Pigment Epithelium-Derived Factor Protects Retinal Neural Cells and Prevents Pathological Angiogenesis in an Ex Vivo Ischemia Model.

Ocular ischemia/hypoxia is a severe problem in ophthalmology that can cause vision impairment and blindness. However, little is known about the changes occurring in the existing fully formed choroidal blood vessels. We developed a new whole organ culture model for ischemia/hypoxia in rat eyes and investigate the effects of pigment epithelium derived factor (PEDF) protein on the eye tissues. The concentration of oxygen within the vitreous was measured in the enucleated rat eyes and living rats. Then, ischemia was mimicked by incubating the freshly enucleated eyes in medium at 4°C for 14 h. Eyes were fixed immediately after enucleation or were intravitreally injected with PEDF protein or with vehicle before incubation. After incubation, light and electron microscopy (EM) as well as Tunel staining was performed. In the living rats, the intravitreal oxygen concentration was on average at 16.4% of the oxygen concentration in the air and did not change throughout the experiment whereas it was ca. 28% at the beginning of the experiment and gradually decreased over time in the enucleated eyes. EM analysis revealed that the shape of the choriocapillaris changed dramatically after 14 h incubation in the enucleated eyes. The endothelial cells made filopodia-like projections into the vessel lumen. They appeared identical to the labyrinth capillaries found in surgically extracted choroidal neovascular membranes from patients with wet age-related macular degeneration (AMD). These filopodia-like projections nearly closed the vessel lumen and showed open gaps between neighboring endothelial cells. PEDF significantly inhibited labyrinth capillary formation and kept the capillary lumen open. The number of TUNEL-positive ganglion cells and inner nuclear layer cells was significantly reduced in the PEDF-treated eyes compared to the vehicle-treated eyes. The structural changes in the chroidal vessels observed under ischemia/hypoxia conditions can mimic early changes in the process of pathological angiogenesis as observed in wet AMD patients. This new model can be used to investigate short-term drug effects on the choriocapillaris after ischemia/hypoxia and it highlighted the potential of PEDF as a promising candidate for treating wet AMD.

A new rat model of treatment-naive quiescent choroidal neovascularization induced by human VEGF165 overexpression.

Vascular endothelial growth factor (VEGF) is a crucial stimulator for choroidal neovascularization (CNV). Our aim was to develop a reproducible and valid treatment-naive quiescent CNV (i.e. without signs of exudation and with normal visual acuity) rat model by subretinal injection of an adeno-associated virus (AAV)-VEGFA165 vector. The CNV development was longitudinally followed up in vivo by scanning laser ophthalmoscopy/optical coherence tomography, fluorescein and Indocyanine Green angiographies and ex vivo by electron microscopy (EM) and immunohistochemistry. In total, 57 eyes were analysed. In vivo, a quiescent CNV was observed in 93% of the eyes 6 weeks post-transduction. In EM, CNV vessels with few fenestrations, multi-layered basement membranes and bifurcation of endothelial cells were observed sharing the human CNV features. Human VEGF overexpression, multi-layered retinal pigment epithelium (RPE) (RPE65) and macrophages/activated microglia (Iba1) were also detected. In addition, 19 CNV eyes were treated for up to 3 weeks with bevacizumab. The retinal and CNV lesion thickness decreased significantly in bevacizumab-treated CNV eyes compared with untreated CNV eyes 1 week after the treatment. In conclusion, our experimental CNV resembles those seen in patients suffering from treatment-naive quiescent CNV in wet age-related macular degeneration (AMD), and responds to short-term treatment with bevacizumab. Our new model can, therefore, be used to test the long-term effect of new drugs targeting CNV under precisely-defined conditions.

Intussusceptive Vascular Remodeling Precedes Pathological Neovascularization.

Objective- Pathological neovascularization is crucial for progression and morbidity of serious diseases such as cancer, diabetic retinopathy, and age-related macular degeneration. While mechanisms of ongoing pathological neovascularization have been extensively studied, the initiating pathological vascular remodeling (PVR) events, which precede neovascularization remains poorly understood. Here, we identify novel molecular and cellular mechanisms of preneovascular PVR, by using the adult choriocapillaris as a model. Approach and Results- Using hypoxia or forced overexpression of VEGF (vascular endothelial growth factor) in the subretinal space to induce PVR in zebrafish and rats respectively, and by analyzing choriocapillaris membranes adjacent to choroidal neovascular lesions from age-related macular degeneration patients, we show that the choriocapillaris undergo robust induction of vascular intussusception and permeability at preneovascular stages of PVR. This PVR response included endothelial cell proliferation, formation of endothelial luminal processes, extensive vesiculation and thickening of the endothelium, degradation of collagen fibers, and splitting of existing extravascular columns. RNA-sequencing established a role for endothelial tight junction disruption, cytoskeletal remodeling, vesicle- and cilium biogenesis in this process. Mechanistically, using genetic gain- and loss-of-function zebrafish models and analysis of primary human choriocapillaris endothelial cells, we determined that HIF (hypoxia-induced factor)-1α-VEGF-A-VEGFR2 signaling was important for hypoxia-induced PVR. Conclusions- Our findings reveal that PVR involving intussusception and splitting of extravascular columns, endothelial proliferation, vesiculation, fenestration, and thickening is induced before neovascularization, suggesting that identifying and targeting these processes may prevent development of advanced neovascular disease in the future. Visual Overview- An online visual overview is available for this article.

The radioprotector ortho-phospho-L-tyrosine (pTyr) attenuates the side effects of fractionated irradiation in retinoblastoma mouse models but also decreases the local tumour control.

BACKGROUND: Radiotherapy (RT) is used to treat retinoblastoma (Rb), the most frequent ocular tumour in children. Besides eradicating the tumour, RT can cause severe side effects including secondary malignancies. This study aimed to define whether the radioprotector ortho-phospho-L-tyrosine (pTyr) prevents RT-induced side effects and affects local tumour control in a xenograft and a genetic orthotopic Rb mouse model. METHODS: B6;129-Rb1tm3Tyj/J (Rb+/-) and Y79-Rb cell-xenografted nude mice were fractionated external beam irradiated (15 fractions of 5Gy 6MV photons during 3weeks) with or without pTyr pre-treatment (100mg/kg BW, 16h prior to each irradiation). One, three, six and nine months after RT, tumour control and RT toxicity were evaluated using in vivo imaging and histology. We also analysed pTyr dependant post irradiation cell survival and p53 activity in vitro. RESULTS: In vitro pTyr pre-treatment showed no radioprotection on Y79 cells, but led to p53 stabilisation in unirradiated Y79 cells and to a facilitation of radiation-induced p21 up-regulation, confirming a modulation of p53 activity by pTyr. In both mouse models, secondary tumours were undetectable. In Rb+/- mice, pTyr significantly lowered RT-induced greying of the fur, retinal thickness reduction and photoreceptor loss. However, in the xenografted Rb model, pTyr considerably decreased RT-mediated tumour control, which was observed in 16 out of 22 control eyes but in none of the 24 pTyr treated eyes. CONCLUSIONS: In Rb+/- mice pTyr significantly prevents RT-induced greying of the fur as well as retinal degeneration. However, since non-irradiated control mice were not used in our study, a formal possibility exists that the effect shown in the retina of Rb+/- mice may be due to ageing of the animals and/or actions of pTyr alone. Unfortunately, as tested in a xenograft model, pTyr treatment reduced the control of Rb tumours.

Effects of intravitreally injected Fc fragment on rat eyes.

PURPOSE: Anti-vascular endothelial growth factor (VEGF) drugs are used to treat neovascular eye diseases. Some of these drugs contain Fc fragments (Fc), but it is unknown how their mode of action is influenced by Fc. Therefore, this study investigated the effects of Fc on rat eyes after intravitreal injection. METHODS: Eighteen Long-Evans rats were intravitreally injected with sterile, biotin-labeled rat Fc (9.1 µg in 5 µl PBS). For control, 5 µl PBS was injected in another nine rats. Animals were sacrificed between 1 and 3 days (group 1), 7 days (group 2), and 14 days (group 3) after injection. The right eyes were examined by electron microscopy (EM). The left eyes were stained by immunohistochemistry to investigate the distribution of Fc and the presence of macrophages. RESULTS: After 1 day, Fc had penetrated into the anterior chamber and the retina up to the inner nuclear layer, and was located especially in retinal vessels. High numbers of infiltrating cells were present within the vitreous, around the ciliary body, anterior chamber and inside the retina 1-3 days after Fc injection (p < 0.02 group 1 vs. control). Immunohistochemistry and EM showed that they were macrophages or granulocytes in close association with Fc. Ultrastructurally, there were effects on the blood vessels such as thrombocyte activation and fibrin formation. CONCLUSIONS: Biotin labeling is ideal for investigating the distribution of intravitreally injected proteins in ocular tissue. Fc fragments at a dose corresponding to their concentration in standard AMD treatments induced inflammation, and particularly the attraction of immune-competent cells. This may be associated with the risk of inflammation or endophthalmitis after anti-VEGF treatment, and needs further investigation.

Establishment of a novel retinoblastoma (Rb) nude mouse model by intravitreal injection of human Rb Y79 cells - comparison of in vivo analysis versus histological follow up.

Retinoblastoma (Rb) is the most frequent primary intraocular tumour in children and, if left untreated, can cause death. Preclinical animal models that mimic molecular, genetic, and cellular features of cancers are essential for studying cancer and searching for promising diagnosis and treatment modalities. There are several models described for Rb, but none of them fully meet our requirements. The aim of this study was to create a novel xenograft-nude mouse-model with broad application possibilities, which closely resembles the clinical observations of Rb patients and which could be used to investigate the development and spread of the tumour by using scanning laser ophthalmoscopy/optical coherence tomography (SLO/OCT) as well as histology methods. We injected human retinoblastoma Y79 cells intravitreally in both eyes of immune-deficient nude mice. The incidences of retinoblastoma as well as growth velocity were analysed 3, 6, 9 and 12 weeks after cell injection in vivo by SLO/OCT as well as ex vivo by electron microscopy (EM) and hematoxylin/eosin (HE) staining. Moreover, internal organs were histologically screened for potentially occurring metastases. Three weeks post-injection, animals developed a retinoblastoma, and after five weeks tumour growth resulted in swelling of the eyes in individual animals, showing a similar phenotype to that of untreated Rb patients at advanced stages of tumour-development. After 12 weeks, 67.5% of all analysed eyes (29 of 42) contained a retinoblastoma. At early stages of Rb development, the SLO/OCT analysis correlated with the histology results. If the tumours were too large, only histological investigations were feasible. The ultrastructural characteristics of the xenograft-tumours were very similar to those described for patient's tumours. In one mouse, brain metastases were observed. Our retinoblastoma mouse model closely resembles the human disease. SLO/OCT can be used for the detection of Rb at early stages of development and could be used for monitoring the success of future therapies.

The effects of VEGF-A-inhibitors aflibercept and ranibizumab on the ciliary body and iris of monkeys.

PURPOSE: To investigate the effects of intravitreal ranibizumab (Lucentis®) and aflibercept (Eylea®) on the ciliary body and the iris of 12 cynomolgus monkeys with regard to the fenestrations of their blood vessels. MATERIALS AND METHODS: Structural changes in the ciliary body and in the iris were investigated with light, fluorescent, and transmission electron microscopy (TEM). The latter was used to specifically quantify fenestrations of the endothelium of blood vessels after treatment with aflibercept and ranibizumab. Each of the two ciliary bodies treated with aflibercept and the two treated with ranibizumab and their controls were examined after 1 and 7 days respectively. Ophthalmological investigations including funduscopy and intraocular pressure measurements were also applied. RESULTS: Ophthalmological investigations did not reveal any changes within the groups. Both drugs reduced the VEGF concentration in the ciliary body pigmented epithelium. The structure of the ciliary body was not influenced, while the posterior pigmented epithelium of the iris showed vacuoles after aflibercept treatment. Ranibizumab was mainly concentrated on the surface layer of the ciliary epithelium, in the blood vessel walls and the lumen of some of the blood vessels, and in the cells of the epithelium of the ciliary body. Aflibercept was more concentrated in the stroma and not in the cells of the epithelium, but as with ranibizumab, also in the blood vessel walls and some of their lumina, and again on the surface layer of the epithelium. Both aflibercept-and ranibizumab-treated eyes showed a decreased number of fenestrations of the capillaries in the ciliary body compared to the untreated controls. On day 1 and day 7, aflibercept had fewer fenestrations than the ranibizumab samples of the same day. CONCLUSIONS: Both aflibercept and ranibizumab were found to reach the blood vessel walls of the ciliary body, and effectively reduced their fenestrations. Aflibercept might eliminate VEGF to a greater extent, possibly due to a higher elimination of fenestrations in a shorter time. Moreover, the vacuoles found in the iris need further research, in order to evaluate whether they carry a possible pathological potential.

Iron accumulation in Bruch's membrane and melanosomes of donor eyes with age-related macular degeneration.

Iron (Fe) accumulation in cytoplasmic storages of the retina and retinal pigment epithelium (RPE) with age has been reported to be a contributing factor to the onset and progression of Age-related Macular Degeneration (AMD). This work investigated whether iron can also be stored in specialized metal-binding melanosomes of the RPE and choroid and in age pigments of the RPE (lipofuscin and melanolipofuscin). As accumulation of debris in Bruch's membrane is an additional hallmark of AMD, the elemental composition of Bruch's membrane was also investigated. Perimacular sections of the retina-choroid complex of six eyes of AMD donors and of seven age-matched healthy controls were investigated using Analytical Electron Microscopy (AEM). The melanosomes of the RPE and choroidal melanocytes of all AMD donors contained about two times higher iron mole fractions (0.06-0.07 at%) compared to the controls, which showed only minor iron mole fractions at or below the detection limit of 0.02 at%. Only melanosomes that contained iron, showed also significant lead peaks (both AMD and control about 0.08 at%). In addition, the electron-dense part of melanolipofuscin granules in the RPE accumulated iron and lead, both for control and AMD donors. Iron in lipofuscin was below the detection limit. The elastic layer of Bruch's membrane of all AMD donors also contained significantly higher iron mole fractions compared to controls (about 0.08 at% Fe), predominantly in areas that were also rich in calcium (Ca) and phosphorus (P), suggesting calcification. Indeed, five of the six AMD donors but only one of the seven controls showed nanocrystalline hydroxyapatite calcifications. Note that such nanocrystalline material can only be detected in EM samples without heavy metal (osmiumtetroxide, uranylacetate) staining. In conclusion, iron accumulation in melanosomal storages and within calcified Bruch's membrane is more pronounced in donors suffering from AMD compared to age-matched controls. This work underlines the common hypothesis that heavy metal homeostasis plays an important role in age-related neuropathy.

A new kind of labyrinth-like capillary is responsible for leakage from human choroidal neovascular endothelium, as investigated by high-resolution electron microscopy.

PURPOSE: This study reports the clinicopathologic findings of leaky sites in pathological vessels after submacular removal of choroidal neovascular membranes (CNV). As the site that causes fluid exudation from neovascular vessels is unknown, specific attention was focused on the formation of fenestrations, cellular junctions, and morphologic alteration which can cause endothelial leakage. METHODS: Choroidal neovascular membranes of 15 patients who underwent submacular surgery for CNV were investigated. Five patients received bevacizumab treatment before surgery, and another five received photodynamic therapy before surgery. The remaining five did not receive any other treatment before surgery. All membranes were embedded for transmission electron microscopy. CNVs were analyzed for pathological cell-to-cell connections, fenestrations, or other pathological conditions which can cause leakage of plasma. RESULTS: The morphology of the newly formed blood channels was very variable, and in principle was not different in treated and untreated patients. The sources of leakage in neovascular choroidal vessels were caused by insufficient endothelial cell connections and by capillaries with microvillar projections into the vessel lumen which blocked cellular perfusion but still allowed the flow of plasma. Fenestrations were only infrequently observed. CONCLUSIONS: A newly discovered type of pathological capillary, called a labyrinth capillary, is very likely responsible for the permanent leakage of fluid. Due to the small vessel lumen, thrombocytes cannot enter these capillaries to close the leakages. Fenestrations did not appear to play a significant role in vascular leakiness.

Effects of a single intravitreal injection of aflibercept and ranibizumab on glomeruli of monkeys.

PURPOSE: It is known that endothelial cells in the kidney are also strongly VEGF-dependent. Whether intravitreal drugs can be detected within the glomeruli or affect VEGF in glomerular podocytes is not known. Therefore, the aim of this pilot study was to investigate the effects of a single intravitreal injection of aflibercept and ranibizumab on glomeruli of monkeys. METHODS: The kidneys of eight cynomolgus monkeys, which were intravitreally injected either with 2 mg of aflibercept or with 0.5 mg of ranibizumab, were investigated one and seven days after injection. Two animals served as controls. The distribution of aflibercept, ranibizumab and VEGF was evaluated using anti-Fc- or anti-F(ab)-fragment and anti-VEGF antibodies respectively. The ratio of stained area/nuclei was calculated using a semi-quantitative computer assisted method. Glomerular endothelial cell fenestration was quantified in electron microscopy using a systematic uniform random sampling protocol and estimating the ratio of fenestrae per µm. RESULTS: Compared to the controls, the anti-VEGF stained area/nuclei ratio of the ranibizumab-treated animals showed no significant changes whereas the stained areas of the aflibercept-treated monkeys showed a significant decrease post-treatment. Immune reactivity (IR) against aflibercept or ranibizumab was detected in aflibercept- or ranibizumab treated animals respectively. The number of fenestrations of the glomerular endothelial cells has shown no significant differences except one day after aflibercept injection in which the number was increased. CONCLUSION: Surprisingly, both drugs could be detected within the capillaries of the glomeruli. After a single intravitreal injection of aflibercept, VEGF IR in the podocytes was significantly reduced compared to controls. Ranibizumab injection had no significant effect on the glomeruli's VEGF level. Whether this is caused by aflibercept's higher affinity to VEGF or because it is used in a higher stoichiometric concentration compared to ranibizumab remains to be investigated.

Different effects of intravitreally injected ranibizumab and aflibercept on retinal and choroidal tissues of monkey eyes.

BACKGROUND: Since there is evidence that the Fc domain of antivascular endothelial growth factor drugs may cause unexpected consequences in retinal and choroidal vessels, the effects of intravitreal ranibizumab and aflibercept on monkey eyes were investigated. METHODS: Four cynomolgus monkeys were intravitreally injected with 0.5 mg of ranibizumab and another four with 2 mg of aflibercept. Two untreated monkeys served as controls. Funduscopy, fluorescein angiography (FA), spectral-domain-optical coherence tomography (SD-OCT) and measurement of intraocular pressure (IOP) were performed. The eyes were inspected by light, fluorescence and electron microscopy. The diameter of the choriocapillaris (CC) was measured by morphometry, and the areas of the CC with free haemoglobin, CC fenestrations and endothelial thickness were quantified. RESULTS: Analysis showed ranibizumab permeated the retina via intercellular clefts, whereas aflibercept was taken up by ganglion cells, cells of the inner and outer retinal layers and the retinal pigment epithelium (RPE). Stasis and haemolysis in the choriocapillaris and choroidal vessels were more frequent after aflibercept treatment, which caused hypertrophy and death of individual RPE cells. The area of the CC was significantly reduced after both drugs compared with controls, but the reduction of the CC endothelium thickness, number of fenestrations and the areas with haemolysis were more pronounced after aflibercept. CONCLUSIONS: Ranibizumab permeated the retina through intercellular spaces, whereas aflibercept was taken up by neuronal and RPE cells. Aflibercept induced protein complex formation and more haemolysis in the choriocapillaris, leading to individual RPE cell death. The clinical significance and relation of these findings to the Fc domain or to other characteristics of aflibercept remain to be investigated.

In vitro induction of protein complexes between bevacizumab, VEGF-A¹65 and heparin: explanation for deposits observed on endothelial veins in monkey eyes.

PURPOSE: By investigating the effects of intravitreal bevacizumab on retinal vessels of monkeys, we found that bevacizumab accumulated locally at high concentration within individual blood vessels. It formed electron-dense fibrous deposits between endothelial cells and erythrocytes or granulocytes inducing retinal vein thrombosis. To better characterise the observed deposits, we investigated in vitro whether these deposits result from a complex between bevacizumab, vascular endothelial growth factor (VEGF)-A(165) and heparin. METHODS: Cynomolgus monkeys were intravitreally injected with 1.25 mg bevacizumab. The eyes were enucleated between 1 and 14 days after injection and investigated by electron microscopy and immunohistochemistry. Human umbilical vein endothelial cells (HUVEC) were incubated with bevacizumab, VEGF-A(165) and heparin at different concentrations. Treatments with ranibizumab served as control. Bevacizumab and ranibizumab were detected immunohistochemically using Cy-3 or immunogold labelled antibodies. RESULTS: Treated animals showed bevacizumab locally at high concentration within retinal blood vessels. Electron-dense deposits inside retinal vessels and between erythrocytes were detected in three out of four treated monkeys. In vitro, many globular aggregates heavily stained with anti-human IgG were only observed with equimolar amounts (240 nM) of bevacizumab and VEGF-A(165) and 0.2 U/ml heparin and not after ranibizumab treatment. The immunogold labelling specifically localised ultrastructurally the complexes formed between bevacizumab, VEGF-A(165) and heparin at the surfaces of HUVEC cells. CONCLUSIONS: Heparin promotes bevacizumab immune complex deposition on to endothelial cells. Our in vitro results could explain the presence of deposits observed on endothelial veins in monkey eyes intravitreally injected with bevacizumab.

Lens Injury Has a Protective Effect on Photoreceptors in the RCS Rat.

Lens injury induced activation of retinal glia, and subsequent release of ciliary neurotrophic factor (CNTF) and leukaemia inhibitory factor (LIF) potently protect axotomised retinal ganglion cells from apoptosis and promotes axon regeneration in the injured optic nerve. The goal of the current study was to investigate if similar effects may also be applicable to rescue photoreceptors from degeneration in a model of retinitis pigmentosa. Lens injury was performed in the Royal College of Surgeons (RCS) rats at the age of one month. The survival of photoreceptors was evaluated histologically, and retinal function was analysed by electroretinography (ERG). Expression of CNTF was also analysed. Lens injury significantly enhanced the survival of photoreceptors 1 month after surgery compared to untreated controls, which was associated with an enhanced ERG response. In addition, lens injury significantly protected photoreceptors from degeneration in the contralateral eye, although to a much lesser extent. We could show that lens injury is sufficient to transiently delay the degeneration of photoreceptors in the RCS rat. The observed neuroprotective effects may be at least partially mediated by an upregulation of CNTF expression seen after lens injury.

Effects of bevacizumab in retina and choroid after intravitreal injection into monkey eyes.

OBJECTIVE: Due to its low price, bevacizumab, which binds vascular endothelial growth factor, is currently used off-label for the treatment of over 50 different eye diseases and has been adopted worldwide despite the absence of serious preclinical data. This study examines the effects of intravitreal bevacizumab on monkey eyes with particular focus on choroidal and retinal vessels. METHODS: Cynomolgus monkeys received an intravitreal injection of 1.25 mg bevacizumab with or without (125)I labeling. The eyes were enucleated between 1 and 14 days after injection and were investigated by electron microscopy, immunocytochemistry, histochemistry or autoradiography. Untreated and phosphate buffered saline (PBS)-injected monkeys were used as controls. RESULTS: Bevacizumab locally accumulated at high concentration within individual blood vessels. It formed electron-dense deposits inside retinal veins and between red and white blood cells, activated thrombocytes and induced retinal vein thrombosis. Retinal cells like Müller cells, astrocytes and microglia were also activated. High amounts of bevacizumab were found in retinal and choroidal vessels which may interfere with blood flow. CONCLUSIONS: The deposits on the retinal vein walls may provide a mechanistic basis for the observed retinal blood flow alterations after bevacizumab treatment in patients.

Formation of immune complexes and thrombotic microangiopathy after intravitreal injection of bevacizumab in the primate eye.

BACKGROUND: In this study, the effect of intravitreal injection of bevacizumab on choroidal blood vessels was examined in primate eyes. METHODS: Four Cynomolgus monkeys received an intravitreal injection of 1.25 mg bevacizumab. The eyes were enucleated on days 1, 4, 7 and 14. For each animal, one eye was embedded in paraffin whereas the other eye was embedded for electron microscopy. Seven untreated or PBS (phosphate buffered saline)-injected monkeys were used as controls. RESULTS: Thrombotic microangiopathy was found in the choriocapillaris and choroidal vessels of all eight injected eyes. Acute microangiopathy was characterized ultrastructurally as swelling of the endothelium, loss of fenestrations and complete collapse of the capillaries, and was commonly observed in bevacizumab-treated eyes. Quantitative analysis showed reduction of the lumina of the choriocapillaris in the eyes of three of the monkeys. Bevacizumab was frequently localized inside the blood vessels, often filling the entire breadth of the vessels, and formed clusters with blood cells. Death of photoreceptors occurred in two monkeys. CONCLUSIONS: This study indicate that intravitreal injection of bevacizumab in monkeys induces activation of platelets, degranulation of thrombocytes and neutrophils, formation of immune complexes, thrombotic microangiopathy and alteration of the blood flow in choroidal vessels.

Lipofuscin can be eliminated from the retinal pigment epithelium of monkeys.

Lipofuscin is a cytologic hallmark of aging in metabolically active postmitotic cells including neurons, cardiac muscle cells, and the retinal pigment epithelium (RPE). High levels of lipofuscin are involved in the pathogenesis of age-related macular degeneration (AMD), the main cause of blindness in the elderly population in the western world. Degradation and exocytosis of lipofuscin by RPE cells have not been observed in vivo until now, and no drug is known to eliminate the intracellular amount of lipofuscin. Here, we show that in monkeys treated with a small molecule belonging to the tetrahydropyridoethers class (n = 36 of 48 monkeys), RPE cells significantly release lipofuscin. In 4 eyes, macrophages were detected which had taken up lipofuscin. They were located between the Bruch's membrane and the RPE, and in the choroid. The quantification of pigment granules was performed by transmission electron microscopy. Our findings open the way to develop therapeutic strategies to remove lipofuscin from RPE cells, which may have implications for the treatment of age-related macular degeneration in which lipofuscin accumulation in cells is a causative factor.

The classical pathway of melanogenesis is not essential for melanin synthesis in the adult retinal pigment epithelium.

Premelanosomes are presumed to be essential for melanogenesis in melanocytes and pre-natal retinal pigment epithelium (RPE) cells. We analysed melanin synthesis in adenoviral-transduced tyrosinase-gene-expressing amelanotic RPE (ARPE) 19 cells to determine whether premelanosome formation is needed for post-natal melanogenesis. The synthesis of melanogenic proteins and melanin granules was investigated by immunocytochemistry and light and electron microscopy. The occurrence of tyrosinase was analysed ultrastructurally by dihydroxyphenylalanine histochemistry. The viability of transduced cell cultures was examined via MTT assay. We found active tyrosinase in small granule-like vesicles throughout the cytoplasm and in the endoplasmic reticulum and nuclear membrane. Tyrosinase was also associated with multi-vesicular and multi-lamellar organelles. Typical premelanosomes, structural protein PMEL17, tyrosinase-related protein 1 and classic melanosomal stages I-IV were not detected. Instead, melanogenesis took place inside multi-vesicular and multi-lamellar bodies of unknown origin. Viability was not affected up to 10 days after transduction. We thus demonstrate a pathway of melanin formation lacking typical hallmarks of melanogenesis.

Ultrastructural analysis of the pigment dispersion syndrome in DBA/2J mice.

PURPOSE: To characterise ocular pigment abnormalities associated with iris atrophy in DBA/2J mice as a model for human pigment dispersion syndrome. METHODS: Immunohistochemistry, electron and light microscopy were performed to examine the eyes of DBA/2J mice ranging in age from 2.5 to 18 months old. The focus of our study was the description of the ultrastructural modifications in the irides of DBA/2J mice. RESULTS: The DBA/2J mice presented modifications in the melanosomes in all the pigmented parts of the eye, including the retinal pigment epithelial cells and choroidal melanocytes of the ciliary pigment epithelium. The extracellular matrix of the iris stroma disappeared with ageing. Pigmented cells detached from the iris and migrated into the trabecular meshwork exclusively on the anterior iris surface. These cells were identified as macrophages by immunohistochemistry and electron microscopy. There was no evidence that melanocytes or iris pigment epithelial cells migrated into the trabecular meshwork, but they became more and more depigmented. The aqueous outflow was blocked by pigment-laden cells, but not by cellular debris or melanosomes. No substantial amount of extracellular melanosomes was observed. CONCLUSION: The morphology of melanosomes is aberrant in all pigment cells in the eyes of DBA/2J mice. We conclude that the disease process begins with the transfer of both immature melanosomes from the iris pigment epithelium (IPE) and melanocytes to macrophages, which subsequently migrate into the trabecular meshwork. Accumulating macrophages cause a blockade of the chamber angle. As the disease progresses, the IPE, melanocytes and iris stroma, including blood vessels, disappear, leading to iris atrophy. It is speculated that the loss of these pigment cells is partly caused by reduction of the iris stroma.

A reproducible and quantifiable model of choroidal neovascularization induced by VEGF A165 after subretinal adenoviral gene transfer in the rabbit.

PURPOSE: To determine the effects of the vascular endothelial growth factor (VEGF)-A(165) delivered using a high capacity adenoviral vector (HC Ad.VEGF-A) on vascular growth and pathological changes in the rabbit eye. To combine different detection methods of VEGF-A(165) overexpression-induced neovascularization in the rabbit. METHODS: HC Ad.VEGF-A(165) was constructed and injected at 5 x 10(6) infectious units (iu) into the subretinal space of rabbit eyes. Two and four weeks postinjection, the development of neovascularization and the expression of HC Ad-transduced VEGF-A(165) protein were followed up in vivo by scanning laser ophthalmoscopy, fluorescein and indocyanine green angiographies and ex vivo by electron microscopy and immunohistochemistry RESULTS: We observed a choroidal neovascularization (CNV) with leakage in 83% of the rabbit eyes. Our findings present clear indications that there is a significant effect on the endothelial cells of the choriocapillaris after subretinal transduction of the retinal pigment epithelium (RPE) with VEGF-A(165) vector. The choroidal endothelial cells were activated, adherent junctions opened, and the fenestration was minimized, while the extracellular matrix localized between the RPE and the endothelium of the choriocapillaris was enlarged toward the lumen of the vessels, inducing a deep invagination of the endothelial cells into the vessel lumen. They also proliferated and formed pathological vessels in the subretinal space. Moreover,there was an increased expression of basic fibroblast growth factor and VEGF-A accompanied by macrophage stimulation, retinal edema, and photoreceptor loss. CONCLUSIONS: This is the first model of VEGF-induced CNV in the rabbit in which the pathological events following overexpression of VEGF by RPE cells have been described in detail. Many of the features of our experimental CNV resemble those observed clinically in patients having wet age-related macular degeneration.

Bevacizumab (avastin) does not harm retinal function after intravitreal injection as shown by electroretinography in adult mice.

PURPOSE: Scavenging of VEGF by specific antibodies is a promising way to treat ocular conditions connected with neovascularization. Intravitreal injections of Avastin (bevacizumab) are performed frequently as a treatment of such conditions. In this study, the authors examine whether the retinal function in wild-type mice is affected by an intravitreal injection of Avastin. METHODS: Electroretinography was performed in four different experimental groups of wild-type C57BL/6 mice before treatment and 1, 4, 12, and 25 days afterwards. The first group was injected intravitreally with BSS, the second one received injections of a vehicle solution, and the third group was injected with the commercial Avastin solution. In a fourth group, sham surgery was performed. Immunohistochemistry was performed in some eyes to evaluate penetration of the bevacizumab molecule through the retina. RESULTS: In all four groups, a similar behavior of the ERG parameters could be detected. One day after the injections, the amplitudes showed a clear decrease. Later on, they recovered gradually. No difference could be seen between eyes injected with Avastin or vehicle solution. Bevacizumab immunoreactivity was already present in the whole retina half an hour after the intravitreal injection and was not detectable 25 days later. Moreover, binding of bevacizumab to endogenous mouse VEGF could be shown. CONCLUSIONS: Based on the electroretinographic findings, the authors conclude that bevacizumab does not have any toxic effects on the mouse retina and its function. The bevacizumab molecule penetrates the retina quickly. Therefore, it can act safely and very quickly, also in deeper retinal layers after its injection.