Apoptosis of vascular cells plays an important role in remodeling and regression of the developing vasculature and it is tightly regulated by expression of pro- and anti-apoptotic factors (Pollman et al., 1999
; Walsh et al., 2000
; Wang et al., 2003
). Bcl-2 family members are major regulators of apoptosis, either acting to positively or negatively influence apoptosis. Aberrant regulation of apoptotic pathways impacts retinal vascular development and retinal diseases including ROP, diabetic retinopathy and age-related macular degeneration.
Although coordinated regulation of anti- and pro-apoptotic factors occur during retinal vascular development, only bcl-2’s role has been studied in any detail with regards to its modulation by angiogenic and anti-angiogenic factors. For example, basic fibroblast growth factor (FGF2) and VEGF mediate their proangiogenic effects, partially, through enhanced expression of bcl-2 (Folkman, 2003
; Iervolino et al., 2002
; Jimenez et al., 2000
; Karsan et al., 1997
; Sorenson and Sheibani, 2002
), while most anti-angiogenic factors, including thrombospondin-1 and endostatin, inhibit angiogenesis through down-regulation of bcl-2 expression (Dias et al., 2002
; Giancotti and Ruoslahti, 1999
). In addition, over-expression of bcl-2 in EC not only enhances formation of blood vessels but also promotes progressive maturation of vasculature by recruitment of perivascular supporting cells (Giancotti and Ruoslahti, 1999
; Kim et al., 2000
). However, less is known regarding bim’s role. Both insulin-like growth factor-1 and erythropoietin, which are protective during OIR (Chen et al., 2008
; Lofqvist et al., 2006
; Lofqvist et al., 2009
), down-regulate bim expression (Abutin et al., 2009
; Linseman et al., 2002
). This is also consistent with the protective role of erythropoietin during the first phase of OIR (Chen et al., 2008
; Lofqvist et al., 2006
), and support a role proposed here for bim in vessel obliteration. Administration of erythropoietin during the second phase of OIR promoted angiogenesis (Chen et al., 2008
; Chen et al., 2009
). Therefore, bcl-2 family members may function not only as modulators of apoptosis, but also as important regulators of vascular function.
Here, we show that formation of the superficial layer of retinal vasculature appears to proceed similarly in bim +/+ and bim -/- mice. We observed a similar relative distance that the retinal vasculature spread from the optic nerve during the first week of life, as well as the spreading of astrocytes, and appearance and density of tip cells. However, formation of the deep vascular plexus was enhanced consistent with the increased retinal vascular density in the absence of bim. Interestingly, vascular cell proliferation and apoptosis was precociously down-regulated prior to P14 in retinas from bim -/- mice. The decreased apoptosis and proliferation in retinas from P14 bim -/- mice was in line with their stage of retinal vascular development rather than postnatal age.
Bcl-2 and bim have been shown to impact not only apoptosis but also cell adhesion, migration and extracellular matrix production (Grutzmacher et al., 2010
; Kondo et al., 2008
; Sheibani, 2007
; Ziehr et al., 2004
). It is tempting to speculate that, in the absence of bim, the extracellular milieu changes such that vascular sprouting and elongation is accelerated leading to enhanced formation of the deep vascular plexus and perhaps its maturation. This hypothesis is supported by increased levels of collagen IV detected in the retinal vasculature of bim -/- mice.
The development of retinal vasculature is tightly coupled to its oxygen needs and exhibits an inherent sensitivity to changes in oxygen levels. This predisposes the developing retina to ROP, a major cause of blindness in immature infants exposed to high levels of oxygen and then brought to room air. In the mouse OIR model, P7 mice are exposed to 75% oxygen for 5 days, and then returned to room air for 5 days. The exposure of developing retinal vasculature to high oxygen prevents growth of additional vessels and promotes loss of existing vessels due to diminished expression of VEGF, and perhaps the anti-apoptotic protein bcl-2 (Wang et al., 2005
). When these mice are returned to room air, the retina becomes ischemic and promotes growth of new vessels, which grow into the vitreous. VEGF expression is maximal 3 days following return to room (P15) and its expression thought to drive abnormal neovascularization in the ischemic retina (Pierce et al., 1996
Little is known about the cellular and molecular mechanisms involved in inherent sensitivity of the developing retinal vasculature to high oxygen, and more specifically what role bim plays during this process. Our studies demonstrated that lack of bim expression relieves the retina’s inherent sensitivity to high oxygen and ischemia-mediated neovascularization. Hyperoxia-mediated vessel obliteration did not occur in the absence of bim suggesting that bim expression may aid this apoptosis driven process. VEGF expression increases following ischemia and is thought to drive normal retinal vascularization and ischemia-mediated retinal neovascularization (Pierce et al., 1996
; Stone et al., 1996
; Stone et al., 1995
). We had previously observed in bcl-2 -/- significant vessel obliteration but decreased neovascularization even though VEGF levels were similar to that of their wild-type counterpart (Wang et al., 2005
). Here we show that despite similar VEGF levels in retinas of both bim +/+ and bim -/- mice during OIR, neovascularization did not occur in the absence of bim. Thus, during OIR in bim -/- mice, VEGF expression appears to only drive normal retinal vascularization, while during OIR in bim +/+ mice VEGF expression appears to drive abnormal vascularization (). Bim and bcl-2 expression influences EC migration (Kondo et al., 2008
) (Grutzmacher et al., 2010
). Thus, it is tempting to speculate that expression of these proteins may influence or be influenced by directional cues such that either normal retinal vascularization or pathological neovascularization are favored during OIR. These data begin to suggest that VEGF expression, on its own, is not sufficient to drive neovascularization following OIR in the presence or absence of retinal vessel obliteration. Although VEGF has been shown to induce bcl-2 expression little is known regarding its impact on bim expression. Our data suggest that bcl-2 family members may act downstream of VEGF in modulating angiogenesis. To the best of our knowledge, bim is the only gene whose deficiency is shown to protect the developing retinal vasculature from sensitivity to hyperoxia.
Exposure to high oxygen prevents formation of the retinal deep vascular plexus during OIR. However in the absence of bim, vascularization of the inner retina proceeded rather normally. This is perhaps due to the lack of hyperoxia-mediated vessel obliteration or a unique ability of bim -/- EC to circumvent hyperoxia-mediated inhibition of normal angiogenesis. In addition, these data may begin to hint at the mechanism by which retinal vascular pruning is modulated by bim. Normally vascular pruning occurs when an overabundance of oxygen triggers a drop in VEGF expression. Perhaps, hyperoxia-mediated vessel obliteration could be considered retinal vascular pruning gone arye. Our data suggests that bim expression facilitates vascular pruning and remodeling. In the absence of bim, changes in oxygen and/or VEGF levels are not sufficient to induce pruning as evidenced by the increased vascular density, EC numbers and attenuation of hyaloid vessel regression in bim -/- mice.
The retina can undergo two types of neovascularization: retinal neovascularization and CNV, as occurs in retinopathy of prematurity and age-related macular degeneration, respectively. Unfortunately, the molecular and cellular mechanisms involved in CNV have not been studied to the same degree as retinal neovascularization. Although these processes are thought to share molecular signals, the role hypoxia plays during CNV remains questionable (Campochiaro, 2000
). Retinal neovascularization was not observed during OIR in bim -/- mice while laser-induced CNV occurred in the absence of bim. Thus, our data is consistent with retinal neovascularization and CNV being regulated in a different manner. Whether these differences in neovascularization are due to choroidal EC fenestration, hypoxia, or stage of development require further delineation. Alternatively, different bcl-2 family member death effectors may modulate choroidal versus retinal neovascularization. Thus, gaining a better understanding of the role bim plays in modulating ocular vascular homeostasis will give us new points of intervention.
- Lack of bim leads to increased retinal vascular density.
- Bim is responsible for retinal vascular sensitivity to hyperoxia.
- Vessel obliteration does not occur during OIR in the absence of bim.