Since the cellular processes underlying neovascular sprout formation remain incompletely understood [45
], increased attention to pericytes and their interaction with endothelial cells will be required not only to attain a better understanding of neovascularization in general, but also to realize the full potential of anti-angiogenic therapy. The critical contribution of pericytes during angiogenesis has been well established by observation of the pathological phenotypes of mice in which pericyte development is blocked [11
]. The functional importance of pericytes has been attributed largely to their ability to stabilize and provide structural support to pre-existing endothelial tubes. They are thought to accomplish this by controlling endothelial cell proliferation and motility, and by contributing to the establishment of a permeability barrier and the regulation of blood flow [36
]. However, it is now becoming clear that pericytes can play a much earlier role in microvascular development than previously realized. The use of NG2 and other markers for nascent pericytes has revealed the participation of these cells in the earliest stages of angiogenesis [6
]. Pericytes may even be important for the stimulation and guidance of nascent vascular tubes. Strategies for targeting pericytes may therefore be able to affect not only existing vessels, but also the formation of new vessels.
Our current studies show that intrinsic targeting of NG2 (by genetic ablation) leads to decreased ischemic angiogenesis in the mouse retina in response to hypoxia. The wild type mouse retina contains more than twice as many pathological vascular tufts as the retina of the NG2 null mouse. Since HIF-1α induction is similar in wild type and knockout retinas, we know that the defect in the null mouse lies not in the initial response of retinal cells to hypoxia, but probably in later stages of vascular cell responsiveness to HIF-1-induced factors such as VEGF. This seems reasonable in light of the fact that NG2 is not expressed by cells of the retina per se, but instead by pericytes in the microvasculature [17
A major factor in the sub-normal angiogenic response of the NG2 null retina appears to be reduced vascular cell proliferation. Only 41% as many mitotic pericytes are present in the ischemic vasculature of the NG2 null retina as in the wild type retina. These data represent the first direct in vivo
evidence in support of a role for NG2 in cell proliferation. The ability of NG2 to sequester growth factors such as bFGF and PDGF-AA and possibly assist in presentation of these factors to their respective signaling receptors could represent one mechanism by which the proteoglycan promotes cell proliferation [21
Interestingly, the absence of NG2 and the decreased number of mitotic pericytes is accompanied by a 1.7-fold decrease in the number of mitotic endothelial cells, suggestive of a stimulatory effect of pericytes on endothelial cell proliferation. This idea is somewhat at odds with previous reports that pericytes can inhibit endothelial cell proliferation in cell culture models [48
] and that the absence of pericytes is accompanied by endothelial cell hyperplasia in vivo [11
]. However, the pericyte/endothelial cell relationship is a complex, dynamic one that is likely to vary depending on the specific model under investigation. An excellent example of this is provided by a recent study of the proliferative retinopathy that results from endothelial cell-specific ablation of PDGF-B [47
]. The general conclusion from this work was that reduction of pericyte density below 50% of normal, invariably led to the development of proliferative retinopathy. Nevertheless, localized instances were also encountered in the same investigation [47
] in which increased pericyte density promoted the formation of chaotic, endothelial cell-rich vasculature, demonstrating that under certain conditions pericytes can have pro-angiogenic properties. The ability to use pericytes as effective anti-angiogenic targets also is suggestive of the pro-angiogenic nature of these cells [9
Our current data support a pro-angiogenic role for pericytes in the formation of ischemic retinal microvessels. Our results with wild type mice show that endothelial cells are richly invested by NG2-positive, PDGF β-receptor-positive pericytes in this pathological vasculature (see also [18
]). Coupled with our documentation of the early participation of NG2-positive pericytes during neovascularization [17
], these observations suggest the possibility that pericyte-derived factors or NG2-dependent sequestration of growth factors might act to promote the proliferation of endothelial cells. Alternatively, the ability of NG2 to neutralize the growth-inhibitory effects of angiostatin [22
] may promote endothelial cell proliferation in the wild type mouse, an effect that would be absent in the NG2 null mouse.
In addition to the quantitative reduction of ischemic angiogenesis in the NG2 knockout mouse, capillaries in the null mouse also have an altered cellular composition. The pericyte:endothelial cell investment ratio in ischemic vessels of the wild type retina is 0.86, or almost one pericyte per endothelial cell. This high investment ratio is characteristic of capillaries in the central nervous system in general, and the retina in particular, possibly contributing to the integrity of the blood-brain barrier and the high metabolic needs of neural tissues [5
]. This investment ratio falls to 0.24 in the ischemic neovasculature of the NG2 null retina. The relative changes in pericyte and endothelial cell proliferation in the NG2 knockout mouse would not appear to account for the magnitude of this decrease. Thus other factors that we have not yet investigated, such as decreased cell motility or increased apoptosis, may contribute to the large decrease in pericyte number relative to that of endothelial cells. While a specific role for NG2 in apoptosis has not been explored, there are numerous indications of NG2 involvement in cytoskeletal reorganization and cell motility [23
]. In future work it will therefore be important for us to investigate the impact of NG2 on these processes in the context of neovascularization.
Intrinsic targeting of NG2 by genetic ablation leads to an even more pronounced decrease in bFGF-induced corneal angiogenesis. Neovasculature covers a 4.4-fold greater surface area in the wild type cornea than in the NG2 null cornea, once again supporting the idea that NG2 plays a role in pericyte development and/or function, and in the development of new vasculature. Interestingly, extrinsic targeting of NG2 through the use of a neutralizing antibody also produces a significant decrease in corneal angiogenesis (2.7-fold). While our data do not allow us to determine which aspects of pericyte function are blocked by the antibody, previous studies have shown that anti-NG2 antibodies are capable of blocking growth factor-induced cell proliferation [58
] and both growth factor-induced and extracellular matrix-induced cell motility [27
] in cell culture models.
Our demonstration of the functional importance of NG2 during pathological ocular angiogenesis logically raises the question of the proteoglycan's function during normal developmental neovascularization. How can we rationalize the observation that the NG2 knockout mouse possesses functional vasculature? More than one answer is possible. First, pathological angiogenesis may differ in some respects from normal angiogenesis. During pathological angiogenesis, the vasculature may be responding to combinations of signals that are not normally experienced during development or else are occurring out of their normal sequence (multiple factors released by tumor cells would be a good example). The role of NG2 may be magnified under these abnormal circumstances. Additional experiments with wild type and NG2 knockout mice are planned in order to examine NG2-dependent aspects of angiogenesis in other types of pathological models such as tumor progression and wound healing. Second, both of our pathological angiogenesis models have utilized postnatal animals, whereas the bulk of developmental neovascularization takes place during embryogenesis. It seems possible that embryonic development involves a higher degree of plasticity than events that occur postnatally. In other words, the ability to compensate for the loss of NG2 may be greater during embryogenesis. In response to postnatal challenges, such compensatory mechanisms may not be available, thus facilitating our ability to detect the contribution of NG2. Examination of normal angiogenic events that occur postnatally (for example in normal retinal development) may therefore reveal the effects of NG2 ablation. It has required detailed and careful experimentation to detect changes in pathological retinal and corneal angiogenesis in the NG2 null mouse. The same type of painstaking analysis may be required to detect subtle deficiencies in developmental neovascularization in the knockout mouse. Such studies remain to be undertaken, but in light of our current results would appear to offer great promise.