Methylglyoxal and high glucose inhibit VEGFR2 phosphorylation at specific tyrosine residues.

Diabetes is characterized by hyperglycemia and a significant risk of vascular complications. Vascular endothelial growth factor (VEGF) and its main receptor VEGFR2 (KDR), which is highly expressed in vascular endothelial cells, are essential mediators of vascular maintenance and angiogenesis. During glycolysis after high calorie food intake, methylglyoxal (MGO) is formed and MGO blood levels are elevated in diabetes. MGO reacts with arginine residues to generate MG-H1 or with lysine residues to carboxyethyl lysine which are common components of advanced glycation end-products. Therefore, the question arises whether hyperglycemic conditions affect VEGF signaling via a ligand-independent direct modification of signaling components. As a first step, the effect of MGO on VEGFR2 activation was investigated in cultured endothelial cells from human umbilical vein by determination of VEGFR2 phosphorylation at selected tyrosine residues by ELISA and immunoblotting using phospho-specific antibodies. Phosphorylation of VEGFR2-Y996, VEGFR2-Y1054, or VEGFR2-Y1175 reached a maximum 5 min after stimulation of endothelial cells with VEGF. Phosphorylation was significantly inhibited by 100 µM MGO and to a lesser extent by high glucose treatment. 2,3-Pentanedione and glyoxal were investigated for comparison. In summary, VEGFR2 phosphorylation is sensitive to MGO or high glucose concentrations which may be relevant in the pathophysiology of microvascular disease in diabetes.

Viral S protein histochemistry reveals few potential SARS-CoV-2 entry sites in human ocular tissues.

Despite the reported low expression of the primary SARS-CoV-2 receptor ACE2 in distinct ocular tissues, some clinical evidence suggests that SARS-CoV-2 can infect the eye. In this study, we explored potential entry sites for SARS-CoV-2 by viral S protein histochemistry on various ocular tissues and compared the staining patterns with RNA and protein expression of TMPRSS2 and ACE2. Potential viral entry sites were investigated by histochemistry using tagged recombinant viral S protein on 52 ocular tissue samples including specimens of the cornea, conjunctiva, lid margin, lacrimal gland tissue, retina, choroid, and RPE. In addition, ACE2 and TMPRSS2 immunohistochemistry were performed on the same ocular tissue, each with distinct antibodies binding to different epitopes. Lung tissue samples were used as positive controls. Finally, bulk RNA sequencing (RNA-Seq) was used to determine the expression of ACE2 and its auxiliary factors in the tissues mentioned above. S protein histochemistry revealed a positive staining in lung tissue but absent staining in the cornea, the conjunctiva, eye lid samples, the lacrimal glands, the retina and the optic nerve which was supported by hardly any immunoreactivity for ACE2 and TMPRSS2 and scarce ACE2 and TMPRSS2 RNA expression. Negligible staining with antibodies targeting ACE2 or TMPRSS2 was seen in the main and accessory lacrimal glands. In contrast, ocular staining (S protein, ACE2, TMPRSS2) was distinctly present in pigmented cells of the RPE and choroid, as well as in the ciliary body and the iris stroma. S protein histochemistry revealed hardly any SARS-CoV-2 entry sites in all ocular tissues examined. Similarly, no significant ACE2 or TMPRSS2 expression was found in extra- and intraocular tissue. While this study suggest a rather low risk of ocular infection with SARS-CoV-2, it should be noted, that potential viral entry sites may increase in response to inflammation or in certain disease states.

Arteriovenous crossing in retinal vessels of mice, rats, and pigs.

Purpose: Retinal vein occlusions (RVOs) are a common disease, but there are no animal models for spontaneous RVO formation. The critical sites of predilection, especially for branch RVO (BRVO), are the arteriovenous crossing sites in the inner retina. To gain more insight into possible animal models, the anatomic structure of retinal arteriovenous crossings was investigated in mice, rats, and pigs and compared to the human situation. Methods: Retinal flat mounts and paraffin sections of eyes from mice, rats, pigs, and humans were stained with GS lectin, Masson's trichrome, or immunohistochemistry for ACTA2 and GFAP. Serial sections of arteriovenous crossing sites were investigated. Results: Mice usually do not show retinal arteriovenous crossings. Rats have a mean of 2.8±1.4 crossings per eye at a mean distance from the optic nerve head of 2.79±0.53 mm, though the diameters of the crossing vessels are small. The situation in pigs is similar to that in humans, with many arteriovenous crossings of vessels and with similar diameters as found in humans. A mean of 28.4±3.5 crossings per retina was found, and 23% of these were arterial overcrossings. Serial paraffin sections showed that the tunica media of the artery touched that of the vein, but they did not fuse. Conclusions: While the retinal arteriovenous crossings of mice and rats are absent or comprised of rather thin vessels, those in the porcine retina are similar to adult humans. Therefore, the porcine retinal vascular bed may serve as a model to assess early steps in the formation of RVOs.

Expression of Angiogenic and Inflammatory Factors in Choroidal Neovascularisation-Derived Retinal Pigment Epithelium.

PURPOSE: Anti-angiogenic treatment is well established in the management of exudative age-related macular degeneration (AMD), but not sufficient in all patients. The characterisation of factors driving this chronic disease could serve to identify additional treatment options. The purpose of this study was to assess gene expression patterns and distinct changes in cells derived from surgically extracted choroidal neovascularisation (CNV) membranes. MATERIALS AND METHODS: The expression of >11,000 genes was analysed by means of a microarray in cells cultured from 2 late-stage CNV membranes compared to primary human retinal pigment epithelium (RPE) and ARPE-19 cells. A pathway analysis was performed to identify gene expression patterns associated with exudative AMD. RESULTS: The analysis revealed significant alterations in gene sets associated with inflammatory processes in CNV-derived cells, involving the upregulation of pro-inflammatory factors IL6, C3, and C5, and downregulation of anti-inflammatory complement factor B and complement factor I. Factors associated with angiogenesis, such as VEGFA or ANGPT2, were not significantly regulated in the 2 RPE-derived cell lines. CONCLUSION: In late-stage CNV membrane-derived RPE, gene expression was shifted towards a pro-inflammatory state. Angiogenesis-associated factors were regulated differently in the 2 CNV-derived RPE membranes. While inflammation seems to be continuously stimulated by RPE associated with late exudative AMD, this appears not to be the case with regard to angioregulatory mechanisms.

Gene expression profiling in a mouse model of retinal vein occlusion induced by laser treatment reveals a predominant inflammatory and tissue damage response.

PURPOSE: Retinal vein occlusion (RVO) has been investigated in several laser-induced animal models using pigs, rabbits and rats. However, laser-induced RVO has been rarely reported in mice, despite the impressive number of available mutants, ease of handling and cost effectiveness. The aim of this study was to further assess the feasibility of a RVO mouse model for gene expression analysis and its possible use to investigate effects of hypoxia. METHODS: C57Bl/6J mice were injected with eosin Y for photo-sensitization. Subsequently, large retinal veins were laser-treated in one eye to induce vascular occlusion. Contralateral control eyes received non-occlusive retinal laser treatment sparing large vessels. The animals were followed for up to eight days and assessed by funduscopy, angiography, hypoxyprobe staining, histopathology and gene expression analysis by qPCR and RNA sequencing (RNAseq). Another group of mice was left untreated and studied at a single time point to determine baseline characteristics. RESULTS: Laser-induced RVO persisted in half of the treated veins for three days, and in a third of the veins for the whole observation period of 8 days. Funduscopy revealed large areas of retinal swelling in all laser-treated eyes, irrespective of vascular targeting or occlusion status. Damage of the outer retina, retinal pigment epithelium (RPE), and even choroid and sclera at the laser site was observed in histological sections. Genes associated with inflammation or cell damage were highly up-regulated in all laser-treated eyes as detected by RNAseq and qPCR. Retinal hypoxia was observed by hypoxyprobe staining in all RVO eyes for up to 5 days with a maximal extension at days 2 and 3, but no significant RVO-dependent changes in gene expression were detected for angiogenesis- or hypoxia-related genes. CONCLUSION: The laser-induced RVO mouse model is characterized by a predominant general inflammatory and tissue damage response, which may obscure distinct hypoxia- and angiogenesis-related effects. A non-occlusive laser treatment control is essential to allow for proper data interpretation and should be mandatory in animal studies of laser-induced RVO to dissect laser-induced tissue damage from vascular occlusion effects.

Association of treatment adherence with real-life VA outcomes in AMD, DME, and BRVO patients.

PURPOSE: Real-life clinical outcomes of patients treated with anti-VEGF drugs for neovascular age-related macular degeneration (nAMD), diabetic macular edema (DME), or macular edema secondary to branch retinal vein occlusion (BRVO) are often inferior to results from randomized clinical trials. This observational cohort study investigates treatment adherence and real-life clinical outcomes within the first year of treatment. PATIENTS AND METHODS: A total of 708 treatment-naïve patients (466 nAMD, 134 DME, and 108 BRVO) were included. Patients were followed with a PRN treatment protocol with three intravitreal injections (IVIs) and a series of 3 monthly injections in case of persistent or recurrent disease activity, as determined by monthly follow-up exams including optical coherence tomographies. Occurrence of gaps of >56 days between treatments or follow-up (nonadherence [NA]) and the reasons for NA (patient- or center-associated) as well as disease activity within the first 12 months of treatment were analyzed. Visual acuity (VA) as well as numbers and dates of optical coherence tomography and IVI were extracted from medical records. RESULTS: NA occurred significantly more often in patients with DME (44%) than nAMD (32%) or BRVO (25%, p<0.01 between groups). NA was mainly patient-associated (nAMD: 80.0%, DME: 83.1%, BRVO: 70.4%, p=0.38 between groups). Patients with nAMD and DME and appropriate treatment/follow-up adherence had a better chance of significantly gaining or maintaining VA, respectively (19.9% vs 12.0% with 3-line-gain in nAMD and 1.3% vs 15.3% 3-line loss in DME; each p<0.05). NA did not correlate with VA outcomes in BRVO (3-line gain 30.9% vs 48.1% and 3-line loss 8.6% vs 7.4%; p>0.05). CONCLUSION: NA to treatment and follow-up regimens is a common problem in the management of patients with AMD and DME and limits clinical treatment outcomes under real-life conditions. Patients with DME have the highest risk of patient-associated NA, associated with a higher risk for significant VA loss.

TREATMENT OF NEOVASCULAR AGE-RELATED MACULAR DEGENERATION PATIENTS WITH VASCULAR ENDOTHELIAL GROWTH FACTOR INHIBITORS IN EVERYDAY PRACTICE: Identification of Health Care Constraints in Germany-The PONS Study.

PURPOSE: The PONS study was conceived to analyze the extent of nonpersistence (NP) and nonadherence (NA) in the treatment of patients with neovascular age-related macular degeneration in everyday clinical practice in Germany. Further objectives were to identify factors that can affect NP and NA and to analyze clinical outcomes under everyday conditions. METHODS: Nonpersistence (no contact with doctor for at least 3 months) and NA (no treatment or follow-up for at least 6 weeks) as well as clinical data were analyzed up to 24 months retrospectively and 12 months prospectively in 480 patients with neovascular age-related macular degeneration in 23 treatment centers. Patients were interviewed for factors possibly affecting NP and NA. RESULTS: One third of patients fulfilled criteria of NA in the first 3 months and two thirds after 6 months. The NP was 18.8% after 12 months. Treatment exclusively at one center, a higher number of patients with neovascular age-related macular degeneration at the treating center, and fixed appointments were associated with a lower risk for NP. An initial gain in visual acuity after upload was not preserved after 12 months (mean change -0.5 Early Treatment Diabetic Retinopathy Study letters). Whereas visual acuity declined by 7.5 Early Treatment Diabetic Retinopathy Study letters in patients with good baseline visual acuity >20/40, visual acuity improved by 8.5 letters in patients with baseline visual acuity of ≤20/200. Only 7.5% of patients underwent an optical coherence tomography scan after 3 upload injections, and only 2.0 optical coherence tomographies were performed in the first 12 months. CONCLUSION: The NP and NA were high in our study population and are likely to have contributed to a suboptimal clinical outcome compared with randomized clinical trials. Shortcomings in the management of patients with neovascular age-related macular degeneration, including restrictions in the timely and adequate follow-up (including optical coherence tomography) and retreatment, appear to be constraining factors in Germany.

Potential selection bias in candidates for stereotactic radiotherapy for neovascular AMD.

PURPOSE: Stereotactic radiotherapy (SRT, IRay) was able to reduce the need for intravitreal injections of anti-VEGF (IVI) in patients with neovascular AMD (nAMD) in a phase II randomized clinical trial. Certain morphologic characteristics, such as lesion size < 4 mm2 or lack of fibrosis, were associated with a better response. The purpose of this cross-sectional study was to investigate eligibility for SRT in a clinical routine setting and to compare clinical features of eligible and non-eligible patients. METHODS: Cross-sectional study of 468 patients treated for nAMD in one study center within a period of 4 months. Clinical features, such as visual acuity or number of IVI since diagnosis and within 6/12 months, as well as the presence for exclusion criteria for SRT were analyzed. Exclusion criteria were sub-divided into lesion-associated (relevant fibrosis, lesion size > 4 mm2, PE tear), ocular comorbidity (e.g., macular comorbidity, vascular disease) and systemic comorbidity (e.g., dementia or tremor). RESULTS: Exclusion criteria were met by 255 patients (54.5%). Exclusion was most dominantly associated with lesion-associated criteria (80.0%) and less often with ocular (20.8%) or systemic (9.4%) comorbidity. A total of 213 patients (45.5%) fulfilled eligibility criteria. Eligible patients had a better VA at time of analysis (0.36 vs. 0.56 logMAR, p < 0.0001) and at baseline (0.38 vs. 0.56 logMAR, p < 0.0001) compared to non-eligible patients. The numbers of previous intravitreal injections since diagnosis in strictly PRN-treated patients served as a surrogate marker for lesion activity and was comparable within the last 6/12 months. Non-eligible patients had a higher number of different anti-VEGF drugs (1.8 vs. 1.6, p = 0.038). CONCLUSIONS: SRT in addition to anti-VEGF can be an option in every second patient with nAMD. Due to morphological exclusion criteria, patients eligible for SRT had a better VA and a better clinical response compared to non-eligible patients.

Value of Optical Coherence Tomography Angiography Imaging in Diagnosis and Treatment of Hemangioblastomas in von Hippel-Lindau Disease.

BACKGROUND AND OBJECTIVE: The introduction of optical coherence tomography angiography (OCTA) provides new insights into the retinal vasculature. The aim of this study was to explore the value of OCTA in imaging retinal hemangioblastomas and monitoring laser treatment in patients with von Hippel-Lindau (VHL) disease. PATIENTS AND METHODS: Ten eyes of 10 patients with VHL disease were included in this retrospective case series. All patients underwent complete ophthalmological work-up including OCTA for retinal and optic nerve head hemangioblastoma. RESULTS: Two patients showed retinal scars and no recurrence of hemangioblastoma in OCTA. Three patients revealed recurrent hemangioblastomas. Two patients demonstrated a new hemangioblastoma. Three patients showed hemangioblastomas of the optic nerve head. Successful laser photocoagulation could be monitored with OCTA. CONCLUSIONS: Screening for retinal hemangioblastomas with OCTA alone is not possible. OCTA may help to distinguish hemangioblastomas and other lesions in VHL disease, especially after treatment and allows the differentiation from harmless non-vascular lesions in questionable cases. [Ophthalmic Surg Lasers Imaging Retina. 2016;47:935-946.].

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.

Intra- and postoperative risks and complications of small-gauge (23-G) versus conventional (20-G) vitrectomy for macular surgery.

PURPOSE: To compare the complication spectrum and rate of 23-G and 20-G vitrectomy for macular surgery. METHODS: This was a retrospective comparative analysis of 20-G and 23-G vitrectomy (introduced in 2007) for macular surgery due to macular pucker or macular hole performed between 2006 and 2010 in 61 and 59 eyes, respectively, by 2 experienced surgeons and 2 trainees. We assessed the adjusted hazard ratio for vitrectomy 23-G vs 20-G with a Cox proportional hazard model. We counted retinal detachment, vitreous hemorrhage, endophthalmitis (as early postoperative complications), or cataract progression (as late postoperative complication) as endpoint. We adjusted for indication, surgeon, retinopexy method, and the endotamponade. RESULTS: Follow-up averaged 712 days. Median time to first event was 385 days in the 23-G group and 342 days in the 20-G group. Cox proportional hazard analysis showed no significant difference between vitrectomy 23-G vs 20-G with regard to postoperative complications (hazard ratio 0.79, 95% confidence interval 0.41-1.52). The other covariates did not exert a statistically significant effect on the risk of adverse events. Looking at individual complications, retinal detachment was exclusively found after 20-G. CONCLUSIONS: In this homogenous large cohort, we did not find a statistically significant difference in rates of complications between 23-G and 20-G vitrectomy techniques for macular surgery. Trainees performed equally well as experienced surgeons.

Hyperoxia causes reduced density of retinal astrocytes in the central avascular zone in the mouse model of oxygen-induced retinopathy.

The mouse model of oxygen-induced retinopathy (OIR) is commonly used to investigate various aspects of the pathogenesis of the retinopathy of prematurity (ROP) as well as angiogenesis in general. Retinal astrocytes were suggested to be involved in retinal angiogenesis. This study aimed to describe their localization and cell density during the course of physiological vascularization and pathological revascularization. Mice expressing H2B-GFP (green fluorescent protein fused to histone 2B) from the endogenous Pdgfra promoter were kept in 75% oxygen from P7 (post natal day 7) to P12 (mouse model of OIR). Retinal flatmounts or cryosections were immunostained for glial fibrillary acidic protein (Gfap), glutamine synthetase (Glul), collagen IV (Col IV), desmin (Des), caspase 3 (Casp3), paired box 2 (Pax2), or Ki67. Astrocytic nuclei were counted with the ImageJ macro AuTOCellQuant. The hypoxic state of the retina was investigated by Hypoxyprobe. The GFP signal of the Pdgfra reporter mice co-localized with Pax2, a nuclear marker for retinal astrocytes. This bright label was much easier to quantify than Gfap or Pax2 staining. Quantification of the cell density of astrocytes during physiological development specified the spreading of astrocytes in a concentrical wave from the optic nerve head towards the periphery. Astrocyte density was reduced during the remodelling of the primary vascular plexus into a hierarchical vascular tree (maximal astrocyte density at P1: 2800 astrocytes/mm2, final astrocyte density: 800 astrocytes/mm2). In the OIR model, cell density of astrocytes was elevated in the peripheral vascularized zone. In contrast, astrocyte density dropped to a half (400 astrocytes/mm2) of the normal value in the central avascular zone during the hyperoxic phase between P8 and P10 by apoptosis and rose only after P17 as the retinal network normalized. An additional drop of astrocyte density was observed within the angles between the large vessels of the central avascular zone during hypoxia between P12 and P17. Astrocyte density was not altered at vascular tufts. The hyperoxia effect on astrocytes including the reduced astrocyte density is not the reason for vascular tuft formation. Hypoxia-affected astrocytes in combination with a reduced astrocytic network in the central avascular zone during the hypoxic phase are important determinants in the formation of pathological features during retinal revascularization.

Semaphorin 3F forms an anti-angiogenic barrier in outer retina.

Semaphorins are known modulators of axonal sprouting and angiogenesis. In the retina, we identified a distinct and almost exclusive expression of Semaphorin 3F in the outer layers. Interestingly, these outer retinal layers are physiologically avascular. Using functional in vitro models, we report potent anti-angiogenic effects of Semaphorin 3F on both retinal and choroidal vessels. In addition, human retinal pigment epithelium isolates from patients with pathologic neovascularization of the outer retina displayed reduced Semaphorin 3F expression in 10 out of 15 patients. Combined, these results elucidate a functional role for Semaphorin 3F in the outer retina where it acts as a vasorepulsive cue to maintain physiologic avascularity.

Choroidal neovascularization reduced by targeted drug delivery with cationic liposome-encapsulated paclitaxel or targeted photodynamic therapy with verteporfin encapsulated in cationic liposomes.

PURPOSE: Intravitreal antivascular endothelial growth factor (anti-VEGF) application has revolutionized the treatment of choroidal neovascularization (CNV), a hallmark of wet age-related macular degeneration. However, additional treatment options are desirable as not all CNV lesions respond to anti-VEGF injections. Here, we assessed the feasibility of targeted delivery of cationic liposome-encapsulated paclitaxel (EndoTAG-1) in treating CNV. Furthermore, we investigated whether a new formulation of verteporfin encapsulated in cationic liposomes (CL-VTP) enhances the effect of photodynamic therapy (PDT). METHODS: EndoTAG-1, LipoSPA, and CL-VTP were produced by encapsulating paclitaxel, succinyl-paclitaxel, or verteporfin in cationic liposomes (CL). Mice underwent argon laser coagulations at day 0 (D0) to induce CNV. EndoTAG-1 and LipoSPA were injected into the tail vein at D1, D3, D5, D7, and D9. Taxol, CL, or trehalose buffer alone was injected in control animals. At D10, all animals were perfused with fluorescein isothiocyanate (FITC)-dextran. Flatmounts comprising the retinal pigment epithelium, choroid, and sclera were prepared for quantifying the CNV by measuring the area of lesions perfused with FITC-dextran. For PDT, mice received an injection with CL-VTP or Visudyne at D10. One eye was treated with PDT while the other served as a control. Evaluation of RPE-choroid-scleral and retinal flatmounts was performed at D12, D14, or D17. Perfusion with FITC-dextran and tetramethylrhodamine-5-(and 6)-isothiocyanate-lectin staining was used to distinguish between perfused and non-perfused choroidal vessels. RESULTS: EndoTAG-1 or LipoSPA significantly reduced CNV size to 15% compared to trehalose controls. The mean CNV area of mice treated with CL was reduced (though not significantly) to about one-half of the value of the trehalose control group. The same was observed for paclitaxel. Thus, the reduction in the CNV size between treatment with CL and treatment with EndoTAG-1 or LipoSPA was 40%, which was not significant. PDT using either CL-VTP or Visudyne reduced CNV size to 65% (D17) of trehalose control size. CNV size was further diminished to 56% with Visudyne and 53% with CL-VTP when PDT was repeated twice. Most importantly, PDT-associated retinal damage was less pronounced using CL-VTP compared to Visudyne. CONCLUSIONS: Systemic intravenous injection of paclitaxel (EndoTAG-1)- or succinyl-paclitaxel (LipoSPA)-loaded CL had a significant antiangiogenic effect in a CNV mouse model. PDT with CL-VTP was as effective as Visudyne in neovascular obliteration but induced less tissue damage. Our data suggest that systemic application of cationic liposome formulations may serve to treat ocular neovascular diseases. This approach may reduce the need for intraocular injections and may benefit patients with neovascular lesions irresponsive to anti-VEGF treatment.

Highly potent VEGF-A-antagonistic DARPins as anti-angiogenic agents for topical and intravitreal applications.

The next-generation ophthalmic anti-VEGF therapeutics must aim at being superior to the currently available agents with regard to potency and improved drug delivery, while still being stable and safe to use at elevated concentrations. We show here the generation of a set of highly potent VEGF-A antagonistic DARPins (designed ankyrin repeat proteins) delivering these properties. DARPins with single-digit picomolar affinity to human VEGF-A were generated using ribosome display selections. Specific and potent human VEGF-A binding was confirmed by ELISA and endothelial cell sprouting assays. Cross-reactivity with VEGF-A of several species was confirmed by ELISA. Intravitreally injected DARPin penetrated into the retina and reduced fluorescein extravasation in a rabbit model of vascular leakage. In addition, topical DARPin application was found to diminish corneal neovascularization in a rabbit suture model, and to suppress laser-induced neovascularization in a rat model. Even at elevated doses, DARPins were safe to use. The fact that several DARPins are highly active in various assays illustrates the favorable class behavior of the selected binders. Anti-VEGF-A DARPins thus represent a novel class of highly potent and specific drug candidates for the treatment of neovascular eye diseases in both the posterior and the anterior eye chamber.

In vivo imaging of choroidal angiogenesis using fluorescence-labeled cationic liposomes.

PURPOSE: Precise monitoring of active angiogenesis in neovascular eye diseases such as age-related macular degeneration (AMD) enables sensitive use of antiangiogenic drugs and reduces adverse side effects. So far, no in vivo imaging methods are available to specifically label active angiogenesis. Here, we report such a technique using fluorophore-labeled cationic liposomes (CL) detected with a standard clinical in vivo scanning laser ophthalmoscope (SLO). METHODS: C57Bl/6 mice underwent laser coagulations at day 0 (d0) to induce choroidal neovascularization (CNV). Liposomes labeled with Oregon green, rhodamine (Rh), or indocyanine green (ICG) were injected into the tail vein at various time points after laser coagulation, and their fluorescence was observed in vivo 60 min later using an SLO, or afterwards in choroidal flatmounts or cryosections. RESULTS: SLO detected accumulated fluorescence only in active CNV lesions with insignificant background noise. The best signal was obtained with CL-ICG. Choroidal flatmounts and cryosections of the eye confirmed the location of retained CL in CNV lesions. Neutral liposomes, in contrast, showed no accumulation. CONCLUSIONS: These results establish fluorophore-labeled CL as high affinity markers to selectively stain active CNV. This novel, non-invasive SLO imaging technique could improve risk assessment and indication for current intraocular antiangiogenic drugs in neovascular eye diseases, as well as monitor therapeutic outcomes. Labeling of angiogenic vessels using CL can be of interest not only for functional imaging in ophthalmology but also for other conditions where localization of active angiogenesis is desirable.

Activation of retinal microglia rather than microglial cell density correlates with retinal neovascularization in the mouse model of oxygen-induced retinopathy.

BACKGROUND: Retinal neovascularization has been intensively investigated in the mouse model of oxygen-induced retinopathy (OIR). Here, we studied the contribution of microglial cells to vascular regression during the hyperoxic phase and to retinal neovascularization during the hypoxic phase. METHODS: Mice expressing green fluorescent protein (GFP) under the Cx3cr1 promoter labeling microglial cells were kept in 75% oxygen from postnatal day 7 (P7) to P12. Microglial cell density was quantified at different time points and at different retinal positions in retinal flat mounts. Microglial activation was determined by the switch from ramified to amoeboid cell morphology which correlated with the switch from lectin negative to lectin positive staining of GFP positive cells. RESULTS: Microglial cell density was constant in the peripheral region of the retina. In the deep vascular layer of the central region, however, it declined 14 fold from P12 to P14 and recovered afterwards. Activated microglial cells were found in the superficial layer of the central avascular zone from P8 to P12 and from P16 to P18. In addition, hyalocytes were found in the vitreal layer in the central region and their cell density decreased over time. CONCLUSION: Density of microglial cells does not correlate with vascular obliteration or revascularization. But the time course of the activation of microglia indicates that they may be involved in retinal neovascularization during the hypoxic phase.

Levels of VEGF but not VEGF(165b) are increased in the vitreous of patients with retinal vein occlusion.

PURPOSE: To determine the concentration of the pro-angiogenic vascular endothelial growth factor VEGF(165) (VEGF) and the anti-angiogenic VEGF(165b) in vitreous samples of patients with branch retinal vein occlusion (BRVO) and central retinal vein occlusion (CRVO) in comparison to patients without retinal occlusive disease. DESIGN: Experimental laboratory investigation. METHODS: Vitreous samples were collected from patients undergoing surgery for arteriovenous dissection after BRVO, radial optic neurotomy after CRVO in the occlusion group, or macular pucker or macular hole in the control group. Concentrations of VEGF and VEGF(165b) were determined by ELISA and an ELISA-type antibody microarray. RESULTS: Average vitreal concentration of VEGF was 8.6 ng/mL in the CRVO group and 2.0 ng/mL in the BRVO group as compared to 0.26 ng/mL in the control group. Average vitreal concentration of VEGF(165b) was 27 pg/mL in the CRVO group, 42 pg/mL in the BRVO group, and 49 pg/mL in the control group. In patients with CRVO and BRVO, the angiogenic balance was shifted towards angiogenic stimulation. CONCLUSION: The severity of RVO from BRVO to CRVO correlates with an increase of VEGF and the decrease of VEGF(165b), indicating a pro-angiogenic shift. Altering the ratio of VEGF(165b)/VEGF(165) might be a feasible approach for treating retinal occlusive diseases.

Therapeutic interference with EphrinB2 signalling inhibits oxygen-induced angioproliferative retinopathy.

PURPOSE: To investigate whether EphrinB2 (EfnB2) or EphB4 influence retinal angiogenesis under physiological or pathological conditions. METHODS: Using the mouse model of oxygen-induced proliferative retinopathy (OIR), the expression of EfnB2, EphB4, vascular endothelial growth factor (VEGF), VEGFR1 and VEGFR2 was quantified by quantitative polymerase chain reaction (qPCR) and localized in EfnB2- and EphB4-lacZ mice. Angioproliferative retinopathy was manipulated by intravitreal injection of dimeric EfnB2 and monomeric or dimeric EphB4. RESULTS: Dimeric EphB4 (EphB4-Fc) and EfnB2 (EfnB2-Fc) enhanced hypoxia-induced angioproliferative retinopathy but not physiological angiogenesis. Monomeric EphB4 (sEphB4) reduced angiogenesis. The messenger RNA (mRNA) level of EfnB2 increased significantly in the hyperoxic phase (P7-P12), while EphB4, VEGF, VEGFR1 and VEGFR2 showed a significant - up to fivefold - increased expression at P14, the start of morphologically visible vasoproliferation caused by relative hypoxia. CONCLUSION: The ephrin/Eph system is involved in angioproliferative retinopathy. Stimulation of EphB4 and EfnB2 signalling using EfnB2-Fc and EphB4-Fc, respectively, enhanced hypoxia-induced angiogenesis. In contrast, sEphB4 inhibited hypoxia-induced angiogenesis. Therefore, angiogenesis is enhanced by signalling through both EphB4 (forward) and EfnB2 (reverse). The distinction in the expression kinetics of EphB4 and EfnB2 indicates that they govern two different signalling pathways and are regulated in diverse ways. sEphB4 might be a useful drug for antiangiogenic therapy.

Vitreal levels of erythropoietin are increased in patients with retinal vein occlusion and correlate with vitreal VEGF and the extent of macular edema.

PURPOSE: This study compares vitreal levels of erythropoietin (EPO) in patients with retinal vein occlusion (RVO) with control subjects. In addition, it investigates different RVO disease parameters (time of vein occlusion, patient age, vitreal vascular endothelial growth factor (VEGF) levels, and extent of central macular edema) for possible correlations with vitreal EPO levels. METHODS: Serum and vitreal EPO were measured from 6 patients with branch retinal vein occlusion, 6 patients with central retinal vein occlusion, and 12 control subjects (10 macular puckers and 2 macular holes). RESULTS: Serum EPO levels (9.8 ± 4.9 mU/mL) did not differ between the RVO and control groups and were significantly lower than vitreal EPO levels in all groups. Vitreal EPO was elevated both in branch RVO (91 ± 59 mU/mL) and central RVO (182 ± 70 mU/mL) compared with controls (35 ± 24 mU/mL). Increased vitreal EPO correlated with higher vitreal VEGF (r = 0.64, P = 0.0008) and more pronounced central macular edema (r = 0.66, P = 0.001). CONCLUSION: The results from this study indicate that EPO is locally expressed in the retina and that it is upregulated together with VEGF in RVO eyes. Because of its role both in neuroprotection and angiogenesis, ocular EPO might represent an interesting target to investigate in patients with RVO, especially in light of the current anti-VEGF treatments.