Retinal NV occurs as a complication of ischemic retinopathies, a group of diseases in which there is damage to retinal vessels, causing them to close and resulting in retinal ischemia. The most prevalent member of the group is diabetic retinopathy,
16 but retinopathy of prematurity (ROP) and retinal vein occlusions are other types of ischemic retinopathies that lead to vision loss. Regardless of the inciting event leading to retinal vessel closure, once a sufficient area of the retina becomes hypoxic, hypoxia inducible factor-1 (HIF-1) levels become elevated,
17 which is sufficient to cause retinal NV.
18 HIF-1 stimulates the expression of genes that contain a
hypoxia response element (
HRE) in their promoter region, and two HIF-1–regulated genes that are necessary for sprouting of retinal NV are
Vegf and
angiopoietin-2. Several studies that helped to establish this were performed in the murine model of oxygen-induced ischemic retinopathy.
19 20 21 22 23 24 Many other molecular targets involved in retinal NV have also been identified in this model.
12 25 26 27 28 29 These studies require quantitative comparisons of the amount of retinal NV in eyes with ischemic retinopathy exposed to different experimental conditions. To quantify NV, it is necessary to distinguish new from preexisting vessels. Normally, there are no vessels above the inner limiting membrane (ILM), so any vessels above the ILM must represent NV. One approach is to perform serial sections, stain every tenth section with a marker for vascular cells, and measure the average vascular cell area above the ILM per section per eye. Given that the only cells invading beyond the ILM are vascular cells, some investigators have counted nuclei above the ILM. These approaches provide a high level of confidence in the measurements but are labor intensive and time consuming, primarily because of the need to cut serial sections through the entire eye. Another approach is to dissect retinas intact, stain whole retinas for vascular markers, and mount the whole retina. Because the normal retinal vasculature is generally stained along with neovascularization, a masked observer must distinguish new from preexistent vessels and outline the new vessels for measurement.
In this study, we sought to determine whether labeling of retinal cells before death facilitated their study in retinal flat mounts. We found that injection of a labeled anti-PECAM1 antibody stained endothelial cells in new and preexistent vessels. Vascular sprouting was observed from superficial vessels, as is widely recognized to occur, and also occurred from vessels deep within the retina. Quantification of intraretinal NV in ocular sections has been previously described.
30 High magnification provided exquisite structural detail, showing bulbous networks of vessels emanating from capillaries and large vessels. Even filopodia, previously described using high-magnification confocal microscopy,
31 were visualized at the tips of sprouts using standard fluorescence microscopy. The fine detail allows easy distinction between new and preexistent vessels by experienced observers, but not by image analysis software. However, injection of unlabeled anti-PECAM1 antibody into the vitreous, followed by postmortem incubation of whole retinas with secondary antibody, resulted in selective staining of new vessels with minimal staining of preexistent vessels or other background. It appears that use of a labeled primary antibody provides greater sensitivity and stains all vessels, whereas use of an unlabeled primary antibody is less sensitive, which allows it to recognize something that is different about the new vessels compared with preexistent vessels. Perhaps the upregulation of PECAM-1 or a structural difference allows for greater accessibility, but whatever the difference that allows for selective staining of new vessels, it is a major advantage given that it allows software recognition of NV and completely objective measurement of its area.
In addition to hypoxia-induced gene expression in endogenous retinal cells, mounting evidence indicates that circulating cells from outside the eye, particularly hematopoietic progenitor cells and leukocytes, may also contribute to retinal NV.
32 33 The role of macrophages in the pathogenesis of ischemia-induced retinal NV is uncertain, though it has been postulated that they to contribute to choroidal NV.
34 35 Coinjection into the vitreous of labeled anti-PECAM1 and anti-F4/80 showed that, compared with normal retinas, ischemic retinas show an increased number of macrophages that localize to regions of vascular sprouting but also to areas of vascular regression. These data support previous studies implicating macrophages in the regression of hyaloid vessels,
36 37 and they suggest that macrophages may also participate in the growth of new vessels in the eye. In other settings, subpopulations of macrophages have been shown to promote opposing effects
38 ; a similar phenomenon may help explain the apparent paradox of opposing macrophage-induced effects on different vessels in proximity in the eye. The fine detail provided by in vivo labeling will be useful to explore potential differences in these two populations of macrophages. The in vivo immunostaining technique can also be used to evaluate for cell surface receptors, as shown by staining for CXCR4, which showed labeling of a subpopulation of the bone marrow–derived cells.
Localization of secreted proteins can be particularly challenging because their constant release from the cells producing them may reduce levels below the limit of detection. However, in vivo immunostaining for PlGF showed a strong signal that colocalized with endothelial cells in new vessels and regressing vessels. Because VEGFR1 is upregulated on endothelial cells participating in NV,
39 40 the increase in staining for PlGF could be attributed to its interaction with the increased population of VEGFR1 on these cells or PlGF produced by the cells. It is intriguing, however, that PlGF, a chemoattractant for bone marrow–derived cells, localizes to growing and regressing vessels surrounded by bone marrow–derived cells. Additional studies are needed to determine whether PlGF contributes to the dense infiltration of macrophages in these locations.
In summary, we have described a new technique of in vivo immunostaining in the eye that will facilitate quantification of retinal NV and a related technique that shows exquisite structural detail of new vessels. This has allowed us to show that new vessels sprout from deep and from superficial capillaries in ischemic retina and that increased numbers of macrophages associate with growing and regressing vessels. The improved resolution enhances localization of cell surface and secreted proteins, suggesting that molecular characterization of macrophages may determine whether different populations of macrophages are involved in these opposing effects.