Differences in activation of intracellular signaling in primary human retinal endothelial cells between isoforms of VEGFA 165.

Purpose: There are reports that a b-isoform of vascular endothelial growth factor-A 165 (VEGFA165b) is predominant in normal human vitreous, switching to the a-isoform (VEGFA165a) in the vitreous of some diseased eyes. Although these isoforms appear to have a different ability to activate the VEGF receptor 2 (VEGFR2) in various endothelial cells, the nature of their ability to activate intracellular signaling pathways is not fully characterized, especially in retinal endothelial cells. We determined their activation potential for two key intracellular signaling pathways (MAPK, AKT) over complete dose-response curves and compared potential effects on the expression of several VEGFA165 target genes in primary human retinal microvascular endothelial cells (HRMECs). Methods: To determine full dose-response curves for the activation of MAPK (ERK1/2), AKT, and VEGFR2, direct in-cell western assays were developed using primary HRMECs. Potential differences in dose-response effects on gene expression markers related to endothelial cell and leukocyte adhesion (ICAM1, VCAM1, and SELE) and tight junctions (CLDN5 and OCLN) were tested with quantitative PCR. Results: Activation dose-response analysis revealed much stronger activation of MAPK, AKT, and VEGFR2 by the a-isoform at lower doses. MAPK activation in primary HRMECs displayed a sigmoidal dose-response to a range of VEGFA 165 a concentrations spanning 10-250 pM, which shifted higher into the 100-5,000 pM range with VEGFA 165 b. Similar maximum activation of MAPK was achieved by both isoforms at high concentrations. Maximum activation of AKT by VEGFA 165 b was only half of the maximum activation from VEGFA 165 a. At a lower intermediate dose, where VEGFA 165 a activated intracellular signaling stronger than VEGFA 165 b, the changes in VEGFA target gene expression were generally greater with VEGFA 165 a. Conclusions: In primary HRMECs, VEGFA 165 a could maximally activate MAPK and AKT at lower concentrations where VEGFA 165 b had relatively little effect. The timing for maximum activation of MAPK was similar for the isoforms, which is different from that reported for non-retinal endothelial cells. Although differences in VEGFA 165 a and VEGFA 165 b are limited to the sequence of their six C-terminal six amino acids, this results in a large difference in their ability to activate at least two key intracellular signaling pathways and VEGF-target gene expression in primary human retinal endothelial cells.

Effects of anti-VEGF treatment on the recovery of the developing retina following oxygen-induced retinopathy.

PURPOSE: Inhibition of VEGF is widely used in patients to control neovascularization and decrease vascular permeability. To date, the effect of VEGF inhibition has not been evaluated in the developing retina such as that seen in premature infants. The goal of this study was to address the effect of anti-VEGF treatment on retinal development of a mouse model of retinopathy. METHODS: C57BL/6J mice were evaluated using a model of oxygen-induced retinopathy. Test animals were treated at postnatal day (P) 14 with intravitreal injections of the VEGF inhibitor aflibercept (2.5 or 10 µg) in one eye. Control animals were treated with injection of PBS in one eye. The noninjected fellow eyes were used as internal controls. Areas of avascular retina and neovascular tufts in injected (treated) eyes and noninjected fellow eyes were determined at P17, and the difference related to these characteristics was obtained among them. To evaluate the effect of VEGF inhibition on neurogenesis, focal ERG was performed at P21 and P42. Histologic evaluation of the retinal structure was also evaluated at P42. RESULTS: Aflibercept treatment reduced the amount of neovascular tufts but significantly increased the area of avascular retina (low dose and high dose) at P17. The delayed vascular growth corresponded to decreased ERG amplitudes (at P21 and P42) and structural changes in the retinal layers that persisted (at P42), despite vascular recovery. CONCLUSIONS: Inhibition of VEGF in developing eyes has the short-term effect of delayed vascular growth and the long-term effects of decreased function with persistent changes in the neuroretinal structures.

Retinal vascular rescue of oxygen-induced retinopathy in mice by norrin.

PURPOSE: Wnt-signaling has been implicated in retinal development. The aim of this study was to investigate the possibility of improving retinal vasculature in an animal model of retinopathy by activating Wnt-signaling. METHODS: C57BL/6J mice were evaluated using a model of oxygen-induced retinopathy (OIR). Test animals were divided in three groups and treated at postnatal day (P) 14 with intravitreal injections of Wnt-signaling modulators (respectively, norrin, Dickkopf-related protein 1 [DKK1], and norrin + DKK1) in one eye. A fourth group of animals were treated with injection of PBS in one eye as well and used as a control group. Areas of avascular retina and neovascular tufts in injected (treated) eyes and noninjected fellow eyes were determined in each of the four groups at P17 (3 days after intravitreal injection) and the difference related to these characteristics was obtained among them. To evaluate the effect of norrin on progression of retinopathy, a fifth litter (eight animals) was also treated with norrin and these retinas were evaluated at different time points. RESULTS: Modulation of Wnt-signaling consistently shows a statistically significant decrease in the avascular area of the retinas. Treatment with norrin (Wnt-signaling activator) or DKK1 (canonical signaling inhibitor) results in a statistically significant reduction of retinal avascular area compared with control eyes. Neovascular tufts were also reduced in treated eyes, albeit to a lesser extent. CONCLUSIONS: Modulation of Wnt-signaling improves retinal vascularization and accelerates vascular recovery after induction of retinopathy in the OIR mouse. Activation of Wnt-signaling (norrin) and inhibition of Wnt-canonical signaling (DKK1) result in similar improvement, indicating that norrin promotes improved vascularization, at least in part, by way of noncanonical Wnt-signaling.

A disposable system for rapid purification of autologous plasmin as an adjunct to vitrectomy - performance and safety profile.

BACKGROUND: The generation of an atraumatic posterior vitreous detachment (PVD), a common goal in vitreoretinal surgery, is a challenge particularly in children and young trauma patients. Plasmin has been proposed as a surgical adjunct to enzymatically generate a PVD. This study assesses the performance and safety of a new system for rapid purification of plasmin as an adjunct to vitrectomy. METHODS: Plasminogen was isolated from human plasma by affinity chromatography using a disposable rapid purification kit, and activated to plasmin with streptokinase. Activities were assessed spectrophotometrically. For safety studies, 38 rabbits received intravitreal injections of one of the following compounds in 0.1 ml respectively: 4.7, 12.7 and 24 IU plasmin, 15 mg dextran, 4,100 U streptokinase, 500 µg ε-aminocaproic acid, 0.1 M potassium phosphate or balanced salt solution (BSS). Thirty min after injection, a two-port vitrectomy was performed. Rabbits were followed clinically and with bright flash electroretinography (ERG) for up to 9 months. The eyes were investigated by light and transmission electron microscopy. RESULTS: The specific plasmin activity obtained from blood of healthy volunteers averaged 42.3 ± 6.6 IU/ml (range 21.6 IU/ml to 54.5 IU/ml). The identity and purity of the enzyme was confirmed by several methods. Clinically, a mild to moderate inflammatory response was seen in most eyes on day 1, but had disappeared by day 7. ERG showed moderate depressions of a- and b-wave amplitudes on day 2, particularly in the potassium phosphate (a: -29.16 ± 4.56, b: -21.23 ± 6.31), 4.7 (a: -34.38 ± 6.64, b: -26.66 ± 6.06) and 24 IU (a: -38.25 ± 4.05, b: -23.38 ± 4.29) plasmin groups, but also in the BSS- (a: -11.19 ± 21.78, b: -11.41 ± 15.47) and dextran- (a: -17.86 ± 14.18, b: -6.67 ± 18.14) treated eyes. ERG changes recovered during follow-up. One rabbit each from the 12.7 and the 24 IU plasmin groups showed a minimal discoloration of one medullary ray after 9 months. Histology did not reveal morphologic signs of toxicity. CONCLUSION: The isolation system generated plasmin with a high degree of purity. A failure-mode analysis did not reveal significant risks of toxicity. A single preparation can provide a maximum dose of 10.9 IU/200 µl, the likely target clinical dose being 1.88 IU. Plasmin doses of at least 12.7 IU appear be safe when injected into rabbit eyes, followed by vitrectomy.

Clinical presentation and genetic correlation of patients with mutations affecting the FZD4 gene.

OBJECTIVE: To correlate the ophthalmic findings of patients with pediatric vitreoretinopathies with mutations occurring in the FZD4 gene. METHODS: A total of 123 patients diagnosed with autosomal-dominant familial exudative vitreoretinopathy (AdFEVR) or retinopathy of prematurity (ROP) and 42 control patients were enrolled in the study. Diagnoses were based on retinal findings at each patient's first examination or during ROP screening. Genomic DNA was isolated and polymerase chain reaction and direct sequencing of the FZD4 gene performed. RESULTS: FZD4 gene mutations were discovered in 13 of the 123 (10.6%) patients. Nine of the 63 patients with AdFEVR (14.3%) has mutations in the FZD4 gene. Four heterozygous mutations were identified: C117R, C181Y, Q505X, and P33S/P168S. Four of the 60 patients with ROP (6.7%) have a double missense mutation P33S/P168S that was also found in the patients with FEVR. No other FZD4 mutations were found in the patients with ROP. Additionally, patients expressing the double mutation had clinical presentations that overlapped, making it difficult to assign a definitive diagnosis. None of the mutations found in the patients with FEVR or ROP were seen in the control chromosomes. CONCLUSION: Mutations occurring in the FZD4 gene affect patients diagnosed with both FEVR and ROP. The clinical picture often overlaps and may require a detailed birth and family history for diagnosis. Genetic testing confirms inherited vitreoretinopathy and helps direct clinical management. Clinical Relevance Patients diagnosed with ROP may have a mutation in the FZD4 gene and display characteristics consistent with FEVR. Analysis of the FZD4 gene should be considered.

Norrin attenuates protease-mediated death of transformed retinal ganglion cells.

PURPOSE: To investigate the effects of norrin, a nonconventional ligand for Wingless-Int (Wnt)-beta-catenin signaling pathway, on protease-mediated death of transformed rat retinal ganglion cells (RGC-5). METHODS: Transformed RGC-5 cells were treated with 2.0 microM staurosporine (SS), a broad-spectrum protein kinase-C inhibitor, to induce growth arrest, differentiation, and elevated levels of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). RGC-5 cells were also treated with 2.0 microM SS and varying doses of recombinant norrin (3.125 to 100 ng/ml). Activation of Wnt pathway was assessed by nuclear translocation of beta-catenin. Proteolytic activity of tPA and uPA was determined by zymography assays and cell viability was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays. Expression and phosphorylation of the low-density lipoprotein-related receptor-1 (LRP-1), a cell surface receptor for tPA and uPA, was determined by immunoprecipitation and western blot analysis. RESULTS: Compared to RGC-5 cells left untreated, cells treated with either SS alone or SS and norrin secreted elevated levels of tPA and uPA. A significant number of RGC-5 cells treated with only SS underwent cell death, whereas cells treated with SS and norrin did not, even though RGC-5 cells secreted elevated levels of tPA and uPA under both treatment conditions. Although norrin activated the Wnt pathway, Dickkopf related protein 1 (Dkk1), an inhibitor of Wnt/beta-catenin pathway, failed to completely block norrin's neuroprotective effects. Assays for expression and phosphorylation of LRP-1 indicated that tPA and uPA cause RGC-5 cell death, in part, by reducing phosphorylation of LRP-1, whereas norrin attenuated tPA and uPA-mediated RGC cell death, in part, by restoring phosphorylation of LRP-1. CONCLUSIONS: Our results suggest that norrin attenuates tPA- and uPA-mediated death of RGC-5 cells by activating Wnt/beta-catenin pathway and by regulating phosphorylation of LRP-1.

Elevated levels of cystatin C and tenascin-C in schisis cavities of patients with congenital X-linked retinoschisis.

PURPOSE: To describe the finding of tenascin C and cystatin-C in the intraschisis cavities of pediatric patients with intraretinal schisis cavities. METHODS: Three patients with congenital X-linked retinoschisis (CXLRS) and one girl with clinical retinoschisis underwent vitrectomy for vision-threatening schisis cavities. At the time of surgery undiluted samples of intraschisis fluid and vitreous fluid from four eyes (three male and one female) were obtained and analyzed by gel electrophoresis and protein sequencing for the presence of tenascin C and cystatin-C. RESULTS: Tenascin C and cystatin-C were found in all four samples of fluid from the intraschisis cavities, including a girl with a clinical presentation of CXLRS. The vitreous samples did not have detectable levels of either protein as determined by gel electrophoresis. CONCLUSIONS: Tenascin C and cystatin-C levels are elevated in intraschisis cavity fluid. Interestingly, this was also found in a girl not carrying a mutation in the retinoschisin gene, indicating that elevated concentrations of tenascin C and cystatin-C result from pathologic changes in the retina and not from the presence of aberrant retinoschisin.