Cathepsin proteases promote angiogenic sprouting and laser-induced choroidal neovascularisation in mice.

Cysteine cathepsins are a family of proteases involved in intracellular protein turnover and extracellular matrix degradation. Cathepsin B (Ctsb) and cathepsin Z (Ctsz) promote tumorigenesis and Ctsb is a known modulator of tumor angiogenesis. We therefore investigated the angiomodulatory function of these cathepsins in vitro as well as in a mouse model of laser-induced choroidal neovascularization (laser-CNV). Ctsb(-/-), Ctsz(-/-), Ctsb/Ctsz double-knockout (Ctsb/z DKO), and wild type (WT) mice underwent argon laser treatment to induce choroidal neovascularization (CNV). The neovascularized area was quantified individually for each lesion at 14 days after laser coagulation. In vitro the effects of cathepsin inhibitors on angiogenesis were analysed by endothelial cell (EC) spheroid sprouting and EC invadosome assays. Retinas from cathepsin KO mice did not show gross morphological abnormalities. In the laser CNV model, however, Ctsb/z DKO mice displayed a significantly reduced neovascularized area compared to WT (0.027 mm(2) vs. 0.052 mm(2); p = 0.012), while single knockouts did not differ significantly from WT. In line, VEGF-induced EC spheroid sprouting and invadosome formation were not significantly altered by a specific cathepsin B inhibitor alone, but significantly suppressed when more than one cathepsin was inhibited. Our results demonstrate that laser-CNV formation is significantly reduced in Ctsb/z DKO mice. In line, EC sprouting and invadosome formation are blunted when more than one cathepsin is inhibited in vitro. These results reveal an angiomodulatory potential of cathepsins with partial functional redundancies between different cathepsin family members.

Kinetics of retinal vaso-obliteration and neovascularisation in the oxygen-induced retinopathy (OIR) mouse model.

PURPOSE: To evaluate the kinetics of peripheral vascularisation, central vessel regression and neovascularisation in the OIR mouse model in order to: i) generate standard kinetics for further studies in this model, and ii) define optimal time points to investigate cellular mechanisms of retinal vascular plasticity. METHODS: From postnatal day 7 (P7) until P12, newborn mice were kept at 75% oxygen. The animals were sacrificed on different time points, during and after O(2) exposure. After intracardial perfusion with FITC-dextran, retinal flatmounts were prepared, and the size of the retinal vascular network, the size of the central avascular area, and the number of blood vessel tufts and clusters were determined. In addition to the fluorescein stain for perfused capillaries, endothelial cells were stained with isolectin. RESULTS: Upon O(2) exposure, there is a rapid depletion of capillaries starting adjacent to the large central arteries. These avascular stripes fuse to form an avascular central area which amounts to 37% of the whole retinal surface after 2 days of hyperoxia. The peripheral capillary network remains intact throughout the incubation period, even though the pace of its centrifugal spreading is decelerated compared to room air controls. Already during O(2) exposure, revascularisation of the central avascular area is initiated by peripheral vessels sprouting in a centripetal direction. Revascularisation is accelerated after the return to room air, and is completed at P25. Maximal pathological neovascularisation can be found at P17, at the border between the avascular and vascular retina. CONCLUSION: Hyperoxia leads to a rapid development of a central avascular area of the retina, with its maximum during not at the end of the hyperoxic phase. Central capillary loss and peripheral vascularisation take place simultaneously, indicating different cellular control mechanisms for different areas of the retina. These standard kinetics for peripheral vascularisation and central vessel regression will: 1) help to compare the effects of angio-modulation, and 2) serve as normal baseline for the characterization of knock-out mice strains with regard to gene-specific vascular changes in the OIR-model.

Rapamycin reduces VEGF expression in retinal pigment epithelium (RPE) and inhibits RPE-induced sprouting angiogenesis in vitro.

Anti-VEGF treatment has become accepted first-line treatment for choroidal neovascularisation (CNV) in age-related macular degeneration. However, VEGF-inhibition does not always lead to sustained CNV-reduction. In this study, the effect of rapamycin was superior to VEGF-inhibition in a co-culture assay of endothelial cells (ECs) and retinal pigment epithelium (RPE). Rapamycin reduced EC sprouting in groups that did not respond to anti-VEGF treatment. Rapamycin did not induce EC apoptosis, but reduced both VEGF-production in RPE and the responsiveness of ECs to stimulation. Rapamycin might therefore be a therapeutic option for CNV patients that do not respond sufficiently to the established anti-VEGF treatments.

[Retinal angiomatosis. Ocular manifestation of von Hippel-Lindau disease]. / Angiomatosis retinae. Okuläre Manifestation des Von-Hippel-Lindau-Syndroms.

Von Hippel-Lindau disease (VHL disease) is a rare multisystem disorder of autosomal dominant inheritance with high penetrance. Inactivation of the VHL-protein leads to an increased expression of hypoxia induced growth factors. Predilection sites for tumor growth are the retina, the central nervous system and various visceral organs. Retinal capillary hemangioblastoma is one of the earliest manifestations of VHL disease. The lifetime risk of permanent visual loss defined as a visual acuity of 0.5 or less is about 35% in gene carriers. It increases to 60% if there is already retinal capillary hemangioblastoma. If VHL disease is suspected, a careful ophthalmological examination should be included in the clinical screening program. Having confirmed the diagnosis, regular ophthalmoscopic monitoring is essential in order to detect developing tumors at an early stage. Therapeutic options for small to medium sized peripheral tumors are laser or cryocoagulation; larger- hemangioblastomas can be treated by brachytherapy using ruthenium plaques, while asymptomatic juxtapapillary tumors can be observed at regular intervals.

[Treatment of pseudophakic cystoid macular edema]. / Therapie des zystoiden Makulaödems bei Pseudophakie.

Pseudophakic cystoid macular edema (PCME) is the most common complication following cataract surgery. In 1-3% of cases it is associated with a decrease in visual acuity. However, PCME has a good prognosis, persisting in only 10% of the patients beyond 2 years. The prophylactic therapy of eyes without additional disease with non-steroidal antiphlogistic drugs or steroids does not influence the final visual acuity. Under certain circumstances, prophylaxis can be a reasonable option. Risk factors that promote the formation of PCME are discussed. The course of acute or chronic PCME can be influenced by drug treatment, but in general the level of evidence for the treatment of this widespread problem is low. We would therefore like to present the Freiburg treatment scheme for PCME for discussion.

[Prognosis and treatment of macular bleeding in neovascular age-related macular degeneration]. / Prognose und Behandlung makulärer Blutungen bei neovaskulärer altersabhängiger Makuladegeneration.

Macular bleeding is associated with an acute loss of visual function and is frequently a complication of neovascular age-related macular degeneration. Blood degradation products can lead to permanent retinal neuronal damage over time. The extent of the bleeding is correlated to the coagulation status of the patient. The treatment strategy depends on the age, size and exact location of the bleeding. The spectrum of therapeutic options ranges from watchful waiting to large scale vitrectomy with removal of subretinal mass bleeding.

JC virus as a marker of human migration to the Americas.

JC virus is a ubiquitous human polyomavirus present in populations worldwide. Seven genotypes differing in DNA sequence by approximately 1-3% characterize three Old World population groups (African, European and Asian) as well as Oceania. It is possible to follow Old World populations into the New World by the JC virus genotypes they carried. The first population to settle in the Americas, the Native Americans, brought with them type 2A from northeast Asia. European settlers arriving after Columbus carried primarily type 1 and type 4. Africans brought by the slave trade carried type 3 and type 6.

Human polyomavirus JC variants in Papua New Guinea and Guam reflect ancient population settlement and viral evolution.

The peopling of the Pacific was a complex sequence of events that is best reconstructed by reconciling insights from various disciplines. Here we analyze the human polyomavirus JC (JCV) in Highlanders of Papua New Guinea (PNG), in Austronesian-speaking Tolai people on the island of New Britain, and in nearby non-Austronesian-speaking Baining people. We also characterize JCV from the Chamorro of Guam, a Micronesian population. All JCV strains from PNG and Guam fall within the broad Asian group previously defined in the VP1 gene as Type 2 or Type 7, but the PNG strains were distinct from both genotypes. Among the Chamorro JCV samples, 8 strains (Guam-1) were like the Type 7 strains found in Southeast Asia, while nine strains (Guam-2) were distinct from both the mainland strains and most PNG strains. We identified three JCV variants within Papua New Guinea (PNG-1, PNG-2 and PNG-3), but none of the Southeast Asian (Type 7) strains. PNG-1 strains were present in all three populations (Highlanders and the Baining and Tolai of New Britain), but PNG-2 strains were restricted to the Highlanders. Their relative lack of DNA sequence variation suggests that they arose comparatively recently. The single PNG-3 strain, identified in an Austronesian-speaking Tolai individual, was closely related to the Chamorro variants (Guam-2), consistent with a common Austronesian ancestor. In PNG-2 variants a complex regulatory region mutation inserts a duplication into a nearby deletion, a change reminiscent of those seen in the brains of progressive multifocal leukoencephalopathy patients. This is the first instance of a complex JCV rearrangement circulating in a human population.

Detection of JC virus in two African cases of progressive multifocal leukoencephalopathy including identification of JCV type 3 in a Gambian AIDS patient.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating central nervous system (CNS) infection, affecting mainly oligodendrocytes, but also occasional astrocytes. In the USA, Europe and Asia, PML is caused by the human polyomavirus JC virus (JCV) and in autopsy series occurs in about 4-7% of AIDS patients. In Africa, the prevalence of PML in AIDS patients is uncertain and the causative agent is unknown. This study reports immunocytochemical and PCR confirmation of PML in the CNS of an AIDS patient dying in Uganda, East Africa (case 1). In a Gambian patient infected with HIV-2 who died 3 months after onset of AIDS/PML in Germany (case 2), it was possible to confirm the identity of the virus by DNA sequencing of the PCR amplified JCV product. This African genotype of the virus (type 3) showed an unusual re-arrangement of the regulatory region, and could be distinguished at several sites from East African and African-American JCV strains described previously. This study has confirmed that PML is a complication of African AIDS as it is in Europe and the USA, and that JCV type 3 is pathogenic in African AIDS patients. Furthermore, the finding of an African genotype of JCV in a patient dying in Germany suggests that in this individual JCV represented a latent infection acquired in Africa.

Progressive multifocal leukoencephalopathy and JC virus genotypes in West African patients with acquired immunodeficiency syndrome: a pathologic and DNA sequence analysis of 4 cases.

OBJECTIVE: Progressive multifocal leukoencephalopathy is caused by polyomavirus JC in immunosuppressed patients. JC virus genotypes are identified by sequence analysis of the viral genome. Despite the prevalence of acquired immunodeficiency syndrome in sub-Saharan Africa, few cases of progressive multifocal leukoencephalopathy have been reported from this region. Here we describe 4 African cases and provide an analysis of viral genotypes. METHODS: Immunohistochemical staining by labeled streptavidin-biotin for capsid protein antigen was performed on all cases. Polymerase chain reaction amplification of viral genomic DNA was followed by direct cycle sequencing. RESULTS: JC virus type 3 was identified in 2 cases, and type 6 was isolated in 1 case. The viral regulatory region from 1 case showed an uncommon rearrangement pattern. CONCLUSIONS: Progressive multifocal leukoencephalopathy in West African patients with acquired immunodeficiency syndrome is caused by African genotypes of JC virus (types 3 and 6). The prevalence of disease in this autopsy series from sub-Saharan Africa (1.5%) was less than has been reported from Europe and the United States (4% to 10%) and may be partly due to biological differences in JC virus genotypes. Further studies will be needed to confirm this observation.

[Angioproliferative retinal disease caused by ischemia]. / Angioproliferative Netzhauterkrankungen bei Ischämie.

Ischemia is a major stimulus for angiogenesis, a biological response mechanism that describes the formation of new blood vessels from existing vessels. An ischemic cell communicates with endothelial cells by soluble factors such as VEGF (vascular endothelial growth factor) and its receptors. A major transcriptional factor for VEGF is HIF-1 (hypoxia inducible factor). Proliferation of endothelial cells alone does not result in stable vascular tubes, this is only achieved by recruiting additional cells such as pericytes. The stabilisation and destabilisation of vessels, which are important prerequisites for vascular growth, are in a dynamic equilibrium which can be modified by additional growth factors such as angiopoietins. In this review we discuss some of the molecular mechanisms leading from ischemia to proliferative retinopathy with a special focus on retinopathy of prematurity and the closely related mouse model of hyperoxia-induced retinopathy. This model is very useful when developing new antiangiogenic therapies based on the increasing understanding of the molecular pathogenesis of ischemic proliferative retinopathy.

Switch of anti-VEGF agents is an option for nonresponders in the treatment of AMD.

BACKGROUND: Although anti-VEGF therapy of exudative AMD with bevacizumab and ranibizumab proved efficacious in the majority of patients, CNV activity does not respond to continued treatment after repeated injections in a considerable amount of patients. These are referred to as nonresponders. A change of the drug to bevacizumab or ranibizumab could possibly offer an alternative option for the treatment of nonresponding exudative AMD. METHODS AND MATERIALS: A total of 138 nonresponders who switched therapy from bevacizumab to ranibizumab (n=114) or vice versa (n=24) were included in a retrospective study. Visual acuity (VA) and foveal thickness before and after the switch of therapy were compared. By means of linear regression analysis, we analyzed possible prognostic factors associated with a favorable outcome for visual acuity. RESULTS: Linear regression analysis revealed a statistically significant benefit for nonresponders when treatment was changed to a different anti-VEGF drug (bevacizumab or ranibizumab). VA at the time of the switch was positively correlated with a beneficial development of VA after changing the drug. There was no significant correlation with age, macular thickness, number of injections before the switch, or the development of VA under treatment before the switch. Both patients switching to Avastin and Lucentis benefitted without statistically significant differences. CONCLUSIONS: An exchange of bevacizumab with ranibizumab or vice versa should be considered in nonresponders in the treatment of exudative AMD. Further prognostic factors may help to identify patients who might benefit from a switch. These factors should be investigated in further studies.

[Pathogenesis of retinopathy of prematurity]. / Pathogenese der Frühgeborenenretinopathie.

Retinopathy of prematurity (ROP) is a complex disease with a multifactorial pathogenetic cascade that is still only partially understood. Important pathogenetic factors are gestational age at birth and birth weight. Potent postnatal factors are exposure to supplemental oxygen, slow weight gain and expression of angiogenic growth factors. Some of these crucial aspects of ROP pathogenesis will be discussed in this article and put into clinical context. With the introduction of intravitreal anti-VEGF (vascular endothelial growth factor) treatment into ROP therapy, the pathomechanistic role of VEGF in ROP deserves a special focus. Apart from VEGF, other factors will be discussed that may precede VEGF upregulation and thus may represent targets for an earlier and potentially protective intervention. Among these insulin-like growth factor 1 (IGF-1) appears to be most prominent. Finally, factors such as postnatal weight gain will be discussed in light of their potential role as screening parameters and their ability to predict ROP severity.

[siRNA in macular degeneration]. / siRNA bei Makuladegeneration.

To date aptamers, recombinant antibodies and antibody fragments which interfere specifically in cellular signal transmission by binding transmitters before a signal can be triggered, are the approved therapeutics agents for treatment of ocular angiogenesis. These substances achieve an effective but in most cases temporary inhibition of vascular growth and permeability. Other alternatives to inhibit cellular communication, such as tyrosine kinase inhibition or especially post-transcriptional gene silencing by degradation of messenger RNA (mRNA) induced by small interfering RNA (siRNA) are being evaluated in ongoing studies. In this overview issues related to mechanisms and molecule design, as well as clinical applications and the first clinical experience in ophthalmology reported on siRNA will be discussed.

[Retinal angiomatosis- an ophthalmological challenge]. / Angiomatosis retinae- Eine ophthalmologische Herausforderung.

BACKGROUND: In this report we provide a description of new findings in retinal angiomatosis (RA) and, in particular, of treatment procedures. METHOD: A review of relevant publications in the literature has been carried out and remarks on differential diagnosis are provided. RESULTS: Haemangioblastomas of the retina and the central nervous system are the dominant manifestations in von Hippel-Lindau syndrome (VHL). As in patients with VHL syndrome the danger of new tumours is great, lifelong follow-up examinations are necessary. A genetic counselling with a DNA-based test of index patients and first degree relatives is recommended. The most frequently occurring retinal peripheral small tumours should be treated with the laser, large tumours, however, with kryo. or brachytherapy. Photodynamic therapy was successfully carried out in some patients as described in the literature. Treatment of tumours in the retinal middle periphery may result in central exudates with visual deterioration. Up to now, no sufficient experience exists concerning treatment with VEGF inhibitors, proton therapy, or transpupillary thermotherapy. Large tumours with retinal complications such as retinal detachment should be treated with combined procedures, mainly with vitrectomy. CONCLUSIONS: An early diagnosis and treatment of retinal haemangioblastomas including examination of first-degree family members with a DNA-based test are necessary.

Carboxymethylcellulose as a new carrier substance for intravitreal injection of reproducible amounts of triamcinolone.

BACKGROUND: Intravitreal application of triamcinolone acetonide has become increasingly popular for the treatment of various retinal disorders. However, dosage, mode of preparation and application differ worldwide. The aim of this study was to find a safe vehicle that would allow intravitreal injection of an exact amount of triamcinolone acetonide without potentially retinotoxic preservatives. METHODS: Solutions of triamcinolone acetonide with a theoretical concentration of 4 mg/0.2 ml were prepared following one sedimentation (A) and two filtration (B, C) methods. In addition, a filtration method using carboxymethylcellulose 2% as a carrier (D) was established. During processing and after injection into an eye model, the crystals were quantified by weight and high-performance liquid chromatography (HPLC), and, hence, the rate of crystal loss during this process was determined. RESULTS: The initial preparation contained 93-106% of the calculated quantity. Method A, containing the entire vehicle, delivered 45%+/-7.3% of the target quantity to the eye model, whereas the vehicle-free methods B and C delivered 15%+/-6.9% and 11%+/-3.2%, respectively. Using carboxymethylcellulose 2% as a preservative-free vehicle, we found 93%+/-3.7% of the calculated amount in the eye model. The missing crystals were mainly sticking to the walls of the syringes and needles used for transfer. CONCLUSION: Common methods for preparing triamcinolone acetonide vary in the amount of drug actually injected intravitreally. Carboxymethylcellulose is an ideal carrier substance for intravitreal application of an exact dose of triamcinolone acetonide without preservatives.

Amplification of the complete polyomavirus JC genome from brain, cerebrospinal fluid and urine using pre-PCR restriction enzyme digestion.

A method is described for amplification of the complete genome of the human polyomavirus JC (JCV) from progressive multifocal leukuencephalopathy (PML) brain, cerebrospinal fluid (CSF) of PML patients, and from the urine of controls without neurological disease. Efficient amplification with the 'long PCR' method is achieved with primers overlapping the single BamHI or EcoRI site following digestion of the DNA sample with the restriction enzyme BamHI or EcoRI, respectively. Cutting of the supercoiled JCV genome allows full separation of the strands during the denaturation step, and permits primer annealing to compete successfully with reassociation of the genomic DNA. With this method a single PCR amplification allows restriction fragment length polymorphism (RFLP) analysis and direct cycle sequencing of any part of the viral genome. RFLP analysis of JCV amplified from CSF has identified a new mutant sequence. Sequencing of clinical samples is useful for typing of JC virus isolates as Type 1 or Type 2 and for characterization of the JCV regulatory region as archetypal or rearranged.

Subretinal tumour in a patient with a limited form of Wegener's granulomatosis.

In 1939 F. Wegener published a report concerning a peculiar febrile syndrome with necrotizing inflammation of the upper respiratory tract, focal glomerulonephritis and systemic angiitis (Wegener 1939). Clinically Wegener's granulomatosis can be divided into a limited and a generalized form. We describe a patient presenting with unilateral ocular symptoms suspicious of a neoplasm, one episode of angina pectoris and general malaise without any clinical signs of upper respiratory tract involvement. Before Wegener's granulomatosis was diagnosed by a positive titre of antineutrophil cytoplasmic antibodies and open lung biopsy, immunoscintigraphy for malignant melanoma showed a false positive result. Systemic treatment with corticosteroids and cyclophosphamide resulted in an improvement of the ocular symptoms within weeks.

Genotype profile of human polyomavirus JC excreted in urine of immunocompetent individuals.

The human polyomavirus JC (JCV) causes the central demyelinating disease progressive multifocal leukoencephalopathy in about 5% of AIDS patients. To characterize the type profile of JCV in a control population in the United States, 54 females (10 to 79 years of age; average age, 43.4 years) and 51 males (18 to 94 years of age; average age, 47.9 years) were examined for the excretion of different genotypes of JCV in their urine by PCR followed by direct cycle sequencing. The group consisted of 89 patients of a general medical clinic in addition to 16 healthy volunteers. The overall incidence of JC viruria was 43 of 105 (40.9%) subjects, with a marked increase for those subjects above the age of 30 years. Two men were found to excrete two different types of JCV at the same time, indicating double infections. Of the three different genotypes of JCV identified to date, type 1 strains (European) were the most common in this cohort (64% of total strains) followed by type 2 (East Asian) (18%). No type 3 (East African) strains were detected. Indirect evidence for the existence of JCV type 3 was found in seven individuals (16%) in the form of a type 1/3 recombinant (also called type 4). In addition, a single example of JCV which differs from types 1, 2, and 3 and may represent a phylogenetically older type (type 5) was found in a 59-year-old African-American. Delineation of sequence variations between JCV types is essential for the design of primers for sensitive PCR with clinical samples.