic RGC damage. Potential harm from the pegaptanib carrier solution at very high local concentrations cannot be excluded.

Efficacy of Rho-kinase inhibition in promoting cell survival and reducing reactive gliosis in the rodent retina.

PURPOSE: To analyze the outcomes of Rho-kinase (ROCK) inhibition on retinal cell survival and glial reactivity under adverse conditions. METHODS: Organotypic cultures of mouse retinas were incubated with the specific ROCK-inhibitor H-1152P for 24 to 48 hours under serum deprivation. Cell damage was determined by ethidium homodimer-1 uptake and caspase-3 cleavage. Immunohistochemistry and Western blot were performed to detect reactive gliosis and to confirm the specificity of H-1152P. The cytokine profile of the culture medium was analyzed using a membrane-based array. H-1152P was administered intravitreally into rats before optic nerve crush (ONC) and the extent of apoptosis and reactive gliosis was determined after 7 days. RESULTS: Cell damage in cultured retinas was significantly reduced in response to 1 microM H-1152P, particularly in the ganglion cell layer. This was associated with a decrease in the levels of glial fibrillary acidic protein (GFAP) isoforms and the number of reactive astrocytes, Müller cells, and microglia. The release of proinflammatory cytokines including TNF-alpha, interferon-gamma, and IL-6 was also reduced, which likely contributed to the significantly lower toxicity of the conditioned media collected from retinas incubated with H-1152P. H-1152P (1 microM) suppressed the ROCK-dependent phosphorylation of adducin without a considerable interference with the protein kinase A/C-mediated phosphorylation events, indicating the specificity of the inhibitor for ROCK. H-1152P also resulted in a significant decrease in the extent of apoptosis and reactive gliosis after ONC. CONCLUSIONS: These results demonstrate the neuroprotective effect of H-1152P-mediated ROCK-inhibition on retinal cells under stress, which may rely partly on the attenuation of glial cell reactivity.

Vitreous levels of bevacizumab and vascular endothelial growth factor-A in patients with choroidal neovascularization.

PURPOSE: To investigate the vitreous levels of bevacizumab and vascular endothelial growth factor-A (VEGF-A) after intravitreal injection of the drug in patients with choroidal neovascularization (CNV). DESIGN: Interventional case series. PARTICIPANTS: Eleven eyes of 11 patients with submacular hemorrhage and CNV due to age-related macular degeneration (n = 10) or angioid streaks (n = 1). METHODS: All patients were treatment naïve except for a single dose of intravitreal injection of bevacizumab (1.25 mg/50 muL dose) and subsequent vitrectomy after various intervals (1-101 days) because of active and progressive lesion. Intravitreal free bevacizumab and VEGF-A levels were measured using enzyme-linked immunosorbent assay and microsphere-based immunoassay, respectively. Vitreous VEGF-A isoforms were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting. MAIN OUTCOME MEASURES: Intravitreal bevacizumab and VEGF-A levels were measured and pharmacokinetic parameters were calculated. RESULTS: Pharmacokinetics of intravitreal bevacizumab followed a 2-compartment model with initial and terminal half-lives of 0.5 and 6.7 days, respectively. Bevacizumab could be detected in all cases, ranging from 2.63 ng/ml to 165 microg/ml. The peak concentration was observed on the second day after intravitreal bevacizumab injection. Vitreous free VEGF-A levels ranged from 0.2 to 33.9 pg/ml and showed a negative correlation with the bevacizumab concentration (P<0.001; r = -0.955) and a positive correlation with time (P<0.001; r = 0.964). However, the percentage expression of VEGF-A(165) exhibited a positive correlation with the bevacizumab concentration (P = 0.032, r = 0.645) and a negative correlation with time (P = 0.007, r = -0.755). A time-dependent increase was found for the percentage expression of VEGF-A(189) (P = 0.023, r = 0.673). Neither bevacizumab- nor time-related alterations were found for VEGF-A(121). CONCLUSIONS: Based on pharmacokinetics, the interval of 6-7 weeks would be appropriate for efficacy, although clinical trials should guide dosing recommendations. Vitreous levels of free VEGF-A showed a negative correlation with the bevacizumab concentration, which confirmed the in vivo binding affinity of bevacizumab to VEGF-A. The analysis of the VEGF-A isoforms suggests differences of interaction between bevacizumab and individual VEGF-A isoforms.

Suppression of melanoma-associated neoangiogenesis by bevacizumab.

BACKGROUND: Bevacizumab, a potent antibody against the vascular endothelial growth factor (VEGF), has been shown to be effective for treatment of colorectal cancer. Recently, high effectiveness of bevacizumab in combination with paclitaxel has been reported in a single metastatic melanoma case. To our knowledge, we demonstrate for the first time the antiangiogenetic effect of bevacizumab in a patient with a vitreous melanoma metastasis. OBSERVATIONS: A 68-year-old man with a vitreous melanoma metastasis of the left eye was treated with a revitrectomy combined with intravitreal bevacizumab application because of iris neovascularization and progressive epiretinal tumor plaques. Four days after the treatment, the melanoma-associated neovascularization completely disappeared, but it recurred after 6 weeks. Although repetitive administration of local bevacizumab produced the same antiangiogenetic effect, progression of the epiretinal tumor plaques could not be stopped with the local bevacizumab treatment. CONCLUSIONS: Intraocular administration of the anti-VEGF drug bevacizumab causes immediate and complete regression of melanoma-associated angiogenesis. The rationale for the therapeutic strategy in our patient was an elevated level of VEGF in the vitreous cavity. Because we could not demonstrate a direct antiproliferative effect of bevacizumab on melanoma metastasis, bevacizumab seems most promising if evaluated in combination with antiproliferative agents.

Safety, penetration and efficacy of topically applied bevacizumab: evaluation of eyedrops in corneal neovascularization after chemical burn.

PURPOSE: That vascular endothelial growth factor (VEGF) plays a major role in inflammatory angiogenesis has been well established. This pilot study was designed to evaluate experimental treatment with bevacizumab eyedrops in corneal neovascularization induced by alkali burn. The feasibility of topical administration, corneal cell viability and corneal penetration were investigated in an animal model. METHODS: Eighteen chinchilla bastard rabbit corneas injured with 1 m NaOH were divided into three groups: untreated, early and late treatment groups. Eyedrops of bevacizumab solution (25 mg/ml) were administered five times daily. Clinical examination under stereoscopic microscope was performed to evaluate corneal opacity, neovascularization, vessel size and oedema. Histopathology was analysed for vessel density and apoptotic reaction. Additionally, intracameral bevacizumab concentration was measured with enzyme-linked immunosorbent assay (ELISA) after repeated topical applications. RESULTS: A fast increase in aqueous bevacizumab concentration was achieved when the solution was instilled every minute onto a healthy eye surface. As well as clear anti-angiogenic effects, anti-fibrotic effects were also seen after corneal burn, maintaining corneal transparency. Early treatment of actively growing vessels showed a significantly better outcome, although apoptosis of pre-existing vessels could also be induced by the late treatment. No specific toxicity was seen regarding epithelium, keratocytes or endothelium. CONCLUSIONS: The data from this pilot study suggest that bevacizumab eyedrops can sufficiently penetrate the corneal stroma and anterior chamber. When administered soon after alkali burn, bevacizumab seems to significantly reduce corneal damage. Combinations of established treatment regimens with topical bevacizumab might be considered in severe injuries with otherwise devastating prognoses.

Purkinje cell survival in organotypic cultures: implication of Rho and its downstream effector ROCK.

Organotypic cultures of postnatal day 1 (P1) to P7 mouse cerebella are well-established models for studying cell survival. In the present work, we investigate the involvement of the Rho/ROCK intracellular pathway in Purkinje cell survival by using organotypic cultures of P3 Swiss mice. Specific inhibitors of Rho or ROCK were applied at different concentrations to the slice cultures, which were maintained for 5 days in vitro. We show that the bacterial exoenzyme C3 transferase, a specific inhibitor of the small GTPase Rho, increases Purkinje cell survival. There is a 4.5- and 2.5-fold increase in Purkinje cell survival when C3 intracellular uptake is promoted either by the PEP-1 peptide or by the C2IN carrier protein, respectively, and not with the commonly used TAT peptide. Moreover, treatment with Y27632 and H-1152, two specific inhibitors of the Rho kinase ROCK, also strongly reduces apoptotic cell death and results in 6.5- and 8.5-fold increases in cell survival, respectively. In immunohistochemical analysis, we also show that H-1152 did not change either glial fibrillary acidic protein or isolectin-B4 staining, indicating that this compound did not alter the cellular composition in our cultures. Thus, our data demonstrate that inhibition of Rho and its downstream effector ROCK may be used to enhance cell survival in neurodegenerative diseases.

Gene expression of the repulsive guidance molecules/neogenin in the developing and mature mouse visual system: C57BL/6J vs. the glaucoma model DBA/2J.

We used in-situ hybridization to analyze the expression patterns of three known members (a, b and c) of the RGM ("repulsive guidance molecule") gene family and of the RGMa receptor neogenin in a glaucoma mouse model (DBA/2J strain) and the C57BL/6J strain, which served as a control. In order to understand the role of the RGMs and neogenin in glaucoma, we characterized their expression patterns in the developing and mature mouse retina and in the optic nerve. In all investigated stages from post-natal day (P) 0 to 15 months (M) RGMa, RGMb and neogenin expression was detected in the ganglion cell layer (GCL). From P10 to 15M, we found RGMa, RGMb and neogenin expression in the inner nuclear layer (INL) and the outer nuclear layer (ONL). In P10- and older mice, the expression patterns of RGMa and its receptor neogenin were similar, while that of RGMb differed from both. As expected, no specific retinal expression of RGMc was detected in any of the age groups investigated. C57BL/6J mice and DBA/2J mice displayed no differences in the expression pattern of RGMa, RGMb, RGMc and neogenin in the developing retina (gestational age 14.5 days (E14.5), P0 & P10). Interestingly, we found a higher expression of RGMa, RGMb and neogenin in the retinas of all glaucoma-affected mice than in the age-matched control strain. Furthermore, we detected a higher RGMa and RGMb expression in the optic nerves of glaucoma-affected DBA/2J-mice older than 11M than in C57BL/6J mice of the same age.

The Rho-kinase inhibitor H-1152P suppresses the wound-healing activities of human Tenon's capsule fibroblasts in vitro.

PURPOSE: To analyze the role of Rho-kinase signaling in the wound-healing activities of human Tenon's capsule fibroblasts by using H-1152P, a potent inhibitor of this kinase, in vitro. METHODS: The optimal concentration of H-1152P was determined by MTT test. Cell proliferation was measured by BrdU incorporation and Ki-67 immunostaining, whereas cell viability was investigated by ethidium homodimer-1 dye exclusion. The actin cytoskeleton organization was demonstrated by alpha-smooth muscle actin (SMA) immunostaining and Alexa 488-phalloidin staining. Cell migration was studied on restrained collagen gels and in a scratch-wound assay followed by Ki-67 and fibronectin immunostaining. The effect of H-1152P on contraction was analyzed in floating collagen gels populated with fibroblasts, which were subsequently processed for fibronectin immunostaining. The levels of adducin and the protein kinase A (PKA)-dependent phosphorylation of this protein were detected by immunoblot analysis, to rule out interference with PKA. RESULTS: Incorporation of BrdU and upregulation of Ki-67 were reduced by 80% to 90% in cells incubated with 10 microM of this inhibitor for 4 days (P < 0.01). H-1152P caused the disassembly of stress fibers in a dose-dependent manner without exerting toxic effects and without a considerable interference with the PKA-pathway. H-1152P also significantly suppressed cell migration 3- to 3.5-fold and the contraction of collagen lattices fivefold with a dose-dependent impairment in the assembly of the fibronectin network. CONCLUSIONS: These findings imply a role for Rho-kinase in the wound-healing activities of human Tenon's capsule fibroblasts and show the potential of H-1152P as a safe and specific means to suppress these events.

Differential toxic effect of dissolved triamcinolone and its crystalline deposits on cultured human retinal pigment epithelium (ARPE19) cells.

The aim of the study was to evaluate the antiproliferative and cytotoxic properties of triamcinolone acetonide (TA) on human retinal pigment epithelium cells (ARPE19) and the role of epicellular crystalline deposits. Monolayer cultures of ARPE19 cells were used. Purified or unpurified crystalline TA suspension (0.01-1.0 mg/ml) or the vehicle alone (benzyl alcohol, 0.025%-0.00025%), diluted in culture medium, were added to the cells that were either grown on cell culture dishes covered by a protecting membrane filter insert or without a filter. After 1, 3, 5 and 7 days mitochondrial activity was measured using the MTT assay and the morphology assessed microscopically. Cellular proliferative activity was monitored by BrdU-incorporation into cellular DNA. For cytotoxicity assays ARPE19 cells were grown to confluence and then cultured in a serum-deficient medium to ensure a static milieu. Annexin V-FITC and propidium iodide co-staining was performed and analyzed by flow cytometry. Exposure to TA without direct cellular contact showed a moderate antiproliferative activity resulting in a dose-dependent suppression of DNA synthesis (maximum 42.7%), but not a cytotoxic effect. In contrast, adherent deposits of crystalline TA particles on top of the cell layer caused a rapid-progressive and dose-dependent cell death preceded by an early phosphatidylserine externalization to the outer leaflet of the plasma membrane. Within a healthy, confluent cell layer the number of viable cells decreased by 14.2, 20.8 and 68.8%, respectively, after one day of direct exposure. Exposure to the vehicle alone caused only a slight growth inhibitory effect in a proliferating cell layer, but early signs of cell death were detected even at the lowest concentration tested. In conclusion, the effect of the vehicle is less pronounced than formerly assumed, but not negligible, thus indicating a beneficial effect of purification. While non-adherent TA, if purified, appears to be safe in clinically used concentrations, direct physical contact with crystalline particles might cause a local, rapid-progressive cytotoxicity that involves the induction of the apoptotic cascade. Therefore, epiretinal deposits after intravitreal TA administration might be critical in terms of long-term biocompatibility.

Expression of endostatin in human choroidal neovascular membranes secondary to age-related macular degeneration.

Endostatin is an endogenous angiogenesis inhibitor which requires E-selectin for its antiangiogenic activity. The aim of this study was to investigate the expression of endostatin in human choroidal neovascular membranes (CNV) secondary to age-related macular degeneration (AMD) with regard to vascularization and proliferative activity. An interventional case series of 36 patients who underwent removal of CNV were retrospectively investigated. Thirty-six CNV were analyzed by light microscopic immunohistochemistry for the expression of CD34 (endothelial cells, EC), CD105 (activated EC), Ki-67 (cell proliferation), Cytokeratin 18 (epithelial cells), VEGF (vascular endothelial growth factor), E-selectin and endostatin. Donor eyes (n=7) including one with AMD were used as controls. Endostatin immunoreactivity was present in choroidal vessels of five as well as in the retinal pigment epithelium (RPE)-Bruch's membrane complex of two donor eyes without AMD. In one eye with AMD, endostatin was detected in RPE, Bruch's membrane and choroidal vessels. Ninety-two percent (33/36) of CNV disclosed endostatin staining. RPE-Bruch's membrane complex, choroidal vessels and stroma were positive in 50% (18/36), 72% (26/36), and 78% (28/36) of the membranes, respectively. Both control eyes and CNV expressed all the investigated markers except E-selectin being positive only in membranes. Endostatin, an endogenous angiogenesis inhibitor, is expressed in CNV and its therapeutic up-regulation may be a new strategy in the treatment of neovascular AMD.

Tyrosinase biosynthesis in adult mammalian retinal pigment epithelial cells.

Tyrosinase (EC 1.14.18.1) is the rate limiting enzyme of melanogenesis and it is unclear whether it is synthesized in postnatal retinal pigment epithelium (RPE). Cultured RPE cells from cattle were fed with isolated rod outer segments (ROS). After phagocytosis, RPE cells were tested for tyrosinase presence and activity with three independent methods: (1) ultrastructural DOPA (l-3,4-dihydroxyphenylalanine) histochemistry (2) immunocytochemistry with anti-tyrosinase antibodies (3) measuring tyrosine hydroxylase activity using [(3)H]tyrosine. With all three methods tyrosinase was found in RPE cells after ROS-feeding but was absent without feeding. In contrast to the classical hypothesis, we demonstrated with three independent methods that the expression of tyrosinase and its enzymatic activity are induced in cultured adult RPE by phagocytosis of rod outer segments (ROS) in vitro.

Expression of endoglin in choroidal neovascularization.

Endoglin (CD105) is a membrane protein involved in the TGF-beta receptor signalling pathway with predominant expression by proliferating endothelial cells. The aim of this study is to analyze the expression of Endoglin in choroidal neovascularization membranes (CNVM) and to compare it to the overall proliferative status of CNVM. Thirty surgically excised CNVM, secondary to age-related macular degeneration, were investigated using light microscopic immunohistochemistry and confocal immunofluorescence microscopy using verified antibodies directed against the endothelial cell markers Endoglin, von Willebrand factor (vWF) and CD34 and the proliferation marker Ki-67. Donor eyes were used as controls. A selective expression of CD34 and vWF as well as Endoglin was found in endothelial cells. Endoglin expression was elevated in vascular endothelial cells contained within CNVM, but a moderate Endoglin expression could also be visualized in quiescent CD34 and vWF positive ocular vasculature. Ki-67 positive cells were detected in CNVM, but these were rarely endothelial cells. Endoglin expression in endothelial cells of CNVM is increased, but rarely associated with a concomitant expression of the proliferation marker Ki-67. The elevated expression of Endoglin in surgically excised CNVM suggests a persisting post-mitotic activation in an advanced stage of this neovascular tissue.

The Rho/ROCK pathway mediates neurite growth-inhibitory activity associated with the chondroitin sulfate proteoglycans of the CNS glial scar.

Axons fail to regenerate in the central nervous system after injury. Chondroitin sulfate proteoglycans (CSPG) expressed in the scar significantly contribute to the nonpermissive properties of the central nervous system environment. To examine the inhibitory activity of a CSPG mixture on retina ganglion cell (RGC) axon growth, we employed both a stripe assay and a nerve fiber outgrowth assay. We show that the inhibition exerted by CSPGs in vitro can be blocked by application of either C3 transferase, a specific inhibitor of the Rho GTPase, or Y27632, a specific inhibitor of the Rho kinase. These results demonstrate that CSPG-associated inhibition of neurite outgrowth is mediated by the Rho/ROCK signaling pathway. Consistent with these results, we found that retina ganglion cell axon growth on glial scar tissue was enhanced in the presence of C3 transferase and Y27632, respectively. In addition, we show that the recently identified inhibitory CSPG Te38 is upregulated in the lesioned spinal cord.