Mitochondrial absorption of short wavelength light drives primate blue retinal cones into glycolysis which may increase their pace of aging.

Photoreceptors have high energy demands and densely packed mitochondria through which light passes before phototransduction. Old world primates including humans have three cone photoreceptor types mediating color vision with short (S blue), medium (M green), and long (L red) wavelength sensitivities. However, S-cones are enigmatic. They comprise <10% of the total cone population, their responses saturate early, and they are susceptible in aging and disease. Here, we show that primate S-cones actually have few mitochondria and are fueled by glycolysis, not by mitochondrial respiration. Glycolysis has a limited ability to sustain activity, potentially explaining early S-cone saturation. Mitochondria act as optical filters showing reduced light transmission at 400-450 nm where S-cones are most sensitive (420 nm). This absorbance is likely to arise in a mitochondrial porphyrin that absorbs strongly in the Soret band. Hence, reducing mitochondria will improve S-cone sensitivity but result in increased glycolysis as an alternative energy source, potentially increasing diabetic vulnerability due to restricted glucose access. Further, glycolysis carries a price resulting in premature functional decline as seen in aged S-cones. Soret band absorption may also impact on mitochondrial rich M and L cones by reducing sensitivity at the lower end of their spectral sensitivity range resulting in increased differentiation from S-cone responses. These data add to the list of unique characteristic of S-cones and may also explain aspects of their vulnerability.

FUNDUS-WIDE SUBRETINAL AND PIGMENT EPITHELIAL ABNORMALITIES IN MACULAR TELANGIECTASIA TYPE 2.

PURPOSE: Macular telangiectasia Type 2 (MacTel) causes glial and photoreceptor cell death in a small, oval patch in the central retina. Beyond this oval area, no disease manifestations have been described so far. Here, we describe a novel pathological aspect of MacTel in the retinal pigment epithelium (RPE) that is not restricted to the clinically affected area but covers the entire retina. METHODS: We have studied postmortem eyes from four patients with MacTel by immunohistochemistry and electron microscopy. RESULTS: We found cellular debris in the subretinal space (between photoreceptor outer segments and RPE), consisting mainly of outer segments and RPE components. In healthy eyes, the RPE normally phagocytoses the tips of the continuously growing outer segments, a process considered to be essential for photoreceptor survival. However, in the patients with MacTel, we found no evidence of ongoing outer segment phagocytosis, and the apical surface of the RPE appeared abnormal throughout most of the retina. CONCLUSION: Reduced outer segment phagocytosis may explain the accumulating debris in the subretinal space but is a surprising finding because visual function in the peripheral retina is normal in patients with MacTel. Nevertheless, the subclinical pathology might induce a specific stress to which the central area is uniquely susceptible.

Stem cells in retinal regeneration: past, present and future.

Stem cell therapy for retinal disease is under way, and several clinical trials are currently recruiting. These trials use human embryonic, foetal and umbilical cord tissue-derived stem cells and bone marrow-derived stem cells to treat visual disorders such as age-related macular degeneration, Stargardt's disease and retinitis pigmentosa. Over a decade of analysing the developmental cues involved in retinal generation and stem cell biology, coupled with extensive surgical research, have yielded differing cellular approaches to tackle these retinopathies. Here, we review these various stem cell-based approaches for treating retinal diseases and discuss future directions and challenges for the field.

Von Hippel-Lindau protein in the RPE is essential for normal ocular growth and vascular development.

Molecular oxygen is essential for the development, growth and survival of multicellular organisms. Hypoxic microenvironments and oxygen gradients are generated physiologically during embryogenesis and organogenesis. In the eye, oxygen plays a crucial role in both physiological vascular development and common blinding diseases. The retinal pigment epithelium (RPE) is a monolayer of cells essential for normal ocular development and in the mature retina provides support for overlying photoreceptors and their vascular supply. Hypoxia at the level of the RPE is closely implicated in pathogenesis of age-related macular degeneration. Adaptive tissue responses to hypoxia are orchestrated by sophisticated oxygen sensing mechanisms. In particular, the von Hippel-Lindau tumour suppressor protein (pVhl) controls hypoxia-inducible transcription factor (HIF)-mediated adaptation. However, the role of Vhl/Hif1a in the RPE in the development of the eye and its vasculature is unknown. In this study we explored the function of Vhl and Hif1a in the developing RPE using a tissue-specific conditional-knockout approach. We found that deletion of Vhl in the RPE results in RPE apoptosis, aniridia and microphthalmia. Increased levels of Hif1a, Hif2a, Epo and Vegf are associated with a highly disorganised retinal vasculature, chorioretinal anastomoses and the persistence of embryonic vascular structures into adulthood. Additional inactivation of Hif1a in the RPE rescues the RPE morphology, aniridia, microphthalmia and anterior vasoproliferation, but does not rescue retinal vasoproliferation. These data demonstrate that Vhl-dependent regulation of Hif1a in the RPE is essential for normal RPE and iris development, ocular growth and vascular development in the anterior chamber, whereas Vhl-dependent regulation of other downstream pathways is crucial for normal development and maintenance of the retinal vasculature.

Quantification of vascular tortuosity as an early outcome measure in oxygen induced retinopathy (OIR).

Oxygen-induced retinopathy (OIR) in mice is a popular model system to study pathological angiogenesis in the retinal vasculature. The system is based on vessel depletion by exposure to hyperoxia, which results in acute retinal hypoxia upon return to room air. This hypoxia then triggers neovascularization in the remaining vessels after 5 days. Here we aimed to establish an additional and earlier experimental readout of the vascular response to hypoxia by quantifying the tortuosity of retinal arteries after 2 days. Mouse pups from three different mouse strains were exposed to hyperoxia from postnatal day (P) 7 to P12 and retinas were analysed at P12, P14 and P17. Hypoxia was assessed by staining with the hypoxia marker EF5 and by measuring Vegf mRNA by qPCR. The retinal vasculature was stained in whole mount retinas and tortuosity of radial arterioles was quantified. C57BL/6J mice were used because the vascular response at P17 is well characterised in this strain. We also used C3H/HeJ mice, which contain the retinal degeneration 1 (Rd1) mutation (Pde6b(Rd1)) and have abnormally thin retinas. These thinner, C3H/HeJ retinas do not become ischemic during the OIR model and do not develop neovascularization. They can therefore be used as a control. In addition, we included C3H/HeJ mice that lack the Rd1 mutation (C3H/He(Rd1-)), with normal thickness retinas, to control for strain differences between C57BL/6J and C3H/HeJ. Quantification of vessel tortuosity at P14 showed tortuous arteries in normal thickness retinas (C57BL/6J and C3H/He(Rd1-)) and straight arteries in the thin C3H/HeJ retinas. This correlated with hypoxia, which was severe in normal thickness retinas and mild in the thin C3H/HeJ retinas. Furthermore, at P17 the normal thickness retinas showed strong neovascularisation whereas in the thin C3H/HeJ retinas the retinal vasculature regenerated normally. In conclusion we have demonstrated that arterial tortuosity can act as an early readout for hypoxia in the OIR model before neovascularisation develops.

Light stimulation of mitochondria reduces blood glucose levels.

Mitochondria regulate metabolism, but solar light influences its rate. Photobiomodulation (PBM) with red light (670 nm) increases mitochondrial membrane potentials and adenosine triphosphate production and may increase glucose demand. Here we show, with a glucose tolerance test, that PBM of normal subjects significantly reduces blood sugar levels. A 15 min exposure to 670 nm light reduced the degree of blood glucose elevation following glucose intake by 27.7%, integrated over 2 h after the glucose challenge. Maximum glucose spiking was reduced by 7.5%. Consequently, PBM with 670 nm light can be used to reduce blood glucose spikes following meals. This intervention may reduce damaging fluctuations of blood glucose on the body.

Making Sense of Electrical Stimulation: A Meta-analysis for Wound Healing.

Electrical stimulation as a mode of external enhancement factor in wound healing has been explored widely. It has proven to have multidimensional effects in wound healing including antibacterial, galvanotaxis, growth factor secretion, proliferation, transdifferentiation, angiogenesis, etc. Despite such vast exploration, this modality has not yet been established as an accepted method for treatment. This article reviews and analyzes the approaches of using electrical stimulation to modulate wound healing and discusses the incoherence in approaches towards reporting the effect of stimulation on the healing process. The analysis starts by discussing various processes adapted in in vitro, in vivo, and clinical practices. Later it is focused on in vitro approaches directed to various stages of wound healing. Based on the analysis, a protocol is put forward for reporting in vitro works in such a way that the outcomes of the experiment are replicable and scalable in other setups. This work proposes a ground of unification for all the in vitro approaches in a more sensible manner, which can be further explored for translating in vitro approaches to complex tissue stimulation to establish electrical stimulation as a controlled clinical method for modulating wound healing.

Loss of Müller's cells and photoreceptors in macular telangiectasia type 2.

PURPOSE: To correlate postmortem histology from a patient with macular telangiectasia (MacTel) type 2 with previously recorded clinical imaging data. DESIGN: Observational clinicopathologic case report. METHODS: The distribution of retinal blood vessels was used to map the location of serial wax sections in color fundus and optical coherence tomography (OCT) images. Fluorescent immunohistochemistry was used to visualize markers for Müller's cells (vimentin and retinaldehyde-binding protein 1), photoreceptors (L-M opsin, rhodopsin, and cytochrome oxidase 2), and the outer limiting membrane (OLM) (zonula occludens 1 and occludin). MAIN OUTCOME MEASURES: Distribution of specific markers in immunohistochemistry on retinal sections through the fovea in relation to clinical data. RESULTS: The clinically recorded region of macular pigment loss in the macula correlated well with Müller's cell depletion. The OCT data showed a loss of the photoreceptor inner segment/outer segment (IS/OS) junction in the central retina, which correlated well with rod loss but not with cone loss. Markers for the OLM were lost where Müller's cells were lost. CONCLUSIONS: We have confirmed our previous finding of Müller's cell loss in MacTel type 2 and have shown that the area of Müller's cell loss matches the area of macular pigment depletion. In this patient, the IS/OS junction seen by OCT was absent in a region where rods were depleted but cones were still present.

Pilot application of iTRAQ to the retinal disease Macular Telangiectasia.

We used the comparative proteomic technique iTRAQ coupled with offline 2DLC-MS/MS to analyze a rare specimen of the poorly understood, potentially blinding ophthalmic condition Macular Telangiectasia type 2 (MacTel type 2). We refined the technique using an internal standard consisting of pooled samples for each iTRAQ experiment to allow for multiple comparisons between different regions of the retina and different tissue donors. A total of 594 nonredundant proteins were identified in the retina and 168 in the vitreous, of which approximately half were found in significantly different abundance in the various comparisons made. The most prominent differences were found within the glycolytic pathway, where 8 proteins were reduced in the diseased macula compared with peripheral retina of the same eye, and 10 were also reduced in comparison with the macula of a control eye. Furthermore, Müller cell-associated proteins, including GFAP, VIME, and GLNA, were also reduced in the diseased macula, consistent with a link between the glycolytic pathway and Müller cells. These changes were validated by Western blotting and immunohistochemical studies. Proteomic analysis of the vitreous revealed an increase of proteins that were reduced in the retina. This supports proteomic analysis of the more easily available vitreous, which may reveal retina-specific protein changes associated with disease. Furthermore, our study has highlighted changes in the glycolytic pathway as a possible component of MacTel type 2 pathobiology.

Systemic glucose levels are modulated by specific wavelengths in the solar light spectrum that shift mitochondrial metabolism.

Systemic glucose levels can be modulated with specific solar wavelengths that influence mitochondrial metabolism. Mitochondrial respiration can be modulated using light that shifts ATP production with exceptional conservation of effect across species, from insects to humans. Known wavelengths have opposing effects of photobiomodulation, with longer wavelengths (660-900 nm red/infrared) increasing ATP production, and 420 nm (blue) light suppressing metabolism. Increasing mitochondrial respiration should result in a greater demand for glucose, and a decrease should result in a reduced demand for glucose. Here we have tested the hypothesis that these wavelengths alter circulating glucose concentration. We first established an oral glucose tolerance test curve in a bumblebee model, which showed sustained increase in systemic glucose beyond that seen in mammals, with a gradual normalisation over eight hours. This extended period of increased systemic glucose provided a stable model for glucose manipulation. Bees were starved overnight and given a glucose load in the morning. In the first group glucose levels were examined at hourly intervals. In the second group, bees were additionally exposed to either 670 nm or 420 nm light and their blood glucose examined. Increasing mitochondrial activity with 670 nm light at the peak of circulating glucose, resulted in a significant 50% reduction in concentration measured. Exposure to 420nm light that retards mitochondrial respiration elevated systemic glucose levels by over 50%. The impact of 670 nm and 420 nm on mitochondria is highly conserved. Hence, different wavelengths of visible light may be used to modulate systemic metabolism bidirectionally and may prove an effective agent in mammals.

The 3D organisation of mitochondria in primate photoreceptors.

Vertebrate photoreceptors contain large numbers of closely-packed mitochondria which sustain the high metabolic demands of these cells. These mitochondria populations are dynamic and undergo fusion and fission events. This activity serves to maintain the population in a healthy state. In the event of mitochondrial damage, sub-domains, or indeed whole mitochondria, can be degraded and population homeostasis achieved. If this process is overwhelmed cell death may result. Death of photoreceptors contributes to loss of vision in aging individuals and is associated with many eye diseases. In this study we used serial block face scanning electron microscopy of adult Macaca fascicularis retinae to examine the 3D structure of mitochondria in rod and cone photoreceptors. We show that healthy-looking photoreceptors contain mitochondria exhibiting a range of shapes which are associated with different regions of the cell. In some photoreceptors we observe mitochondrial swelling and other changes often associated with cellular stress. In rods and cones that appear stressed we identify elongated domains of mitochondria with densely-packed normal cristae associated with photoreceptor ciliary rootlet bundles. We observe mitochondrial fission and mitochondrion fragments localised to these domains. Swollen mitochondria with few intact cristae are located towards the periphery of the photoreceptor inner-segment in rods, whilst they are found throughout the cell in cones. Swollen mitochondria exhibit sites on the mitochondrial inner membrane which have undergone complex invagination resulting in membranous, electron-dense aggregates. Membrane contact occurs between the mitochondrion and the photoreceptor plasma membrane in the vicinity of these aggregates, and a series of subsequent membrane fusions results in expulsion of the mitochondrial aggregate from the photoreceptor. These events are primarily associated with rods. The potential fate of this purged material and consequences of its clearance by retinal pigment epithelia are discussed.

Perifoveal müller cell depletion in a case of macular telangiectasia type 2.

PURPOSE: To assess the histopathologic changes in a postmortem sample derived from an eye donor with macular telangiectasia (MacTel) type 2 to gain further insight into the cause of the disease. DESIGN: Clinicopathological case report. PARTICIPANTS: Postmortem tissue was collected from 5 different donors: 1 MacTel type 2 patient; 1 healthy control; 2 type 2 diabetic patients, 1 with retinopathy and 1 without retinopathy; and 1 patient with unilateral Coat's disease. METHODS: Macular pigment distribution in the posterior part of freshly dissected eyes was documented by macrophotography. Paraffin sections from both the macular and peripheral regions were assessed using antigen retrieval and immunohistochemistry to study the distribution of cell-specific markers. Blood vessels were visualized with antibodies directed against collagen IV and claudin 5; glial cells with antibodies against glial fibrillary acidic protein (GFAP), vimentin, glutamine synthetase (GS), and retinaldehyde binding protein (RLBP1, also known as CRALBP); microglia with an antibody against allograft inflammatory factor 1 (also known as Iba1); and photoreceptors with antibodies against rhodopsin and opsin. Using anatomic landmarks, the sections then were matched with the macular pigment distribution and a fluorescein angiogram of the patient that was obtained before the patient's death. MAIN OUTCOME MEASURES: Presence and distribution of macular pigment and cell-specific markers. RESULTS: Macular pigment was absent in the macula. Furthermore, abnormally dilated capillaries were identified in a macular region that correlated spatially with regions of fluorescein leakage in an angiogram that was obtained 12 years before death. These telangiectatic vessels displayed a marked reduction of the basement membrane component collagen IV, indicating vascular pathologic features. The presence of GFAP was limited to retinal astrocytes, and no reactive Müller cells were identified. Importantly, reduced immunoreactivity with Müller cell markers (vimentin, GS, and RLBP1) in the macula was observed. The area that lacked Müller cells corresponded with the region of depleted macular pigment. CONCLUSIONS: These findings suggest that macular Müller cell loss or dysfunction is a critical component of MacTel type 2, which may have implications for future treatment strategies.

Fundamental differences in patterns of retinal ageing between primates and mice.

Photoreceptors have high metabolic demands and age rapidly, undermining visual function. We base our understanding mainly on ageing mice where elevated inflammation, extracellular deposition, including that of amyloid beta, and rod and cone photoreceptor loss occur, but cones are not lost in ageing primate although their function declines, revealing that primate and mouse age differently. We examine ageing primate retinae and show elevated stress but low inflammation. However, aged primates have a >70% reduction in adenosine triphosphate (ATP) and a decrease in cytochrome c oxidase. There is a shift in cone mitochondrial positioning and glycolytic activity increases. Bruch's membrane thickens but unlike in mice, amyloid beta is absent. Hence, reduced ATP may explain cone functional decline in ageing but their retained presence offers the possibility of functional restoration if they can be fuelled appropriately to restore cellular function. This is important because as humans we largely depend on cone function to see and are rarely fully dark adapted. Presence of limited aged inflammation and amyloid beta deposition question some of the therapeutic approaches taken to resolve problems of retinal ageing in humans and the possible lack of success in clinical trials in macular degeneration that have targeted inflammatory agents.

Assessment of the Complex Refractive Indices of Xenopus Laevis Sciatic Nerve for the Optimization of Optical (NIR) Neurostimulation.

Despite an increasing interest in the use of light for neural stimulation, there is little information on how it interacts with neural tissue. The choice of wavelength in most of the optical stimulation literature is based on already available light sources designed for other applications. This paper is the first one to report the complex refractive index of the sciatic nerve of Xenopus laevis, which is a crucial parameter for identifying the optimal wavelength of optical stimuli. The Xenopus laevis neural tissue is the most widely used tissue type in peripheral neurostimulation studies. In this paper, the reflectance ( ) and the transmittance ( ) of the sciatic nerve were measured over a wavelength range of 860-2250 nm, and the corresponding real ( ) and the imaginary ( ) refractive indices were calculated using appropriate formulae in a novel way. The reported values were between 1.3-1.44 and the values are of the order of over the full wavelength range. The absorption coefficient was found to be 100-500 cm . Several localized wavelength ranges were identified that can offer a maximized power coupling between potential optical stimuli and the neural tissue (1150-1200 nm, 1500-1700 nm, and 1900-2050 nm). The narrower regions of 1400-1600 nm and 1850-2150 nm were found to exhibit maximized absorbance. Separately, three regions were identified, where the penetration depths are the greatest (950-1000 nm, 1050-1350 nm, and 1600-1900 nm). This paper provides, for the first time, the fundamental specifications for optimizing the parameters of optical neurostimulation systems.

Neuropilin 1 Involvement in Choroidal and Retinal Neovascularisation.

PURPOSE: Inhibiting VEGF is the gold standard treatment for neovascular age-related macular degeneration (AMD). It is also effective in preventing retinal oedema and neovascularisation (NV) in diabetic retinopathy (DR) and retinal vein occlusions (RVO). Neuropilin 1 (Nrp1) is a co-receptor for VEGF and many other growth factors, and therefore a possible alternative drug target in intra ocular neovascular disease. Here we assessed choroidal and retinal NV in an inducible, endothelial specific knock out model for Nrp1. METHODS: Crossing Nrp1 floxed mice with Pdgfb-CreERT2 mice produced tamoxifen-inducible, endothelial specific Nrp1 knock out mice (Nrp1ΔEC) and Cre-negative, control littermates. Cre-recombinase activity was confirmed in the Ai3(RCL-EYFP) reporter strain. Animals were subjected to laser-induced CNV (532 nm) and spectral domain-optical coherence tomography (SD-OCT) was performed immediately after laser and at day 7. Fluorescein angiography (FA) evaluated leakage and postmortem lectin staining in flat mounted RPE/choroid complexes was also used to measure CNV. Furthermore, retinal neovascularisation in the oxygen induced retinopathy (OIR) model was assessed by immunohistochemistry in retinal flatmounts. RESULTS: In vivo FA, OCT and post-mortem lectin staining showed a statistically significant reduction in leakage (p<0.05), CNV volume (p<0.05) and CNV area (p<0.05) in the Nrp1ΔEC mice compared to their Cre-negative littermates. Also the OIR model showed reduced retinal NV in the mutant animals compared to wild types (p<0.001). CONCLUSION: We have demonstrated reduced choroidal and retinal NV in animals that lack endothelial Nrp1, confirming a role of Nrp1 in those processes. Therefore, Nrp1 may be a promising drug target for neovascular diseases in the eye.

No evidence for loss of short-wavelength sensitive cone photoreceptors in normal ageing of the primate retina.

In old world primates including humans, cone photoreceptors are classified according to their maximal sensitivity at either short (S, blue), middle (M, green) or long (L, red) wavelengths. Colour discrimination studies show that the S-cone pathway is selectively affected by age and disease, and psychophysical models implicate their loss. Photoreceptors have high metabolic demand and are susceptible to age or disease-related losses in oxygen and nutrient supply. Hence 30% of rods are lost over life. While comparable losses are not seen in cones, S-cones comprise less than 10% of the cone population, so significant loss would be undetected in total counts. Here we examine young and aged primate retinae stained to distinguish S from M/L-cones. We show there is no age-related cone loss in either cone type and that S-cones are as regularly distributed in old as young primates. We propose that S-cone metabolism is less flexible than in their M/L counterparts, making them more susceptible to deficits in normal cellular function. Hypoxia is a feature of the ageing retina as extracellular debris accumulates between photoreceptors and their blood supply which likely impacts S-cone function. However, that these cells remain in the ageing retina suggests the potential for functional restoration.

Rescue of the MERTK phagocytic defect in a human iPSC disease model using translational read-through inducing drugs.

Inherited retinal dystrophies are an important cause of blindness, for which currently there are no effective treatments. In order to study this heterogeneous group of diseases, adequate disease models are required in order to better understand pathology and to test potential therapies. Induced pluripotent stem cells offer a new way to recapitulate patient specific diseases in vitro, providing an almost limitless amount of material to study. We used fibroblast-derived induced pluripotent stem cells to generate retinal pigment epithelium (RPE) from an individual suffering from retinitis pigmentosa associated with biallelic variants in MERTK. MERTK has an essential role in phagocytosis, one of the major functions of the RPE. The MERTK deficiency in this individual results from a nonsense variant and so the MERTK-RPE cells were subsequently treated with two translational readthrough inducing drugs (G418 & PTC124) to investigate potential restoration of expression of the affected gene and production of a full-length protein. The data show that PTC124 was able to reinstate phagocytosis of labeled photoreceptor outer segments at a reduced, but significant level. These findings represent a confirmation of the usefulness of iPSC derived disease specific models in investigating the pathogenesis and screening potential treatments for these rare blinding disorders.

Improving Mitochondrial Function Protects Bumblebees from Neonicotinoid Pesticides.

Global pollination is threatened by declining insect pollinator populations that may be linked to neonicotinoid pesticide use. Neonicotinoids over stimulate neurons and depolarize their mitochondria, producing immobility and death. However, mitochondrial function can be improved by near infrared light absorbed by cytochrome c oxidase in mitochondrial respiration. In flies, daily exposure to 670nm light throughout life increases average lifespan and aged mobility, and reduces systemic inflammation. Here we treat bumble bees with Imidacloprid a common neonicotinoid. This undermined ATP and rapidly induced immobility and reduced visual function and survival. Bees exposed to insecticide and daily to 670nm light showed corrected ATP levels and significantly improved mobility allowing them to feed. Physiological recordings from eyes revealed that light exposure corrected deficits induced by the pesticide. Overall, death rates in bees exposed to insecticide but also given 670nm light were indistinguishable from controls. When Imidacloprid and light exposure were withdrawn, survival was maintained. Bees and insects generally cannot see deep red light so it does not disturb their behaviour. Hence, we show that deep red light exposure that improves mitochondrial function, reverses the sensory and motor deficits induced by Imidacloprid. These results may have important implications as light delivery is economic and can be placed in hives/colonies.

Pleiotropic action of CpG-ODN on endothelium and macrophages attenuates angiogenesis through distinct pathways.

There is an integral relationship between vascular cells and leukocytes in supporting healthy tissue homeostasis. Furthermore, activation of these two cellular components is key for tissue repair following injury. Toll-like receptors (TLRs) play a role in innate immunity defending the organism against infection, but their contribution to angiogenesis remains unclear. Here we used synthetic TLR9 agonists, cytosine-phosphate-guanosine oligodeoxynucleotides (CpG-ODN), to investigate the role of TLR9 in vascular pathophysiology and identify potential therapeutic translation. We demonstrate that CpG-ODN stimulates inflammation yet inhibits angiogenesis. Regulation of angiogenesis by CpG-ODN is pervasive and tissue non-specific. Further, we noted that synthetic CpG-ODN requires backbone phosphorothioate but not TLR9 activation to render and maintain endothelial stalk cells quiescent. CpG-ODN pre-treated endothelial cells enhance macrophage migration but restrain pericyte mobilisation. CpG-ODN attenuation of angiogenesis, however, remains TLR9-dependent, as inhibition is lost in TLR9 deficient mice. Additionally, CpG-ODNs induce an M1 macrophage phenotype that restricts angiogenesis. The effects mediated by CpG-ODNs can therefore modulate both endothelial cells and macrophages through distinct pathways, providing potential therapeutic application in ocular vascular disease.

Evaluation of Nonperfused Retinal Vessels in Ischemic Retinopathy.

PURPOSE: Retinal ischemia has been traditionally assessed by fluorescein angiography, visualizing perfused vessels. However, this method does not provide any information about nonperfused vessels, and although it is often assumed that vessels in ischemic areas regress, we know little about how nonperfused retinal vessels change over time. Here, we aim to learn more about the long-term fate of nonperfused vessels in the retinal vasculature. METHODS: Optical coherence tomography (OCT) was used to visualize perfusion as well as structural properties of the retinal vasculature in patients suffering from retinal vascular occlusions. In addition, postmortem tissue from a patient with long standing (6 years) central retinal vein occlusion (CRVO) was investigated, using immunohistochemistry on whole-mount retina and paraffin sections to visualize blood vessel components. RESULTS: Comparing OCT angiography with enface OCT images revealed that in ischemic areas of the retina, nonperfused, larger vessels could be detected as hyperreflective structures in enface OCT images. Furthermore, analysis of a postmortem tissue sample from a CRVO patient with a large nonperfused region in the macula, revealed preservation of the basement membrane from all retinal vessels, including nonperfused, acellular vessels of all calibers. CONCLUSIONS: Our data suggests long-term preservation of vascular basement membrane in ischemic retina. This has implications for therapeutic approaches aiming to alleviate retinal ischemia via the regeneration of damaged vessels.