Bertan Cakir

Consensus guidelines for the use and interpretation of angiogenesis assays

AbstractThe formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

Sema3f Protects Against Subretinal Neovascularization In Vivo

Pathological neovascularization of the outer retina is the hallmark of neovascular age-related macular degeneration (nAMD). Building on our previous observations that semaphorin 3F (Sema3f) is expressed in the outer retina and demonstrates anti-angiogenic potential, we have investigated whether Sema3f can be used to protect against subretinal neovascularization in two mouse models. Both in the very low-density lipid-receptor knockout (Vldlr−/−) model of spontaneous subretinal neovascularization as well as in the mouse model of laser-induced choroidal neovascularization (CNV), we found protective effects of Sema3f against the formation of pathologic neovascularization. In the Vldlr−/− model, AAV-induced overexpression of Sema3f reduced the size of pathologic neovascularization by 56%. In the laser-induced CNV model, intravitreally injected Sema3f reduced pathologic neovascularization by 30%. Combined, these results provide the first evidence from two distinct in vivo models for a use of Sema3f in protecting the outer retina against subretinal neovascularization.

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

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

A machine learning approach for automated assessment of retinal vasculature in the oxygen induced retinopathy model

Preclinical studies of vascular retinal diseases rely on the assessment of developmental dystrophies in the oxygen induced retinopathy rodent model. The quantification of vessel tufts and avascular regions is typically computed manually from flat mounted retinas imaged using fluorescent probes that highlight the vascular network. Such manual measurements are time-consuming and hampered by user variability and bias, thus a rapid and objective method is needed. Here, we introduce a machine learning approach to segment and characterize vascular tufts, delineate the whole vasculature network, and identify and analyze avascular regions. Our quantitative retinal vascular assessment (QuRVA) technique uses a simple machine learning method and morphological analysis to provide reliable computations of vascular density and pathological vascular tuft regions, devoid of user intervention within seconds. We demonstrate the high degree of error and variability of manual segmentations, and designed, coded, and implemented a set of algorithms to perform this task in a fully automated manner. We benchmark and validate the results of our analysis pipeline using the consensus of several manually curated segmentations using commonly used computer tools. The source code of our implementation is released under version 3 of the GNU General Public License (https://www.mathworks.com/matlabcentral/fileexchange/65699-javimazzaf-qurva).

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

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

Fibroblast Growth Factor 21 Protects Photoreceptor Function in Type 1 Diabetic Mice

Retinal neuronal abnormalities occur before vascular changes in diabetic retinopathy. Accumulating experimental evidence suggests that neurons control vascular pathology in diabetic and other neovascular retinal diseases. Therefore, normalizing neuronal activity in diabetes may prevent vascular pathology. We investigated whether fibroblast growth factor 21 (FGF21) prevented retinal neuronal dysfunction in insulin-deficient diabetic mice. We found that in diabetic neural retina, photoreceptor rather than inner retinal function was most affected and administration of the long-acting FGF21 analog PF-05231023 restored the retinal neuronal functional deficits detected by electroretinography. PF-05231023 administration protected against diabetes-induced disorganization of photoreceptor segments seen in retinal cross section with immunohistochemistry and attenuated the reduction in the thickness of photoreceptor segments measured by optical coherence tomography. PF-05231023, independent of its downstream metabolic modulator adiponectin, reduced inflammatory marker interleukin-1β (IL-1β) mRNA levels. PF-05231023 activated the AKT-nuclear factor erythroid 2–related factor 2 pathway and reduced IL-1β expression in stressed photoreceptors. PF-05231023 administration did not change retinal expression of vascular endothelial growth factor A, suggesting a novel therapeutic approach for the prevention of early diabetic retinopathy by protecting photoreceptor function in diabetes.

Adiponectin Mediates Dietary Omega-3 Long-Chain Polyunsaturated Fatty Acid Protection Against Choroidal Neovascularization in Mice

Purpose Neovascular age-related macular degeneration (AMD) is a major cause of legal blindness in the elderly. Diets with omega3-long-chain-polyunsaturated-fatty-acid (ω3-LCPUFA) correlate with a decreased risk of AMD. Dietary ω3-LCPUFA versus ω6-LCPUFA inhibits mouse ocular neovascularization, but the underlying mechanism needs further exploration. The aim of this study was to investigate if adiponectin (APN) mediated ω3-LCPUFA suppression of neovessels in AMD. Methods The mouse laser-induced choroidal neovascularization (CNV) model was used to mimic some of the inflammatory aspect of AMD. CNV was compared between wild-type (WT) and Apn−/− mice fed either otherwise matched diets with 2% ω3 or 2% ω6-LCPUFAs. Vldlr−/− mice were used to mimic some of the metabolic aspects of AMD. Choroid assay ex vivo and human retinal microvascular endothelial cell (HRMEC) proliferation assay in vitro was used to investigate the APN pathway in angiogenesis. Western blot for p-AMPKα/AMPKα and qPCR for Apn, Mmps, and IL-10 were used to define mechanism. Results ω3-LCPUFA intake suppressed laser-induced CNV in WT mice; suppression was abolished with APN deficiency. ω3-LCPUFA, mediated by APN, decreased mouse Mmps expression. APN deficiency decreased AMPKα phosphorylation in vivo and exacerbated choroid-sprouting ex vivo. APN pathway activation inhibited HRMEC proliferation and decreased Mmps. In Vldlr−/− mice, ω3-LCPUFA increased retinal AdipoR1 and inhibited NV. ω3-LCPUFA decreased IL-10 but did not affect Mmps in Vldlr−/− retinas. Conclusions APN in part mediated ω3-LCPUFA inhibition of neovascularization in two mouse models of AMD. Modulating the APN pathway in conjunction with a ω3-LCPUFA-enriched-diet may augment the beneficial effects of ω3-LCPUFA in AMD patients.

Thrombocytopenia is associated with severe retinopathy of prematurity

Retinopathy of prematurity (ROP) is characterized by abnormal retinal neovascularization in response to vessel loss. Platelets regulate angiogenesis and may influence ROP progression. In preterm infants, we assessed ROP and correlated with longitudinal postnatal platelet counts (n = 202). Any episode of thrombocytopenia (<100 × 109/l) at ≥30 weeks postmenstrual age (at onset of ROP) was independently associated with severe ROP, requiring treatment. Infants with severe ROP also had a lower weekly median platelet count compared with infants with less severe ROP. In a mouse oxygen-induced retinopathy model of ROP, platelet counts were lower at P17 (peak neovascularization) versus controls. Platelet transfusions at P15 and P16 suppressed neovascularization, and platelet depletion increased neovascularization. Platelet transfusion decreased retinal of vascular endothelial growth factor A (VEGFA) mRNA and protein expression; platelet depletion increased retinal VEGFA mRNA and protein expression. Resting platelets with intact granules reduced neovascularization, while thrombin-activated degranulated platelets did not. These data suggest that platelet releasate has a local antiangiogenic effect on endothelial cells to exert a downstream suppression of VEGFA in neural retina. Low platelet counts during the neovascularization phase in ROP is significantly associated with the development of severe ROP in preterm infants. In a murine model of retinopathy, platelet transfusion during the period of neovascularization suppressed retinopathy.