Sascha Fauser

A central role for inflammation in the pathogenesis of diabetic retinopathy

Diabetic retinopathy is a leading cause of adult vision loss and blindness. Much of the retinal damage that characterizes the disease results from retinal vascular leakage and nonperfusion. Diabetic retinal vascular leakage, capillary nonperfusion, and endothelial cell damage are temporary and spatially associated with retinal leukocyte stasis in early experimental diabetes. Retinal leukostasis increases within days of developing diabetes and correlates with the increased expression of retinal intercellular adhesion molecule‐1 (ICAM‐1) and CD18. Mice deficient in the genes encoding for the leukocyte adhesion molecules CD18 and ICAM‐1 were studied in two models of diabetic retinopathy with respect to the long‐term development of retinal vascular lesions. CD18−/− and ICAM‐1−/− mice demonstrate significantly fewer adherent leukocytes in the retinal vasculature at 11 and 15 months after induction of diabetes with STZ. This condition is associated with fewer damaged endothelial cells and lesser vascular leakage. Galactosemia of up to 24 months causes pericyte and endothelial cell loss and formation of acellular capillaries. These changes are significantly reduced in CD18‐ and ICAM‐1‐deficient mice. Basement membrane thickening of the retinal vessels is increased in long‐term galactosemic animals independent of the genetic strain. Here we show that chronic, low‐grade subclinical inflammation is responsible for many of the signature vascular lesions of diabetic retinopathy. These data highlight the central and causal role of adherent leukocytes in the pathogenesis of diabetic retinopathy. They also underscore the potential utility of anti‐inflammatory treatment in diabetic retinopathy.

Restoration of photoreceptor ultrastructure and function in retinal degeneration slow mice by gene therapy

The gene Prph2 encodes a photoreceptor-specific membrane glycoprotein, peripherin-2 (also known as peripherin/rds), which is inserted into the rims of photoreceptor outer segment discs in a complex with rom-1 (ref. 2). The complex is necessary for the stabilization of the discs, which are renewed constantly throughout life, and which contain the visual pigments necessary for photon capture. Mutations in Prph2 have been shown to result in a variety of photoreceptor dystrophies, including autosomal dominant retinitis pigmentosa and macular dystrophy. A common feature of these diseases is the loss of photoreceptor function, also seen in the retinal degeneration slow (rds or Prph2 Rd2/Rd2) mouse, which is homozygous for a null mutation in Prph2. It is characterized by a complete failure to develop photoreceptor discs and outer segments, downregulation of rhodopsin and apoptotic loss of photoreceptor cells. The electroretinograms (ERGs) of Prph2Rd2/Rd2 mice have greatly diminished a-wave and b-wave amplitudes, which decline to virtually undetectable concentrations by two months. Subretinal injection of recombinant adeno-associated virus (AAV) encoding a Prph2 transgene results in stable generation of outer segment structures and formation of new stacks of discs containing both perpherin-2 and rhodopsin, which in many cases are morphologically similar to normal outer segments. Moreover, the re-establishment of the structural integrity of the photoreceptor layer also results in electrophysiological correction. These studies demonstrate for the first time that a complex ultrastructural cell defect can be corrected both morphologically and functionally by in vivo gene transfer.

A rare nonsynonymous sequence variant in C3 is associated with high risk of age-related macular degeneration

Through whole-genome sequencing of 2,230 Icelanders, we detected a rare nonsynonymous SNP (minor allele frequency = 0.55%) in the C3 gene encoding a p.Lys155Gln substitution in complement factor 3, which, following imputation into a set of Icelandic cases with age-related macular degeneration (AMD) and controls, associated with disease (odds ratio (OR) = 3.45; P = 1.1 × 10−7). This signal is independent of the previously reported common SNPs in C3 encoding p.Pro314Leu and p.Arg102Gly that associate with AMD. The association of p.Lys155Gln was replicated in AMD case-control samples of European ancestry with OR = 4.22 and P = 1.6 × 10−10, resulting in OR = 3.65 and P = 8.8 × 10−16 for all studies combined. In vitro studies have suggested that the p.Lys155Gln substitution reduces C3b binding to complement factor H, potentially creating resistance to inhibition by this factor. This resistance to inhibition in turn is predicted to result in enhanced complement activation.

Overexpression of HTRA1 Leads to Ultrastructural Changes in the Elastic Layer of Bruch's Membrane via Cleavage of Extracellular Matrix Components

Variants in the chromosomal region 10q26 are strongly associated with an increased risk for age-related macular degeneration (AMD). Two potential AMD genes are located in this region: ARMS2 and HTRA1 (high-temperature requirement A1). Previous studies have suggested that polymorphisms in the promotor region of HTRA1 result in overexpression of HTRA1 protein. This study investigated the role of HTRA1 overexpression in the pathogenesis of AMD. Transgenic Htra1 mice overexpressing the murine protein in the retinal pigment epithelium (RPE) layer of the retina were generated and characterized by transmission electron microscopy, immunofluorescence staining and Western Blot analysis. The elastic layer of Bruchs membrane (BM) in the Htra1 transgenic mice was fragmented and less continuous than in wild type (WT) controls. Recombinant HTRA1 lacking the N-terminal domain cleaved various extracellular matrix (ECM) proteins. Subsequent Western Blot analysis revealed an overexpression of fibronectin fragments and a reduction of fibulin 5 and tropoelastin in the RPE/choroid layer in transgenic mice compared to WT. Fibulin 5 is essential for elastogenesis by promoting elastic fiber assembly and maturation. Taken together, our data implicate that HTRA1 overexpression leads to an altered elastogenesis in BM through fibulin 5 cleavage. It highlights the importance of ECM related proteins in the development of AMD and links HTRA1 to other AMD risk genes such as fibulin 5, fibulin 6, ARMS2 and TIMP3.

Cumulative Effect of Risk Alleles in CFH, ARMS2, and VEGFA on the Response to Ranibizumab Treatment in Age-Related Macular Degeneration

PURPOSE Intravitreal ranibizumab injections currently are the standard treatment for neovascular age-related macular degeneration (AMD). However, a broad range of response rates have been observed, the reasons for which are poorly understood. This pharmacogenetic study evaluated the impact of high-risk alleles in CFH, ARMS2, VEGFA, vascular endothelial growth factor (VEGF) receptor KDR, and genes involved in angiogenesis (LRP5, FZD4) on the response to ranibizumab treatment and on the age of treatment onset. In contrast to previous studies, the data were stratified according to the number of high-risk alleles to enable the study of the combined effects of these genotypes on the treatment response. DESIGN Case series study. PARTICIPANTS A cohort of 420 eyes of 397 neovascular AMD patients. METHODS The change in visual acuity (VA) between baseline and after 3 ranibizumab injections was calculated. Genotyping of single nucleotide polymorphisms in the CFH, ARMS2, VEGFA, KDR, LPR5, and FZD4 genes was performed. Associations were assessed using linear mixed models. MAIN OUTCOME MEASURES The VA change after 3 ranibizumab injections and the age of neovascular disease onset. RESULTS After ranibizumab treatment, AMD patients without risk alleles in the CFH and ARMS2 genes (4.8%) demonstrated a mean VA improvement of 10 Early Treatment Diabetic Retinopathy Study (ETDRS) letters, whereas no VA improvement was observed in AMD patients with 4 CFH and ARMS2 risk alleles (6.9%; P = 0.014). Patients with 4 high-risk alleles in CFH and ARMS2 were 5.2 years younger than patients with 1 or 2 risk alleles, respectively (63.5%; P<0.0001). The mean age at which the first ranibizumab treatment was carried out among AMD patients with all 6 risk alleles in CFH, ARMS2, and VEGFA was 65.9 years (2%) versus 75.3 years in patients with 0 or 1 high-risk allele (8.8%; P = 0.001). After ranibizumab treatment, patients with 6 high-risk alleles demonstrated a mean VA loss of 10 ETDRS letters (P<0.0001). CONCLUSIONS This study evaluated the largest pharmacogenetic AMD cohort reported to date. A cumulative effect of high-risk alleles in CFH, ARMS2, and VEGFA seems to be associated with a younger age of onset in combination with poor response rates to ranibizumab treatment.

Visual outcome of patients with macular edema after pars plana vitrectomy and indocyanine green-assisted peeling of the internal limiting membrane

PurposeTo evaluate the efficacy of inner limiting membrane (ILM) peeling in persistent macular edema.MethodsThis retrospective review analyzed a series of 23 eyes from 23 patients with persistent macular edema treated by pars plana vitrectomy (PPV) with indocyanine green (ICG)-assisted peeling of the ILM. Thirteen female and 10 male patients with a mean age of 57.2±15.6 (24–77) years underwent operation between May 2000 and October 2001. The main diagnoses were uveitis (anterior, intermediate, posterior and panuveitis) (n=9), central retinal vein occlusion (CRVO) (n=4), diabetic retinopathy (DR) (n=5), vitreoretinal traction syndrome (n=2), and Irvine–Gass syndrome (n=3). Nine eyes had undergone phacoemulsification (PE) previously and two eyes had been subjected to combined PE and ILM peeling. The eyes were tamponaded with gas (3), silicone oil (5) or air (11). In four cases no endotamponade was used. Improvement in visual acuity of 2 lines or more was regarded as significant.ResultsVisual acuity improved after 3 months in 9 of the 23 patients. After 6 months and at the follow-up, a significant improvement was found in 6/21 and 7/21 patients. This improvement was predominantly seen in patients with uveitis (5/9), or diabetic maculopathy (3/5); One patient with Irvine–Gass syndrome showed a significant reduction, one with vitreoretinal traction an improvement in visual acuity. The group of patients with CRVO showed no significant change during the follow-up. The choice of endotamponade did not alter the visual acuity outcome.ConclusionsDifferent patient groups respond differently to ILM peeling. Although overall significant visual acuity improvement was observed in only one third of all cases 12 months after ILM peeling for persistent macular edema, patients with uveitis and nonproliferative diabetic maculopathy demonstrated a benefit. The lack of long-term improvement in the majority of cases is in accordance with the hypothesis that ILM peeling may reduce the intraretinal edema, but does not affect the underlying mechanism causing macular edema. So far, only diabetics have shown improvement (still unproven) from ILM peeling, and this study provides no justification for extending the treatment to macular edema of other causes. Large-scale investigations are needed to evaluate the efficacy in certain diagnosis groups.

Serum concentrations of vascular endothelial growth factor in an infant treated with ranibizumab for retinopathy of prematurity.

ganglion cells were first discovered 10 years ago, and many important aspects of their function remains unknown, not least the important question of how much or how little light is needed to maintain stable photoentrainment. Melanopsin-driven photoentrainment of circadian rhythms and pupillary response to short wavelength light are coupled, and looking at the pupillary light response to blue light is a clinically useful indirect measurement of the function of the melanopsin containing retinal ganglion cells. Melanopsin is a bi-stable molecule (Mure et al. 2009) that is reflected in the enhanced pupillary constriction to blue light after prior exposure to blue light (Hansen et al. 2011). Pupillary blue light response depends on pupil size and is greater after pupillary dilation (Nissen et al. 2011). Yet surprisingly, melanopsinmediated pupillary blue light response is enhanced with age in spite of age-related miosis and reduced lens transmission (Herbst et al. 2012). Speculatively, the system may adapt to decreased blue light levels with age by increasing the sensitivity. These observations demonstrate the complexity of the system and difficulties encountered when extrapolating from the filtering effects of the lens or IOLs to the photoentrainment potential of the aged or even pseudophakic eye. We felt there were too many unknown factors to compute a photoentrainment potential for the aged eye and hence restricted our computations to the effect of the lens. One aspect of the debate is whether blue-blocking IOLs are detrimental to photoentrainment of circadian rhythms or not. Equally important is the question of whether to use blue-blocking IOLs or not. The main rationale for using blueblocking IOLs is the theory that blue light is involved in the pathogenesis of age-related macular degeneration (AMD). As we write in our paper, the theory has not been scientifically proven and concluding that we argue that the ‘chromophores they consider to be optically insignificant to adversely affect circadian photoentrainment might somehow reduce the risk of AMD’ is a twisting of our argumentation. What we do write is that if someone were to conduct a study proving a protective effect of blue-blocking IOLs on AMD, the study would need to have a considerable time perspective. We do not yet have the scientific proof to say whether blue-blocking IOLS do or do not reduce the risk of AMD. Sleep disturbances increase with age, and a correlation with lens yellowing has been found (Kessel et al. 2011). Cataract surgery (with UV-blocking IOLs) leads to better sleep and well-being (Asplund & Lindblad 2004) indicating that short wavelength filtering by the lens is important for the photoentrainment of circadian rhythms in vivo. Our results showed that the difference of yellow-tinted and UV-only blocking IOLs is exceeded by several folds by the natural discoloration of the human lens. How this may affect photoentrainment is – of course – speculation and as Dr. Mainster and Dr. Turner states prospective, randomized clinical trials are needed to determine the clinical significance.

Sequence analysis of the complete mitochondrial genome in patients with Leber's hereditary optic neuropathy lacking the three most common pathogenic DNA mutations.

Lebers hereditary optic neuropathy (LHON) is a maternally inherited disorder characterized by central vision loss in young adults. The majority of LHON cases around the world are associated with mutations in the mitochondrial genome at nucleotide positions (np) 3460, 11,778, and 14,484. Usually, these three mutations are screened in suspected LHON patients. The result is important not only in respect to the diagnosis but also as different LHON mutations lead to variations in expression, severity, and recovery of the disease. There are, however, a significant number of patients without any of these primary mutations. In these situations, genetic counselling of a patient and his family can be difficult. We sequenced the complete mitochondrial DNA (mtDNA) in 14 LHON patients with the typical clinical features but without a primary mtDNA mutation to evaluate the potential of extensive mutation screening for clinical purposes. Our results suggest to include the mutation at np 15,257 in a routine screening as well as the ND6 gene, a hot spot for LHON mutations. Screening for the secondary LHON mutations at np 4216 and np 13,708 may also help in making the diagnosis of LHON as these seem to modify the expression of LHON mutations. Although they do not allow to prove the clinical diagnosis, their presence increases the probability of LHON. Sequencing the complete mitochondrial genome can reveal novel and known rare disease causing mutations. However, considering the effort it adds little value for routine screening.

Chronic Central Serous Chorioretinopathy Is Associated with Genetic Variants Implicated in Age-Related Macular Degeneration

PURPOSE In this study, single nucleotide polymorphisms (SNPs) at 19 loci, previously associated with age-related macular degeneration (AMD), were systematically tested for association in patients with chronic central serous chorioretinopathy (cCSC). In addition, we evaluated the effect of detailed phenotyping on these genetic associations. DESIGN Case-control study. PARTICIPANTS We included 292 cCSC patients, 1147 AMD patients, and 1311 control individuals. METHODS We genotyped SNPs at 19 AMD-associated loci and 6 additional SNPs at the complement factor H (CFH) locus. Phenotyping of all patients was based on fundoscopy, spectral-domain optical coherence tomography, fluorescein angiography (FA), and indocyanine green angiography. MAIN OUTCOME MEASURES We measured the allele frequencies of 25 AMD-associated SNPs and CFH haplotype frequencies in patients with cCSC and the effect of phenotypic subdivision of cCSC on genetic associations. RESULTS One SNP in ARMS2 (rs10490924) was significant after Bonferroni correction (Punadjusted=0.002; odds ratio [OR]=0.64). The SNPs at 3 other AMD loci (CFH, TNFRSF10A, ADAMTS9) showed a trend toward association with typical cCSC. Further analysis of the CFH locus identified 2 SNPs that significantly conferred increased risk for cCSC and 1 that was protective. The CFH-H3 haplotype was also found to be protective (P=0.01; OR=0.54). Using multimodal imaging, 197 patients were classified as having typical cCSC, 52 patients had unilateral abnormalities on FA that were otherwise typical of cCSC, and 43 patients had a clinical picture that could be compatible with cCSC, but with features that could also indicate other macular diseases. Significant differences of the minor allele frequencies of the tested SNPs were observed between these 3 phenotypic subgroups. CONCLUSIONS Chronic CSC is associated with genetic variants in ARMS2 and CFH, indicating a genetic and pathophysiologic overlap between cCSC and AMD. Intriguingly, alleles in ARMS2 and CFH that confer risk of AMD may be protective for cCSC, and alleles in CFH that are protective for AMD confer risk for cCSC. Significant differences in allele frequencies were found among the phenotypic subgroups for several SNPs, illustrating the importance of correct clinical classification.

Complement Factor D in Age-Related Macular Degeneration

PURPOSE To examine the role of complement factor D (CFD) in age-related macular degeneration (AMD) by analysis of genetic association, copy number variation, and plasma CFD concentrations. METHODS Single nucleotide polymorphisms (SNPs) in the CFD gene were genotyped and the results analyzed by binary logistic regression. CFD gene copy number was analyzed by gene copy number assay. Plasma CFD was measured by an enzyme-linked immunosorbent assay. RESULTS Genetic association was found between CFD gene SNP rs3826945 and AMD (odds ratio 1.44; P = 0.028) in a small discovery case-control series (462 cases and 325 controls) and replicated in a combined cohorts meta-analysis of 4765 cases and 2693 controls, with an odds ratio of 1.11 (P = 0.032), with the association almost confined to females. Copy number variation in the CFD gene was identified in 13 out of 640 samples examined but there was no difference in frequency between AMD cases (1.3%) and controls (2.7%). Plasma CFD concentration was measured in 751 AMD cases and 474 controls and found to be elevated in AMD cases (P = 0.00025). The odds ratio for those in the highest versus lowest quartile for plasma CFD was 1.81. The difference in plasma CFD was again almost confined to females. CONCLUSIONS CFD regulates activation of the alternative complement pathway, which is implicated in AMD pathogenesis. The authors found evidence for genetic association between a CFD gene SNP and AMD and a significant increase in plasma CFD concentration in AMD cases compared with controls, consistent with a role for CFD in AMD pathogenesis.