Felicitas Bucher

Hyperoxia causes reduced density of retinal astrocytes in the central avascular zone in the mouse model of oxygen-induced retinopathy

The mouse model of oxygen-induced retinopathy (OIR) is commonly used to investigate various aspects of the pathogenesis of the retinopathy of prematurity (ROP) as well as angiogenesis in general. Retinal astrocytes were suggested to be involved in retinal angiogenesis. This study aimed to describe their localization and cell density during the course of physiological vascularization and pathological revascularization. Mice expressing H2B-GFP (green fluorescent protein fused to histone 2B) from the endogenous Pdgfra promoter were kept in 75% oxygen from P7 (post natal day 7) to P12 (mouse model of OIR). Retinal flatmounts or cryosections were immunostained for glial fibrillary acidic protein (Gfap), glutamine synthetase (Glul), collagen IV (Col IV), desmin (Des), caspase 3 (Casp3), paired box 2 (Pax2), or Ki67. Astrocytic nuclei were counted with the ImageJ macro AuTOCellQuant. The hypoxic state of the retina was investigated by Hypoxyprobe. The GFP signal of the Pdgfra reporter mice co-localized with Pax2, a nuclear marker for retinal astrocytes. This bright label was much easier to quantify than Gfap or Pax2 staining. Quantification of the cell density of astrocytes during physiological development specified the spreading of astrocytes in a concentrical wave from the optic nerve head towards the periphery. Astrocyte density was reduced during the remodelling of the primary vascular plexus into a hierarchical vascular tree (maximal astrocyte density at P1: 2800 astrocytes/mm2, final astrocyte density: 800 astrocytes/mm2). In the OIR model, cell density of astrocytes was elevated in the peripheral vascularized zone. In contrast, astrocyte density dropped to a half (400 astrocytes/mm2) of the normal value in the central avascular zone during the hyperoxic phase between P8 and P10 by apoptosis and rose only after P17 as the retinal network normalized. An additional drop of astrocyte density was observed within the angles between the large vessels of the central avascular zone during hypoxia between P12 and P17. Astrocyte density was not altered at vascular tufts. The hyperoxia effect on astrocytes including the reduced astrocyte density is not the reason for vascular tuft formation. Hypoxia-affected astrocytes in combination with a reduced astrocytic network in the central avascular zone during the hypoxic phase are important determinants in the formation of pathological features during retinal revascularization.

CD44 expression in endothelial colony-forming cells regulates neurovascular trophic effect

Vascular abnormalities are a common component of eye diseases that often lead to vision loss. Vaso-obliteration is associated with inherited retinal degenerations, since photoreceptor atrophy lowers local metabolic demands and vascular support to those regions is no longer required. Given the degree of neurovascular crosstalk in the retina, it may be possible to use one cell type to rescue another cell type in the face of severe stress, such as hypoxia or genetically encoded cell-specific degenerations. Here, we show that intravitreally injected human endothelial colony-forming cells (ECFCs) that can be isolated and differentiated from cord blood in xeno-free media collect in the vitreous cavity and rescue vaso-obliteration and neurodegeneration in animal models of retinal disease. Furthermore, we determined that a subset of the ECFCs was more effective at anatomically and functionally preventing retinopathy; these cells expressed high levels of CD44, the hyaluronic acid receptor, and IGFBPs (insulin-like growth factor-binding proteins). Injection of cultured media from ECFCs or only recombinant human IGFBPs also rescued the ischemia phenotype. These results help us to understand the mechanism of ECFC-based therapies for ischemic insults and retinal neurodegenerative diseases.

Antibody-Mediated Inhibition of Tspan12 Ameliorates Vasoproliferative Retinopathy Through Suppression of β-Catenin Signaling

Background: Anti-angiogenic biologicals represent an important concept for the treatment of vasoproliferative diseases. However, the need for continued treatment, the presence of nonresponders, and the risk of long-term side effects limit the success of existing therapeutic agents. Although Tspan12 has been shown to regulate retinal vascular development, nothing is known about its involvement in neovascular disease and its potential as a novel therapeutic target for the treatment of vasoproliferative diseases. Methods: Rodent models of retinal neovascular disease, including the mouse model of oxygen-induced retinopathy and the very low density lipoprotein receptor knockout mouse model were analyzed for Tspan/&bgr;-catenin regulation. Screening of a phage display of a human combinatorial antibody (Ab) library was used for the development of a high-affinity Ab against Tspan12. Therapeutic effects of the newly developed Ab on vascular endothelial cells were tested in vitro and in vivo in the oxygen-induced retinopathy and very low density lipoprotein receptor knockout mouse model. Results: The newly developed anti-Tspan12 Ab exhibited potent inhibitory effects on endothelial cell migration and tube formation. Mechanistic studies confirmed that the Ab inhibited the interaction between Tspan12 and Frizzled-4 and effectively modulates &bgr;-catenin levels and target genes in vascular endothelial cells. Tspan12/&bgr;-catenin signaling was activated in response to acute and chronic stress in the oxygen-induced retinopathy and very low density lipoprotein receptor mouse model of proliferative retinopathy. Intravitreal application of the Ab showed significant therapeutic effects in both models without inducing negative side effects on retina function. Moreover, combined intravitreal injection of the Ab with a known vascular endothelial growth factor inhibitor, Aflibercept, resulted in significant enhancement of the therapeutic efficacy of each monotherapy. Combination therapy with the Tspan12 blocking antibody can be used to reduce anti-vascular endothelial growth factor doses, thus decreasing the risk of long-term off-target effects. Conclusions: Tspan12/&bgr;-catenin signaling is critical for the progression of vasoproliferative disease. The newly developed anti-Tspan12 antibody has therapeutic effects in vasoproliferative retinopathy and can enhance the potency of existing anti- vascular endothelial growth factor agents.

Interferon-γ is a master checkpoint regulator of cytokine-induced differentiation

Significance The understanding of the molecular mechanisms of activation and checkpoint processes has important therapeutic implications. Here, we show that interferon-γ is a master checkpoint regulator for many cytokines. It operates partially by activating STAT1 signaling. However, most important is the mechanism that allows it to assume master regulator status. To do this, it induces internalization of gp130, a common component of many heterodimeric cytokine receptors. Therefore, this cytokine checkpoint could open a whole new paradigm in cell biology. Cytokines are protein mediators that are known to be involved in many biological processes, including cell growth, survival, inflammation, and development. To study their regulation, we generated a library of 209 different cytokines. This was used in a combinatorial format to study the effects of cytokines on each other, with particular reference to the control of differentiation. This study showed that IFN-γ is a master checkpoint regulator for many cytokines. It operates via an autocrine mechanism to elevate STAT1 and induce internalization of gp130, a common component of many heterodimeric cytokine receptors. This targeting of a receptor subunit that is common to all members of an otherwise diverse family solves the problem of how a master regulator can control so many diverse receptors. When one adds an autocrine mechanism, fine control at the level of individual cells is achieved.

iPSC-Derived Retinal Pigment Epithelium Allografts Do Not Elicit Detrimental Effects in Rats: A Follow-Up Study

Phototransduction is accomplished in the retina by photoreceptor neurons and retinal pigment epithelium (RPE) cells. Photoreceptors rely heavily on the RPE, and death or dysfunction of RPE is characteristic of age-related macular degeneration (AMD), a very common neurodegenerative disease for which no cure exists. RPE replacement is a promising therapeutic intervention for AMD, and large numbers of RPE cells can be generated from pluripotent stem cells. However, questions persist regarding iPSC-derived RPE (iPS-RPE) viability, immunogenicity, and tumorigenesis potential. We showed previously that iPS-RPE prevent photoreceptor atrophy in dystrophic rats up until 24 weeks after implantation. In this follow-up study, we longitudinally monitored the same implanted iPS-RPE, in the same animals. We observed no gross abnormalities in the eyes, livers, spleens, brains, and blood in aging rats with iPSC-RPE grafts. iPS-RPE cells that integrated into the subretinal space outlived the photoreceptors and survived for as long as 2 1/2 years while nonintegrating RPE cells were ingested by host macrophages. Both populations could be distinguished using immunohistochemistry and electron microscopy. iPSC-RPE could be isolated from the grafts and maintained in culture; these cells also phagocytosed isolated photoreceptor outer segments. We conclude that iPS-RPE grafts remain viable and do not induce any obvious associated pathological changes.

Angiogenesis and Eye Disease

The retina consists of organized layers of photoreceptors, interneurons, glia, epithelial cells, and endothelial cells. The economic model of supply and demand used to appropriately determine cost is highly applicable to the retina, in which the extreme metabolic demands of phototransduction are met by precisely localized and designed vascular networks. Proper development and maintenance of these networks is critical to normal visual function; dysregulation is characteristic of several devastating human diseases, including but not limited to age-related macular degeneration and diabetic retinopathy. In this article, we focus on the lessons learned from the study of retinal vascular development and how these lessons can be used to better maintain adult vascular networks and prevent retinal diseases. We then outline the vasculotrophic contributions from neurons, retinal pigment epithelium (RPE) cells, and glia (specifically microglia) before we shift our focus to pathology to provide molecular contexts for neovascular retinal diseases. Finally, we conclude with a discussion that applies what we have learned about how retinal cells interact with the vasculature to identify and validate therapeutic approaches for neurovascular disease of the retina.

Fully automated, deep learning segmentation of oxygen-induced retinopathy images

Oxygen-induced retinopathy (OIR) is a widely used model to study ischemia-driven neovascularization (NV) in the retina and to serve in proof-of-concept studies in evaluating antiangiogenic drugs for ocular, as well as nonocular, diseases. The primary parameters that are analyzed in this mouse model include the percentage of retina with vaso-obliteration (VO) and NV areas. However, quantification of these two key variables comes with a great challenge due to the requirement of human experts to read the images. Human readers are costly, time-consuming, and subject to bias. Using recent advances in machine learning and computer vision, we trained deep learning neural networks using over a thousand segmentations to fully automate segmentation in OIR images. While determining the percentage area of VO, our algorithm achieved a similar range of correlation coefficients to that of expert inter-human correlation coefficients. In addition, our algorithm achieved a higher range of correlation coefficients compared with inter-expert correlation coefficients for quantification of the percentage area of neovascular tufts. In summary, we have created an open-source, fully automated pipeline for the quantification of key values of OIR images using deep learning neural networks.

Correction: Antibody targeting TSPAN12/β-catenin signaling in vasoproliferative retinopathy

[This corrects the article DOI: 10.18632/oncotarget.23401.].

Antibody targeting Tspan12/β-catenin signaling in vasoproliferative retinopathy

Abnormal vessel growth and breakdown of the blood retinal barrier are major causes of vision loss in western countries [1]. Over the past decade, we have seen an increasingly successful use of biological agents targeting vascular endothelial growth factor (VEGF) to treat diabetic retinopathy and age-related macular degeneration via intravitreal injections. However, longterm studies have shown that visual acuity still drops in over 30% of treated patients, while certain patient populations do not responds to anti-VEGF treatment at all [2, 3]. Discoveries of novel complementary targets are thus necessary to improve the therapeutic success. In our recent paper, we identified tetraspanin 12 (TSPAN12) as novel therapeutic target for retinal vascular disease, and developed a TSPAN12-targeting therapeutic antibody [4]. TSPAN12 belongs to the Tetraspanin family, which mainly includes cell surface proteins characterized by four transmembrane domains and two extracellular loops [5]. Tetraspanins interact with various cell surface proteins and regulate their intracellular trafficking, lateral diffusion and clustering at the plasma membrane [6]. They have also been associated with several pathological conditions such as tumor progression and metastasis [7]. In the retina, TSPAN12 is selectively expressed in the retinal vasculature and acts as a key regulator for retinal vascular development by activating β-catenin signaling. In 2009, Junge and colleagues first published results from a large-scale genetic screen suggesting that mutations in the previously uncharacterized TSPAN12 gene caused retinal vascular defects similar to those observed in FZD4, LRP5, and Norrin, which induced the blindness-causing disease familial exudative vitreoretinopathy [8]. With the help of TSPAN12-deficient mice, they proved TSPAN12 to be critical for the development of the retinal vasculature through the activation of β-catenin signaling. In vitro studies showed that TSPAN12 promotes the complex formation of Frizzled-4 (FZD4) and its co-receptor, lowdensity lipoprotein receptor-related protein 5 (LRP5). While past studies focused on the role of TSPAN12 in retinal development, we are the first to show that TSPAN12/β-catenin signaling plays an important role in retinal neovascular disease [4]. In our present study, we developed anti-TSPAN12 antibodies and tested to see if they can reduce FZD4/TSPAN12-mediated β-catenin signaling and thus be used as a treatment for vasoproliferative retinopathy. For the selection of a high affinity antibody, a 48-amino acid peptide antigen encompassing the big extracellular loop of TSPAN12 was designed. Then, a phage library consisting of approximately 109 human combinatorial antibodies was panned against the antigen. The selected TSPAN12 antibody was found to have a significant inhibitory effect on human umbilical vein endothelial cell functions such as migration and cell-cell adhesion. In addition, β-catenin expression was significantly decreased by the TSPAN12 antibody, suggesting an inhibition of TSPAN12/βcatenin signaling. The mechanism appears to involve the disruption of TSPAN12 interaction with FZD4, as was measured as a reduction during a co-immunoprecipitation experiment. To investigate the therapeutic potential of this TSPAN12 antibody, we used two mouse models of vasoproliferative retinopathy, the oxygen-induced retinopathy (OIR) model and the very-low-density lipoprotein receptor (VLDLR) knockout model [4]. In both models, intravitreal injection of the TSPAN12 antibody significantly reduced abnormal vessel growth. The TSPAN12 Ab selectively targeted β-catenin signaling in vascular endothelial cells in vivo without affecting retinal VEGF levels. Combination therapy with a known anti-VEGF agent such as Aflibercept demonstrated significant therapeutic synergy providing the opportunity to decrease therapeutic doses of anti-VEGF agents and reduce unwanted side-effects. These aspects support the potential clinical use of the TSPAN12 antibody. Due to the selective expression of TSPAN12 in retinal vascular endothelial cells the TSPAN12 antibody may also serve as delivery vehicle for endothelial specific therapeutics. It may be possible to develop a novel fusion protein similar to Aflibercept that combines the TSPAN12 and VEGF-receptor antigen domain. Furthermore in the OIR model, the TSPAN12 Ab more strongly supported physiologic revascularization into hypoxic retinal tissue compared to anti-VEGF treatment. Therefore, modulating TSPAN12 activity may also present a promising target to encourage physiologic revascularization of avascular areas in diabetic retinopathy or vein occlusion, a yet unsolved problem.