The results we have obtained in the corneal model of inflammatory neovascularization allow two important conclusions to be drawn regarding the role of VEGF-A in blood and lymphatic vessel growth. First, endogenous VEGF-A can promote lymphangiogenesis, at least in the context of inflammatory forms of neovascularization. Second, signaling via VEGFR1 on leukocytes, particularly monocytes/macrophages, is a critical step in “immune amplification” of signals that promote pathological HA and lymphangiogenesis.
The present observations, that lymphangiogenesis and HA occur contemporaneously in CNV and that both responses are equally blocked by the selective inhibition of endogenous VEGF-A, appear to contradict the established notion that the ligation of VEGF-A to VEGFR2 induces solely HA, while interactions between VEGF-C/VEGF-D and VEGFR3 discretely mediate lymphangiogenesis. Indeed, a substantial literature supports this essential dichotomy in the function of VEGF family proteins and their receptors. For example, when applied to differentiated chick chorioallantoic membrane (CAM), VEGF-A was found to stimulate HA, but not lymphangiogenesis, while VEGF-C induced only lymphangiogenesis (15
). Interestingly, the VEGFR1-selective ligand PlGF was unable to induce either lymphangiogenesis or HA in the CAM assay. Similarly, in the corneal micropocket assay, VEGF-A was reported to induce HA but not lymphangiogenesis (11
), and in several studies using adenoviral overexpression, VEGF-C consistently induced lymphangiogenesis, while VEGF-A did not (12
). While these studies do demonstrate that VEGF-C/VEGFR3 and VEGF-A/VEGFR2 interactions can induce pure lymphangiogenic and hemangiogenic responses, respectively, under certain conditions, more recent studies are beginning to show that this dichotomy is far from complete.
In fact, VEGF-C and -D possess dual lymphangiogenic and hemangiogenic properties (2
), and VEGFR3, while universally expressed by the lymphatic endothelium, is also expressed by vascular endothelial cells under some conditions, particularly during embryonic development and periods of active vessel remodeling, including that occurring in pathology (34
In contrast to VEGF-C and -D, there is comparatively little evidence to support the notion that VEGF-A might be involved in lymphangiogenesis. However, a recent molecular profiling study has shown that lymphatic endothelial cells can express VEGFR2 and that VEGF-A is as effective as VEGF-C in supporting their survival and promoting tube formation in vitro (16
). Another recent study has demonstrated that adenoviral overexpression of VEGF-A164
in the rabbit ear leads to the formation of “giant” lymphatic vessels (20
). These studies raised the possibility that endogenous VEGF-A might, under some circumstances, play a role in promoting lymphangiogenesis — a possibility that we have confirmed in the present studies.
Specifically, we have demonstrated that (a) exogenous VEGF-A alone can induce lymphangiogenesis in the corneal pocket assay (different findings in a previous study [ref. 11
] might be explained by the use of different mouse strains, amounts of VEGF-A and staining techniques); (b) lymphangiogenesis and HA occur contemporaneously in a corneal injury model of inflammatory neovascularization; (c) selective pharmacological neutralization of VEGF-A/PlGF completely inhibited both HA and lymphangiogenesis in this model due to primary inhibition of blood and lymphatic vessel formation rather than via accelerated regression; and (d) following corneal injury, both lymphangiogenesis and HA were equivalently reduced in transgenic mice that expressed only either VEGF-A164
). Taken together, these results demonstrate that endogenous VEGF-A plays a critical role in promoting lymphangiogenesis as well as HA, at least under certain pathophysiological conditions.
We next turned our attention to mechanisms that might explain the coordinate induction of HA and lymphangiogenesis in this model and the effective suppression of both responses by selective inhibition of VEGF-A. Here we noted that in addition to suppressing CNV, administration of VEGF Trap also significantly suppressed the inflammatory response that is induced by the placement of intrastromal corneal sutures. It is well established that VEGF-A is a potent monocyte chemoattractant and that this effect is mediated by ligation of VEGFR1 (27
). Thus, one likely scenario is that VEGF-A indirectly stimulates lymphangiogenesis in CNV by recruiting bone marrow–derived cells, particularly monocytes/macrophages, to the affected site and these cells in turn are the source of one or more lymphangiogenic factors. Activated leucocytes are know to express and secrete a large number of cytokines and other regulatory peptides and proteins, including VEGF-A (31
). Moreover, it has recently been shown that a subfraction of circulating VEGFR3+
monocytes also strongly expresses VEGF-C and VEGF-D upon recruitment to peritumoral sites or in vitro stimulation (8
). Moreover, VEGF-C+
macrophages colocalize with new peritumoral lymph vessels, strongly suggesting a role for these cells in lymphangiogenesis (8
). Furthermore, it is known that proinflammatory cytokines, rather than hypoxia, upregulate VEGF-C expression (43
) and that VEGF-C consequently is highly expressed in inflammatory conditions (44
) suggesting even more strongly that VEGF-A–recruited macrophages upregulate VEGF-C/VEGF-D in response to corneal inflammatory cytokines. Indeed we have demonstrated here that CD11b+
macrophages in the inflamed corneal stroma express VEGF-C (more than VEGF-D) and that bone marrow–derived mouse macrophages transcribe both VEGF-C and -D mRNA.
The results of the present study directly support the concept that VEGF-A–mediated recruitment of inflammatory cells by VEGFR1 ligation is an important step in the initiation of the lymphangiogenic response in CNV. Pharmacological neutralization of VEGF-A significantly inhibited recruitment of inflammatory cells into the cornea after suture placement. Moreover, systemic depletion of bone marrow–derived cells by irradiation significantly attenuated corneal lymphangiogenesis after an inflammatory stimulus. Furthermore, local depletion of macrophages using subconjunctival clodronate liposomes substantially inhibited lymphangiogenesis. Finally, macrophages in inflamed corneas expressed both lymphangiogenic VEGF-C and -D. Taken together, these findings provide strong evidence that macrophage recruitment is an essential mediator of the (indirect) lymphangiogenic effect of VEGF-A (Figure depicts this concept). Here it is also important to note that macrophage depletion not only suppressed lymphangiogenesis following corneal injury but also effectively suppressed concomitant HA. This observation is consistent with a previous study showing that selective macrophage depletion inhibits pathological neovascularization in other disease models (45
), supporting the notion that inflammation is also a requisite component of pathological HA mediated by VEGF-A (45
Figure 11 Proposed concept of an (indirect) lymphangiogenic role of VEGF-A via recruitment of bone marrow–derived macrophages, which in turn can release both hemangiogenic and lymphangiogenic growth factors. Macrophages seem to be important for immune amplification, (more ...)
While VEGF-mediated recruitment of inflammatory cells clearly plays an important and apparently predominant role in promoting pathological neovascularization, it is quite likely that other, more direct actions of VEGF-A contribute to initiating both hemangiogenic and lymphangiogenic responses. For example, VEGF-A acts directly on vascular endothelium to upregulate the expression of adhesion molecules that promote leukostasis (47
). Likewise, rapid VEGF-mediated increases in the permeability of resident vessels and the consequent extravasation of serum proteins also serve to promote the subsequent formation of both blood and lymphatic vessels (17
). It is also possible that VEGF-A acts directly on VEGFR2 to promote the growth and organization of the lymphatic endothelium (16
). Finally, in addition to recruiting inflammatory cells that supply cytokines and growth factors to the site of injury, VEGF-A may also amplify angiogenic responses by recruiting VEGFR1-positive hematopoietic progenitor cells to the affected site and promoting their differentiation into vascular endothelium (for review see refs. 2
While our data strongly support the concept that recruitment of monocytes/macrophages by VEGF-A, through VEGFR1, is an early and essential step in an immune amplification cascade that leads to both inflammatory HA and lymphangiogenesis (see Figure ), it is formally possible that the VEGFR1 ligand PlGF could also be partly responsible for promoting both corneal HA and lymphangiogenesis. Indeed, both VEGF TrapR1R2
and VEGF TrapR1/A40
bind PlGF as well as VEGF-A. Although results of other studies indicate that PlGF can collaborate with VEGF-A in the stimulation of pathological HA (51
), three facts “argue against” the possibility that endogenous PlGF plays a significant role in promoting inflammatory lymphangiogenesis: (a) PlGF binds only to VEGFR1, while the lymphatic endothelium expresses only VEGFR2 and VEGFR3 (53
); (b) overexpression of AD-PlGF in the rabbit ear resulted in the formation of blood vessels, but in contrast to VEGF-A it did not cause lymphangiogenesis (20
); and (c) in the present study, both lymphangiogenesis and HA were comparably reduced in VEGF-A isoform–deficient transgenic mice.
Currently, the most parsimonious mechanistic explanation for VEGF-A–mediated lymphangiogenesis in CNV is that VEGF-A promotes this response indirectly by binding to VEGFR1 and recruiting macrophages that secrete VEGF-C and/or VEGF-D at the site of injury. However, in a previous study, application of an exogenous VEGF-C isoform (156S) was unable to induce lymphangiogenesis in the cornea micropocket assay (in contrast, e.g., to the skin) (11
). Thus, while there is evidence that VEGFR3 signaling is necessary for corneal lymphangiogenesis (11
), the hypothesis that VEGFR3-signalling is sufficient for the initiation of corneal lymphangiogenesis awaits experimental confirmation.
Inflammation is a common feature of diverse conditions characterized by pathological neovascularization, so it is quite possible that VEGF-A may play an important role in promoting lymphangiogenesis as well as abnormal HA in other disease states (42
). If so, the present findings may have important ramifications for “antiangiogenesis” therapies currently in development for the treatment of a variety of diseases. As previously noted, a strong correlation exists between the degree of peritumoral inflammation and lymphangiogenesis in diverse types of human tumors (42
). VEGF-A is highly expressed in most solid tumors and might serve to amplify lymphangiogenesis as well as HA in cancers by recruiting “lymphangiogenic” monocytes/macrophages. Thus, antiangiogenic strategies that target VEGF-A signaling might also prove effective in at least partially suppressing peritumoral lymphangiogenesis. In the context of corneal transplant rejection, recruitment of antigen-presenting cells into afferent lymphatic vessels is an essential step in the process by which the host immune response emerges to foreign transplant antigens. Therapeutic strategies aimed at suppressing newly outgrowing lymphatics should improve transplant survival by inhibiting allosensitization (C. Cursiefen and J.W. Streilein, unpublished observations). As immune rejection is the most important cause of corneal graft failure, our findings suggest that effective inhibitors of VEGF-A signaling have the potential to improve the survival of corneal transplants.
Note added in proof.
J. Wayne Streilein is deceased.