Monica Aronsson

Hypoxia-Inducible Factor-1α Is Associated With Sprouting Angiogenesis in the Murine Laser-Induced Choroidal Neovascularization Model.

PURPOSE To investigate the expression and distribution of neoangiogenic molecules and the role of hypoxia during the development of experimental choroidal neovascularization (CNV). METHODS Lesions were induced on C57Bl6 mice using laser photocoagulation. Animals were euthanized in a timely manner and eyecups were dissected from enucleated eyes. Choroids were immunostained for pericytes, sprouting endothelial cells (EC), or vascular EC. Choroidal neovascularization lesions where analyzed for tissue hypoxia, hypoxia-inducible factors (HIF), and heat-shock proteins (HSP). RESULTS Choroidal neovascularization lesions showed a trend of increased cellular recruitment throughout the time-course and the lesions displayed positive staining for angiogenic markers. Both pericytes and sprouting EC displayed a radial progression, while vascular EC displayed a more uniform distribution across the CNV lesions. Furthermore, positive tissue hypoxia staining was observed and associated with expression of HIF-1α and vascular endothelial growth factor (VEGF). CONCLUSIONS Our data delimitate specific temporal windows during CNV initiation, propagation, maturation, and even recovery in experimental CNV. We show that murine CNV undergoes hypoxia-associated sprouting angiogenesis, and demonstrate involvement of pericytes. Moreover, we have shown expression of HIF-1α to the retinal pigment epithelium surrounding the CNV lesions, together with VEGF upregulation, independently of the HSP response induced by the laser thermal insult.

The Urokinase Receptor-Derived Peptide UPARANT Mitigates Angiogenesis in a Mouse Model of Laser-Induced Choroidal Neovascularization

PURPOSE A mouse model of age-related macular degeneration (AMD) was used to investigate the anti-angiogenic and anti-inflammatory role of UPARANT in laser-induced choroidal neovascularization (CNV). METHODS Choroidal neovascularization was induced by laser photocoagulation, and UPARANT was intravitreally injected. Some experiments were also performed after either intravitreal injection of anti-VEGF drugs or systemic administration of UPARANT. Immunohistochemistry using CD31 antibodies was used to evaluate the area of CNV. Evans blue dye extravasation was quantitatively assessed. Transcripts of markers of outer blood retinal barrier were measured by quantitative RT-PCR, also used to evaluate angiogenesis and inflammation markers. Western blot was used to determine levels of transcription factors encoding genes involved in angiogenesis and inflammation. Levels of urokinase-type plasminogen activator (uPA), its receptor (uPAR), and formyl peptide receptors (FPRs) were determined at the transcript and the protein level. RESULTS Intravitreal UPARANT reduced the CNV area and the leakage from the choroid. The uPA/uPAR/FPR system was upregulated in CNV, but was not influenced by UPARANT. UPARANT recovered laser-induced upregulation of transcription factors encoding angiogenic and inflammatory markers. Accordingly, angiogenic and inflammatory factors were also reduced. UPARANT as compared to anti-VEGF drugs displayed similar effects on CNV area. CONCLUSIONS UPARANT mitigates laser-induced CNV by inhibiting angiogenesis and inflammation through an action on transcription factors encoding angiogenesis and inflammatory genes. The finding that UPARANT is effective against CNV may help to establish uPAR and its membrane partners as putative targets in the treatment of AMD.

In Vivo Imaging of Subretinal Bleb-Induced Outer Retinal Degeneration in the Rabbit.

PURPOSE To analyze the morphologic effects of subretinal blebs in rabbits using real-time imaging by spectral-domain optical coherence tomography (SD-OCT), infrared-confocal scanning laser ophthalmoscopy (IR-cSLO), and blue-light fundus autofluorescence (BAF). METHODS Subretinal blebs of PBS or balanced salt solution (BSS) were induced in albino or pigmented rabbits using a transvitreal pars plana technique. Spectral-domain optical coherence tomography, IR-cSLO, and BAF were done at multiple intervals for up to 12 weeks after subretinal bleb injection. The morphologic effects were compared with histologic analysis on hematoxylin-eosin-stained sections of the neurosensory retina and on flat-mounts of phalloidin-labeled RPE. RESULTS Scans of SD-OCT of the normal rabbit posterior segment revealed 11 bands including six layers of the photoreceptors. Subretinal blebs of PBS or BSS caused acute swelling of the neurosensory retina followed by gradual atrophy. Outer retinal thickness was significantly reduced with pronounced degeneration of all the photoreceptor OCT layers. En face IR-cSLO showed a hyperreflective area corresponding to the progressive photoreceptor degeneration, whereas BAF revealed both hyper- and hypofluorescent changes in the RPE layer. The in vivo results were confirmed by histology and on subretinal flatmounts demonstrating extensive photoreceptor loss and disruption of the RPE mosaic. CONCLUSIONS Subretinal blebs induce pronounced photoreceptor degeneration and RPE changes in the rabbit as demonstrated by in vivo imaging using SD-OCT, IR-cSLO, and BAF.

Integration of Subretinal Suspension Transplants of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells in a Large-Eyed Model of Geographic Atrophy

Purpose Subretinal suspension transplants of human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) have the capacity to form functional monolayers in naive eyes. We explore hESC-RPE integration when transplanted in suspension to a large-eyed model of geographic atrophy (GA). Methods Derivation of hESC-RPE was performed in a xeno-free and defined manner. Subretinal bleb injection of PBS or sodium iodate (NaIO3) was used to induce a GA-like phenotype. Suspensions of hESC-RPE were transplanted to the subretinal space of naive or PBS-/NaIO3-treated rabbits using a transvitreal pars plana technique. Integration of hESC-RPE was monitored by multimodal real-time imaging and by immunohistochemistry. Results Subretinal blebs of PBS or NaIO3 caused different degrees of outer neuroretinal degeneration, RPE hyperautofluorescence, focal RPE loss, and choroidal atrophy; that is, hallmark characteristics of GA. In nonpretreated naive eyes, hESC-RPE integrated as subretinal monolayers with preserved overlying photoreceptors, yet not in areas with outer neuroretinal degeneration and native RPE loss. When transplanted to eyes with PBS-/NaIO3-induced degeneration, hESC-RPE failed to integrate. Conclusions In a large-eyed preclinical model, subretinal suspension transplants of hESC-RPE did not integrate in areas with GA-like degeneration.

Gene Transfer of Prolyl Hydroxylase Domain 2 Inhibits Hypoxia-inducible Angiogenesis in a Model of Choroidal Neovascularization

Cellular responses to hypoxia are mediated by the hypoxia-inducible factors (HIF). In normoxia, HIF-α proteins are regulated by a family of dioxygenases, through prolyl and asparagyl hydroxylation, culminating in proteasomal degradation and transcriptional inactivation. In hypoxia, the dioxygenases become inactive and allow formation of HIF transcription factor, responsible for upregulation of hypoxia genes. In ocular neoangiogenic diseases, such as neovascular age-related macular degeneration (nAMD), hypoxia seems pivotal. Here, we investigate the effects of HIF regulatory proteins on the hypoxia pathway in retinal pigment epithelium (RPE) cells, critically involved in nAMD pathogenesis. Our data indicates that, in ARPE-19 cells, prolyl hydroxylase domain (PHD)2 is the most potent negative-regulator of the HIF pathway. The negative effects of PHD2 on the hypoxia pathway were associated with decreased HIF-1α protein levels, and concomitant decrease in angiogenic factors. ARPE-19 cells stably expressing PHD2 impaired angiogenesis in vitro by wound healing, tubulogenesis, and sprouting assays, as well as in vivo by iris-induced angiogenesis. Gene transfer of PHD2 in vivo resulted in mitigation of HIF-mediated angiogenesis in a mouse model of nAMD. These results may have implications for the clinical treatment of nAMD patients, particularly regarding the use of gene therapy to negatively regulate neoangiogenesis.

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

A novel in vivo model of puncture-induced iris neovascularization

Purpose To assess iris neovascularization by uveal puncture of the mouse eye and determine the role of angiogenic factors during iris neovascularization. Methods Uveal punctures were performed on BalbC mouse eyes to induce iris angiogenesis. VEGF-blockage was used as an anti-angiogenic treatment, while normoxia- and hypoxia-conditioned media from retinal pigment epithelium (RPE) cells was used as an angiogenic-inducer in this model. Iris vasculature was determined in vivo by noninvasive methods. Iris blood vessels were stained for platelet endothelial cell adhesion molecule-1 and vascular sprouts were counted as markers of angiogenesis. Expression of angiogenic and inflammatory factors in the puncture-induced model were determined by qPCR and western blot. Results Punctures led to increased neovascularization and sprouting of the iris. qPCR and protein analysis showed an increase of angiogenic factors, particularly in the plasminogen-activating receptor and inflammatory systems. VEGF-blockage partly reduced iris neovascularization, and treatment with hypoxia-conditioned RPE medium led to a statistically significant increase in iris neovascularization. Conclusions This study presents the first evidence of a puncture-induced iris angiogenesis model in the mouse. In a broader context, this novel in vivo model of neovascularization has the potential for noninvasive evaluation of angiogenesis modulating substances.