Consensus guidelines for the use and interpretation of angiogenesis assays

The 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

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

<div><p>Purpose</p><p>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.</p><p>Methods</p><p>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.</p><p>Results</p><p>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.</p><p>Conclusion</p><p>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.</p></div

Histology of the occlusion site.

<p>Veins were occluded at d0. Two days later, eyes were prepared for serial paraffin sections that were stained with HE. Typical sections from three RVO eyes and three control eyes are shown. A1—A4: Sections at different positions of a single laser site. The RPE is slightly affected and Bruch’s membrane is intact. The inner limiting membrane is detached by a large inner retinal serous exsudation, and the INL is also affected by a serous exsudation. A1 is from the distal part of the laser site with the vein containing erythrocytes. A2 shows a fold (invagination) within the outer retina and a vein that contains a fibrous plug. A3 is from the center of the laser site showing a large retinal invagination and a bleeding vein containing a fibrous plug. A4 is from the proximal part of the laser site showing the retinal fold within the RPE and a decreased vein. The whole series of sections is shown in the supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191338#pone.0191338.s001" target="_blank">S1 Fig</a>. B1—B4: Sections at different positions of another single laser site. The inner limiting membrane is detached by a large inner retinal serous exsudation, and the INL is also affected by a serous exsudation. RPE and Bruch’s membrane are perforated as is seen in B3. B1 shows the distal part of the laser site with a fold of photoreceptor outer segments within the RPE and a vein containing a fibrous plug. B2 shows a fold within the outer retina. B3 is from the central part of the laser site showing a retinal fold and fused material of photoreceptors, RPE, and choroid. B4 shows the proximal vein that is diminished and a lateral part of the laser site with a fold of outer segments of the photoreceptors. The retina shows unequal thickness. The whole series of sections is shown in the supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191338#pone.0191338.s002" target="_blank">S2 Fig</a>. C1—C3: Sections from control laser sites without vein occlusion. The morphology of the laser sites is principally equal to RVO laser sites. C1 shows a fold of the outer retina of a laser site with intact Bruch’s membrane. The choroid is affected as shown by its reduced thickness at the center of the laser site. C2 and C3 are sections from a laser site with disrupted RPE and choroid. The choroid shows a reduced thickness, and even the sclera is affected. The material of the outer retinal fold is partially fused. C4 is a section from an intact retina for comparison. ILM: inner limiting membrane; INL: inner nuclear layer; ONL: outer nuclear layer; POS: photoreceptor outer segments; RGC: retinal ganglion cells.</p

RVO and hypoxic area.

<p>In a preliminary experiment, two veins were occluded in two mice at d0. Fluorescein angiography by SLO showed the occlusion of the veins, and fundus images taken by SLO are shown for comparison. Retinal flatmounts were stained for vessels with lectin (green) and with hypoxyprobe (red) for hypoxic areas. The squares show the positions of the SLO images while the arrows show the position of laser sites. In one mouse at d2, the vein to the left was re-opened while the lower vein was still closed (arrow) and surrounded by a hypoxic area. In the other mouse at d3, the lower vein was re-opened (arrow) but showed some remnants of the hypoxic area. Typical results from a study of 5 mice are shown.</p

Time course of RVO and hypoxia.

<p>RVO was induced at d0 in two mice per time point. Fundus images were taken by a Micron III camera and flatmounts were prepared at the indicated days. Flatmounts were stained for vessels with an antibody raised against collagen IV (Col4, green) and with hypoxyprobe (red) for hypoxic areas. Flatmounts at d0 were not laser-treated. In one eye of each mouse, the veins were occluded (RVO) while the other eye was laser-treated between the large vessels (control). The circle indicates the area of the corresponding fundus image. Hypoxic areas were found from d1 to d3, diminished at d5 and disappeared completely at d8. No hypoxia staining was observed after intervenous laser treatment in the control group. The large edematous areas (gray areas of the fundus images) at the laser sites were observed both in the RVO and the control group. They resulted in weaker hypoxia staining (compare the RVO eye at d2).</p

Primer.

<p>Primer.</p

mRNA expression analysis (qPCR) of genes strongly up-regulated in RVO.

<p>The first 4 factors had an expression maximum at d1 while the others had a maximum at d2 or d3. All of them are involved in inflammation or responses to stress. Most interestingly, the up-regulation found in RVO was also detected in the controls that were laser-treated between the large vessels. Note the logarithmic expression scale that shows equal distances for factors that are up- or down-regulated. Asterisks at the right of the corresponding data point indicate significant differences as compared to d0. Mice at d0 were not laser-treated.</p

Genes up-regulated in RVO versus laser-treated control.

<p>Genes up-regulated in RVO versus laser-treated control.</p