A novel ex vivo murine retina angiogenesis (EMRA) assay

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Methods in eye research

A novel ex vivo murine retina angiogenesis (EMRA) assay Q1

Sara Rezzola a, b, Mirella Belleri a, Domenico Ribatti c, Ciro Costagliola d, Marco Presta a, **, Francesco Semeraro b, * a

Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123 Brescia, Italy Department of Ophthalmology, University of Brescia, Brescia, Italy c Department of Basic Biomedical Sciences, Neuroscience and Sensory Organs, Unit of Human Anatomy and Histology, University of Bari, Italy d Department of Medicine and Health Sciences, University of Molise, Campobasso, Italy b

a r t i c l e i n f o

a b s t r a c t

Article history: Available online xxx

Pathological retinal angiogenesis results from the imbalance of pro-angiogenic and anti-angiogenic factors. In particular, vascular endothelial growth factor (VEGF) plays a pivotal role in retinal neovascularization and various therapeutic VEGF blockers have evolved over time. Nevertheless, new retinal angiogenesis models are crucial for investigating anti-angiogenic therapies and bringing them to patients. Here, we developed a novel ex vivo murine retina angiogenesis (EMRA) assay in which endothelial sprouts originate from mature and quiescent retinal vessels. In this model, retina fragments from adult mice are embedded in a three-dimensional fibrin gel in the presence of human recombinant VEGF. Starting from the 3rde4th day of incubation, endothelial cell sprouts invading the fibrin gel can be observed under an inverted microscope and measured at different time points thereafter. The effect of VEGF is dose-dependent, maximal stimulation being observed at day 7 for retina fragments stimulated with 25e75 ng/ml of the growth factor. To assess whether the EMRA assay is suitable for testing the activity of anti-angiogenic compounds, retina fragments were incubated with VEGF in the presence of the neutralizing anti-VEGF antibodies bevacizumab and ranibizumab. The results demonstrate that both antibodies inhibit VEGF activity in a dose-dependent manner. In conclusion, the EMRA assay represents a new ex vivo model of retinal neovascularization suitable for the rapid screening of novel anti-angiogenic therapeutics. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: angiogenesis bevacizumab endothelium mouse ranibizumab retina VEGF

1. Introduction Angiogenesis, the growth of new blood vessels from preexisting ones, plays a pivotal role in various physiological and pathological conditions (Carmeliet and Jain, 2011). In particular, pathological retinal angiogenesis is a key component of irreversible causes of blindness. Indeed, proliferative diabetic retinopathy accounts for the highest incidence of acquired blindness in the working-age population (Congdon et al., 2004; Klein, 2007), choroidal subretinal neovascularization that occurs in age-related macular degeneration represents the leading cause of blindness in people over the age of 65 (Friedman et al., 2004; Semeraro et al., 2011), and retinopathy of prematurity is a major cause of

* Corresponding author. Department of Ophthalmology, Spedali Civili, Piazzale Spedali Civili 1, 25123 Brescia, Italy. Tel.: þ39 030 3995308; fax: þ39 030 3388191. ** Corresponding author. Tel.: þ39 030 3717311; fax: þ39 030 0303717747. E-mail addresses: [email protected] (M. Presta), [email protected], [email protected] (F. Semeraro).

acquired blindness in children (Mechoulam and Pierce, 2003; Chen et al., 2011). Pathological retinal angiogenesis results from the imbalance of pro-angiogenic and anti-angiogenic factors (Gariano and Gardner, 2005; Siemerink et al., 2010). In particular, vascular endothelial growth factor (VEGF) is a major inducer of retinal/choroid neovascularization [see (Antonetti et al., 2012; Kim and D’Amore, 2012; Miller et al., 2013) and references therein]. Since the seminal discovery of VEGF accumulation in eyes of patients with diabetic retinopathy (Aiello et al., 1994), three different VEGF blockers have evolved over time (pegaptanib, ranibizumab and aflibercept); a fourth agent, bevacizumab, is used off-label (Costagliola et al., 2012; Stewart, 2012). Nevertheless, the study of diabetic retinopathy and other retinal disorders opens new lines of angiogenesis inquiry and experimental models of retinal angiogenesis are crucial for investigating novel anti-angiogenic therapies and bringing them to patients. In vivo mouse models of retinal angiogenesis have been developed to understand the underlying mechanisms of angiogenesisdependent sight-threatening conditions and for the identification

0014-4835/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.exer.2013.04.014

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of therapeutic anti-angiogenic strategies (Montezuma et al., 2009; Stahl et al., 2010). Also, several in vitro and ex vivo angiogenesis assays have been established using endothelial cells of different origin (including macrovascular and microvascular endothelium from human, murine and bovine vessels) for a rapid screening of potential anti-angiogenic compounds (Goodwin, 2007; Staton and Reed, 2009). However, endothelial cells are characterized by a significant heterogeneity (Bastaki et al., 1997; Chi et al., 2003; Stewart et al., 2011) that occurs also within the specific microenvironment to be investigated (Zetter, 1988). It is therefore essential to choose assay conditions and endothelial cell origin that most closely resemble the angiogenic disease being studied. This is especially important when aiming to translate preclinical data to the clinic (Staton and Reed, 2009). Thus, retina-based in vitro and ex vivo angiogenesis assays are eagerly required. To this respect, isolation of endothelial cells from the retina allows their use in various in vitro assays that mimic different steps of the angiogenic process (Su et al., 2003; Klettner and Roider, 2008; Stewart et al., 2011). Also, retinal explant cultures have been prepared from neonatal and adult mice to investigate endothelial tip cell responses to angiogenic stimuli (Murakami et al., 2006; Sawamiphak et al., 2010). Here, we describe a novel murine ex vivo retinal angiogenesis (EMRA) assay in which endothelial cell sprouts are induced from mature, quiescent retinal vessels of adult mice. In this model, murine retina fragments are embedded in a three-dimensional fibrin gel in the presence of increasing concentrations of human recombinant VEGF. Endothelial cell sprouts originating from preexisting retinal vessels invade the fibrin gel in a VEGF dosedependent manner. Accordingly, the anti-VEGF agents bevacizumab and ranibizumab exert a dose-dependent inhibitory effect on endothelial sprouting.

The EMRA assay represents a new model of retinal neovascularization suitable for a rapid ex vivo screening of novel antiangiogenic therapeutics. 2. Materials and supplies Retinae were isolated from 4 to 5 week-old C57BL/6 mice (Charles River, Calco, Italy). All reagents were of analytical grade. Three-dimensional fibrin gels were prepared with bovine fibrinogen and thrombin plus aprotinin (all from SigmaeAldrich, St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM), M199 medium and fetal calf serum (FCS) were from GIBCO Life Technologies (Grand Island, NY). Human recombinant VEGF (VEGF-A165 isoform) was kindly provided by K. Ballmer-Hofer (PSI, Villigen, Switzerland). Endothelial sprouts were stained with biotinconjugated BS-I Isolectin B4 from Bandeiraea simplicifolia (Sigmae Aldrich) and streptavidin, Alexa Fluor 488 conjugateÓ (Molecular Probes, Eugene, OR). Bevacizumab (AvastinÓ) was from Roche (Basel, Switzerland) and ranibizumab (LucentisÓ) from Novartis (Horsham, UK). 3. Detailed methods The EMRA assay is based on the isolation of retina fragments from 4 to 5 week-old adult mice. Next, fragments are embedded in a three-dimensional fibrin gel in the presence of human recombinant VEGF (Fig. 1). Starting from the 3rde4th day of incubation, endothelial cell sprouts originating from pre-existing retinal vessels invade the fibrin gel. Endothelial sprouts can be observed under an inverted microscope (Fig. 2AeC) and counted at different time points thereafter. Experimental procedures, characterization of endothelial sprouts and quantification of the effect of VEGF and

Fig. 1. Schematic depiction of the EMRA assay. The murine retina is dissected from the removed eye (a). Then, the isolated retina is cut with forceps along the edge and across the center (dotted lines) to obtain four fragments (b). Retina fragments are embedded in a three-dimensional fibrin gel and incubated with VEGF (c). After 7 days, endothelial sprouts originating from retinal vessels and invading the surrounding gel are counted under an inverted microscope (d).

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Fig. 2. Characterization of retinal endothelial sprouts. Retina fragments were incubated for 7 days in fibrin gel in the absence (A) or in the presence (BeG) of 75 ng/ml of VEGF. VEGF induces the formation of endothelial sprouts that invade the fibrin gel (B, C). D, E. Staining with the endothelial marker Bandeiraea simplicifolia BS-I isolectin-B4 shows endothelial sprouts invading the gel (arrows) that originate from the retinal vessels (*). A 3D-reconstruction of the endothelial sprouts in panel D is shown in Suppl. Video 1. The video was obtained by 3D reconstruction of the optical sections of the retina fragment along the Z axis generated by an ApoTome Carl Zeiss fluorescence microscopy system. F, G. Semithin sections of retina fragments highlight a solid endothelial sprout rich in vacuoles invading the fibrin gel (rectangle in F) and capillaries within the gel characterized by wide lumina and very thin walls (rectangles in G).

anti-VEGF agents on the neovascular response in the EMRA assay are detailed below. 3.1. Isolation of murine retina fragments Eyes were removed with curved forceps from 4 to 5 week-old mice and transferred into a 10 cm-plate containing serum-free DMEM. Under a stereomicroscope the eye was held in place with

forceps while a pair of fine scissors were inserted at the corneasclera boundary. Then, after a circumferential incision around the limbus, the retina was carefully dissected from the surrounding sclera and the vitreous was removed using fine forceps. Next, the isolated retina was cut along the whole edge to induce a peripheral injury of the retinal tissue and from the rim to the center to obtain four equal quadrants from each explant (Fig. 1). This procedure allows the isolation of retina fragments very homogenous in size

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(1.35  0.19 mg; mean  SD, n ¼ 21). Finally, isolated retina fragments were incubated overnight in serum-free DMEM at 37  C in 5% CO2. 3.2. Retina fragment embedding in fibrin gel and culture conditions Bovine fibrinogen (3.1 mg/mI) was dissolved in DMEM containing antibiotics plus 5.0 mg/ml aprotinin and the solution was filtered through a 0.45 mm-pore filter. Next, 400 mU/mI of bovine thrombin were added to the fibrin solution that was quickly dispensed into 48 well-plates (100 ml per well) and allowed to clot for 5 min at 37  C. In the meantime, 1.5 ml microcentrifuge tubes were prepared, each containing 350 ml of the fibrinogen plus aprotinin solution. Retina fragments were individually transferred to each tube using a Gilson pipet with a cut blue tip and added with bovine thrombin (400 mU/mI). Next, the mixture containing the retina fragment was rapidly transferred on the top of the previously prepared fibrin-coated wells and allowed to gel at 37  C. After clotting, 350 ml of DMEM containing 10% FCS and the molecule(s) under test were added on the top of the gel and replaced after 3 days. 3.3. Endothelial sprout staining After 7 days of incubation with VEGF, fibrin-embedded retina fragments were fixed within the wells by replacing the culture medium with 0.5 ml of 4% paraformaldehyde (PFA) in phosphatebuffered saline (PBS) followed by 2 h of incubation at 4  C. Then, samples were washed 3 times with PBS (10 min each) and incubated for 30 min in blocking solution (1% bovine serum albumin/ 0.5% Tween 20 in PBS). Tissue permeabilization was obtained by washing the samples twice (30 min at room temperature under gentle shaking) with PBlec solution [0.1 mM CaCl2, 0.1 mM MgCl2, 0.1 mM MnCl2, and 1% Triton X-100 in PBS 8 (pH 6.8)]. Next, samples were incubated overnight at 4  C with the biotin-conjugated endothelial marker Bandeiraea simplicifolia BS-I Isolectin B4 (1:500 in PBlec). After 3 washes with PBS at room temperature, samples were incubated overnight at 4  C with streptavidin, Alexa FluorÒ 488 conjugate (1:500 in 0.5% bovine serum albumin/0.25% Tween 20 in PBS) and stored in PBS at 4  C. Finally, fibrin-embedded samples were removed from the wells with a curved spatula and placed on a microscope glass slide. Endothelial sprouts were photographed under an inverted epifluorescence microscope (Zeiss Axiovert 200 M) at 200 magnification. As shown in Fig. 2D,E, the sprouts invading the fibrin gel are positively stained by the endothelial marker Bandeiraea simplicifolia BS-I isolectin-B4, thus confirming their identity as endothelial cell sprouts originating from mature retinal vessels (see Suppl. Video 1). Further studies will be required to understand the distinct contribution of retinal microvascular, arterial and/or venous endothelium to sprout formation in this assay. Supplementary video related to this article can be found at http://dx.doi.org/10.1016/j.exer.2013.04.014. 3.4. Microscopic analysis of endothelial sprouts Fibrin-embedded samples were fixed by replacing the culture medium with 0.5 ml of 1.5% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.4) for 1 h at room temperature. Then, samples were collected from the wells with a curved spatula and the excess of fibrin gel was removed with a surgical blade. Retina fragments were post-fixed in phosphate-buffered 1% osmium tetroxide for 1 h at room temperature, dehydrated in graded ethanol series, and embedded in Epon 812 resin for microscopy (EMbed 812, Electron Microscopy Sciences, Hatfield, PA). Semithin 1 mm sections were

stained with 1% toluidine blue and photographed under a light microscope (Zeiss Axioskop, Oberkochen, Germany). As shown in Fig. 2F,G, solid endothelial sprouts rich in vacuoles and small vascular tubes invading the gel are recognizable in VEGF-treated retina fragments together with capillaries characterized by wide lumina and very thin walls. 3.5. Quantification of neovascular response Retina fragments were embedded in three-dimensional fibrin gel in the absence or in the presence of increasing concentrations of human recombinant VEGF, ranging between 6.25 and 75 ng/ml. The formation of endothelial cell sprouts was followed under an inverted microscope (IX51, Olympus) at 100 magnification and sprouts were counted in a double-blind fashion every 24 h for 7 days. Medium plus or minus VEGF was replaced after 3 days. As shown in Fig. 3A, endothelial cell sprouts invading the fibrin gel are detectable starting from the 3rde4th day of incubation. During the following days sprouts increase in length and number as a function of VEGF concentration, maximal stimulation being observed at day 7 for retina fragments treated with 25e75 ng/ml of VEGF (Fig. 3B). At this time point the average length of endothelial sprouts was equal to 63.8  5.0 and 161.2  15.5 mm for control and VEGFtreated samples, respectively (mean  SEM; n ¼ 100; P < 0.001, Student’s t test). After the first week of incubation, degradation of the retina fragments and of endothelial sprouts may occur. Thus, day 7 was considered as the optimal time point for assay quantification. To assess the capability of the EMRA assay to evaluate the efficacy and potency of anti-angiogenic treatments, fibrin-embedded retina fragments were incubated with 75 ng/ml of VEGF in the presence of the anti-VEGF agents bevacizumab and ranibizumab (both at 1.0 nM). Then, endothelial sprouts were counted throughout the whole experimental period until day 7. In keeping with previous observations (Klettner and Roider, 2008; Stewart et al., 2011), the two antibodies exert a significant inhibitory effect on VEGF-induced neovascularization, bevacizumab being slightly more potent than ranibizumab (Fig. 3C). Doseeresponse experiments confirmed the neutralizing anti-VEGF activity of the two antibodies, with ID50 values equal to 0.1 nM and 0.5 nM for bevacizumab and ranibizumab, respectively (Fig. 3D). No significant inhibition of basal sprouting was instead exerted by bevacizumab or ranibizumab in VEGF-untreated samples at all the doses tested (Fig. 3C and data not shown). 4. Potential pitfalls and troubleshooting 4.1. Isolation of murine retina fragments Isolation of the retina is a critical step and requires a careful handling of the specimen. In order to preserve the retinal structure it is important to avoid any contact of the forceps with its surface. Also, cutting the retina along its edge should be performed holding the specimen in place with forceps that gently grab only the peripheral area to be removed. 4.2. Three-dimensional fibrin gel preparation and retina fragment embedding Fibrin clot will gel in a few seconds after thrombin is added to the fibrinogen solution. It is therefore essential to work rapidly. We recommend to gently resuspend retina fragments in the fibrinogen solution one at the time and to rinse the pipette tip with the fibrinogen solution before transferring the retina fragment from the microcentrifuge tube to the fibrin-coated well. This will prevent

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Fig. 3. Quantification of the EMRA assay. A. Retina fragments were incubated in fibrin gel in the absence (B) or in the presence (C) of 75 ng/ml of VEGF. Endothelial sprouts originating from each fragment were counted under an inverted microscope every 24 h. B. Retina fragments were incubated with increasing concentrations of VEGF ranging between 6.25 and 75 ng/ml and endothelial sprouts were counted at day 7. C. Retina fragments were incubated in the absence (open symbols) or in the presence (closed symbols) of 75 ng/ml of VEGF and added with 1.0 nM of the anti-VEGF antibody bevacizumab (6, :) or ranibizumab (,, -). Fragments incubated with VEGF alone (C) or with no addition (B) were used as positive and negative controls, respectively. Endothelial sprouts were counted every 24 h. D. Retina fragments were incubated with 75 ng/ml of VEGF in the presence of increasing concentrations of bevacizumab (:) or ranibizumab (-) and endothelial sprouts were counted at day 7. Data are the mean  SEM of 3 independent experiments each with 8 retina fragments per experimental point.

a possible tissue damage that may preclude the neovascular response. Once embedded, retina fragments may digest the fibrin clot and the lysis of the gel around the explant may cause the premature loss of matrix support for angiogenic sprouting. Fibrinolytic activity is highest during the first days of incubation and can be blocked by adding 5.0 mg/ml of the serine protease inhibitor aprotinin to the

fibrin gel. Aprotinin has no anti-angiogenic effects at the dose tested. 4.3. Quantification of neovascular response Incubation of retina fragments in M199 medium completely abolishes their capacity to respond to VEGF stimulation. Thus, all

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the experiments are performed in DMEM. Also, overnight starvation of isolated retinae in serum-free DMEM is recommended in order to reduce the spontaneous endothelial cell sprouting that may occur in untreated retina fragments. The age of mice may represent a critical factor to be considered when performing the EMRA assay. Routinely, we isolate retina fragments from 4 to 5 week-old mice. Similar results can be obtained with 2e3 week-old mice. In contrast, retina fragments isolated from 10 or 16 week-old mice show a dramatic decrease in their capacity to respond to VEGF stimulation. In the course of this work we observed a significant intersample variability in the angiogenic response of retina fragments. When all the results obtained during the whole experimental period were analyzed together, the mean  SD of the number of sprouts per fragment measured at day 7 was equal to 5.1  9.2 and 15.1  18.4 for control (n ¼ 166) and VEGF-treated (n ¼ 185) samples, respectively (P < 0.001, Student’s t test). An adequate period of training of the investigator performing retina fragment isolation (usually 2e3 months, processing 8e10 retinae per week) results in a significant reduction of such variability due, at least in part, to erroneous handling of the retinae. At present, in a representative experiment performed on 16 retina fragments per group, we measured 0.8  1.3 and 15.3  9.8 sprouts/fragment in control and VEGF-treated samples, respectively (P < 0.001, Student’s t test). This makes the assay affordable in terms of costs and experimental time and suitable for drug testing using a limited number of retina fragments. 4.4. EMRA assay versus the murine aorta ring assay The EMRA assay is reminiscent of the widely used murine aorta ring assay [see (Nicosia, 2009) and references therein]. Indeed, both assays are based on the formation of endothelial sprouts from a quiescent endothelium; these sprouts invade a three-dimensional fibrin clot, a relevant extracellular matrix component, within 7 days after stimulation with VEGF. In our hands, both assays require experimental skills that can be attained after an adequate period of training, leading to the isolation of 8 retina fragments or 10e15 aorta rings per mice. Thus, costs, timing, reproducibility, and the possibility to use tissues from transgenic and mutant mouse strains represent similar, interesting features of the two assays. Nevertheless, as stated above, the heterogeneity of vascular endothelium and of its microenvironment (Zetter, 1988; Bastaki et al., 1997; Chi et al., 2003; Staton and Reed, 2009; Stewart et al., 2011) requires the use of assay conditions and of endothelial/accessory cell types that most closely resemble the angiogenic disease being studied. To this respect, the EMRA assay appears to be more suitable than the murine aorta ring assay to investigate the mechanisms of pathological retinal angiogenesis and for the identification of specific therapeutic anti-angiogenic strategies. In conclusion, the EMRA assay represents a new ex vivo model of retinal neovascularization suitable for the screening of novel antiangiogenic therapeutics. Moreover, this assay may provide a model system to elucidate the molecular basis of pathological retinal angiogenesis. Acknowledgments We wish to thank Dr. S. Mitola for helpful suggestions and criticisms and Dr. C. Ravelli for video acquisition of retinal sprouts. This work was supported in part by grants from Ministero dell’Istruzione, Università e Ricerca (MIUR, Centro IDET, FIRB project RBAP11H2R9 2011) and Associazione Italiana per la Ricerca sul Cancro (AIRC grant n 10396) to MPand from Ministero dell’Istruzione, Università e

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Please cite this article in press as: Rezzola, S., et al., A novel ex vivo murine retina angiogenesis (EMRA) assay, Experimental Eye Research (2013), http://dx.doi.org/10.1016/j.exer.2013.04.014

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