We have demonstrated that normalized revascularization of the vaso-obliterated retina in the mouse OIR model strongly correlates with the survival of astroglia and microglia during hypoxic insult. In each case, astrocyte and microglia survival within the vaso-obliterated, hypoxic retina correlates with accelerated revascularization of the normal retinal plexuses and reduced pathological intravitreal NV. Vascular degeneration in the central retinal vasculature is similar in both BALB/cByJ and C57Bl/6J mouse retinas after exposure to hyperoxia. Therefore, the neovascular drive is likely to be comparable in the two strains upon return to normoxia. However, in BALB/cByJ mice, the retinal astrocytes and retinal microglia survive within the vaso-obliterated areas of the retina following return to normoxia. Subsequently, normal retinal revascularization occurs much more quickly and without formation of pathological pre-retinal NV. In C57Bl/6J mice, the retinal astrocytes and retinal microglia disappear from the avascular, hypoxic areas of the retina and this is associated with slower recovery and formation of large pathological, pre-retinal vascular tufts.
The correlation between the survival of astrocytes within the hypoxic, vaso-obliterated areas after return to normoxia and subsequent revascularization in the mouse OIR model is also demonstrated by analyzing treatments that preserve the endogenous astrocytic template and subsequently normalize retinal revascularization. We hypothesize that in the absence of astrocytes, no template remains to guide blood vessel growth during the revascularization resulting in disorganized vessels and the formation of pathological, intravitreal NV. However, when astrocytes survive within the central vaso-obliterated retina, the remaining astrocytic template facilitates normalized revascularization of the characteristic retinal vascular plexuses. Endothelial filopodia can be observed extended along the astrocytic template () during revascularization suggesting that the vessels are indeed identifying underlying guidance cues provided by the astrocytes. This facilitates similar retinal vascular guidance mechanisms to those that initially produce the characteristic vascular plexuses during development, a process known to be mediated by endothelial filopodia and astrocytic guidance cues (Dorrell et al. 2002
; Fruttiger et al. 1996
; Gerhardt et al. 2003
). BM-MPCs, astrocytes and astrocyte-conditioned media all protect the endogenous astrocytes and subsequently promote normalized revascularization in C57BL/6J mouse retinas.
Proteomic analysis of the astrocyte-conditioned media was used to identify proteins that may mediate the rescue effect (). Both bFGF and VEGF were identified in the astrocyte-conditioned media () and are known to be secreted by retinal astrocytes and microglia (Scherer and Schnitzer 1994; Yamada et al. 2000
). We have demonstrated that intravitreal injection of either bFGF or VEGF at low doses mimics the astrocyte and astrocyte-conditioned media-mediated rescue effects. It is unlikely that such therapy would be used to treat vascular-associated retinopathies since both bFGF and VEGF are known to contribute to neovascularization in these diseases. In fact, most therapies currently used to treat ischemic retinopathies are designed to inhibit their activity. However, our data demonstrates that at specific doses, these growth factors can actually have beneficial effects, presumably by protecting critical vascular-related glial cells from hypoxia-induced cell death. Our data suggests that it is important to consider possible detrimental effects that treatments blocking similar growth factors might have on vascular-related cells such as microglia and astrocytes. Inhibition of VEGF or bFGF certainly can block endothelial cell proliferation and reduce vascular permeability (Aiello et al. 1995
; Caldwell et al. 2003
; Witmer et al. 2003
), but may also lead to loss of the important microglia and astrocytes as well and long-term repercussions. Thus, it may be important to identify other potential therapies to treat ischemic diseases of the eye.
Cell-based therapy may be one avenue by which ocular vascular diseases can be treated in the future. Recently, a population of Müller glia were found that can function as neuronal stem cells (Bernardos et al. 2007
). Perhaps, in the future, these cells or other glial progenitors may be found that can replace lost astrocytes. In the meantime, cell therapy with astrocytes or myeloid progenitors may be a useful approach since these cells can react to the local environment, potentially secrete appropriate levels of growth factors important for normalized vascular regrowth, and lead to retinal vascular stabilization. Astrocyte injection rescues the vascular phenotype whereas other cell types such as fibroblasts or embryonic stem cells have no effect. However, we do not observe incorporation of the injected astrocytes into the retina. Thus, the use of astrocytes as a long-term cell therapy in cases of continuing ischemia may not be practical. Previous studies from our laboratory have demonstrated that intravitreal injection of bone marrow-derived myeloid progenitor cells (BM-MPCs) leads to accelerated and normalized revascularization, as well as inhibition of pathological NV following hyperoxia-induced vascular obliteration in the mouse OIR model (Ritter et al. 2006
). We now demonstrate that this vascular rescue is preceded by BM-MPC mediated rescue of retinal astrocytes within the vascular obliterated zones of C57Bl/6J mouse retinas, suggesting a potential mechanism of action for the observed rescue activity. By preventing astrocyte cell death in the hypoxic zones, and re-populating the central retina with microglia, the BM-MPCs may help facilitate normalized revascularization of the central retina in C57/Bl-6 mice which would otherwise develop pathological NV. Myeloid progenitors incorporate into the retina and become functionally active microglia and thus may have continued, long-term beneficial effects on the survival of critical vascular-associated glial cells. This may provide a paradigm for long-term therapy which can normalize revascularization of the ischemic retina and alleviate problems associated with the underlying disease.