Historically, VEGFR-1 was assigned a role as a nonsignaling decoy receptor because of the low activity and embryonic dispensability of its tyrosine kinase function. More recently, its role has become more interesting because VEGFR-1 signaling has been reported both to promote (6
) and suppress (8
) VEGF-A–driven angiogenesis. We report not only that PlGF-1 inhibits inflammatory CNV, extending the scope of VEGFR-1 function, but also what we believe is the unprecedented observation that VEGF-A itself can suppress angiogenesis. Multiple lines of evidence emerging from genetic ablation, antibody neutralization, and receptor-selective ligand activation all strongly support the thesis that the antiangiogenic action of VEGF-A is mediated by VEGFR-1. Previously, VEGF-A has been reported to reduce VEGF-E–induced VEGFR-2 tyrosine kinase phosphorylation in capillary endothelial cells in vitro, raising the provocative hypothesis that VEGF-A could limit its own angiogenic activity through VEGFR-1 (29
). We have presented in vivo confirmation of this hypothesis. These findings contribute significantly to our understanding of the consequences of the interaction of VEGFR-1 with its ligands and describe a unique mechanism by which VEGFR-1 regulates angiogenesis.
Prior reports have noted that the biological effects of VEGF-A are dose dependent. These observations are consistent with our data, which indicate that high levels of VEGF-A cannot sustain, and indeed may inhibit, CNV. Exogenous VEGF-A in the myocardium or skeletal muscle can lead to dysregulated vasculogenesis (30
) and disrupt embryonic intersomitic artery formation (32
). In addition, exogenous VEGF-A can inhibit smooth muscle cell proliferation, without affecting angiogenesis (33
). However, we believe our data are the first to demonstrate direct arrest of endothelial cell proliferation and frank inhibition of angiogenesis by VEGF-A.
These data do not contradict the well-described angiogenic properties of VEGF-A. Indeed, our data indicating that intravitreous injection of antibodies against VEGF-A or VEGFR-2 reduces laser-induced CNV confirm findings of other investigators using different antagonists (11
). Rather, they unveil an unrecognized ability of excess VEGF-A in the postinjury setting to suppress angiogenesis by predominantly activating VEGFR-1 signaling and preventing endothelial cells from responding to mitogenic signals. It appears that the system is exquisitely sensitive to the precise level of VEGF-A in the immediate postinjury period and responds differently if that level exceeds the concentration of VEGF-A induced by injury. The “switch” that diverts the injured tissue away from proliferation is driven through VEGFR-1 signaling, which appears to be dominant in an environment of excess VEGF-A, in contrast to the usual dominance of VEGFR-2. Unraveling this antagonistic pathway of excess VEGF-A and comparing it with those of existing angiogenic inhibitors may reveal additional robust and powerful therapeutic targets in the panoply of angiogenesis-driven disorders.
Rakic and colleagues demonstrated that systemic administration of anti–VEGFR-1 antibody inhibits experimental CNV (24
), while we found that intravitreous delivery did not. This divergence could arise from the fact that cell populations are affected differentially by local versus systemic VEGFR-1 blockade. Systemic but not intravitreous VEGFR-1 blockade would interfere with mobilization of bone marrow–derived progenitor cells (35
) that are known to contribute to CNV (36
). This difference is clinically relevant as most anti–VEGF-A therapies for CNV rely on intravitreous delivery to minimize potential adverse effects of systemic VEGF-A antagonism.
Rakic et al. (24
) also demonstrated that laser-induced CNV is inhibited in Plgf–/–
mice, which is consistent with the role of PlGF in the bone marrow, where it mobilizes progenitors either directly by recruiting VEGFR-1+
cells or indirectly by inducing matrix metalloproteinase-9, which increases progenitor cell proliferation and motility via release of soluble Kit ligand (35
). In contrast, intravitreous injection of PlGF, which inhibits CNV when administered after injury, would not execute such effects on the bone marrow. These differences also might have emerged because exogenous and endogenous PlGF may heterodimerize with VEGF-A differently in CNV. The role of these heterodimers is controversial because they have been reported both to promote (38
) and antagonize (39
) neovascularization. An analogous variation is observed in experimental retinal neovascularization, which is inhibited both by genetic ablation of Plgf
) and by intravitreous administration of PlGF (40
), although the latter may be due to increased cell survival. A similar divergence in angiogenic response to endogenous and exogenous proteins exists in the case of other cytokines, such as plasminogen activator inhibitor type I (PAI-1). Indeed, Rakic and colleagues also have shown that laser-induced CNV is inhibited both in Pai–/–
mice and in PAI-1–treated wild-type mice (41
). The apparently different effects of endogenous and exogenous PlGF also might reflect modulation of plasminogen activator activity by VEGFR-1 activation (42
Reduction in CNV in Plgf–/–
mice also is intriguing because these mice express other VEGFR-1 ligands, including VEGF-A (6
). This raises the formal possibility that reduced CNV in Plgf–/–
mice might reflect molecular or developmental plasticity in response to gene disruption rather than PlGF deficiency alone. Secondary developmental effects induced by the altered vascular phenotype account for a similar divergence phenomenon in Alk1–/–
mice, which display enhanced angiogenesis (44
), while enforced activin receptor–like kinase 1 (ALK1) expression stimulates endothelial cell migration and proliferation (45
). It also is possible that subtle defects in ocular vascular development in Plgf–/–
mice reported by Carmeliet and colleagues (6
) might have influenced their CNV responses.
These data add VEGF-A to the group of cytokines, such as angiopoietin-1 (46
), nitric oxide (49
), pigment epithelium– derived factor (51
), and TGF-β (45
), whose modulation of angiogenesis displays context-dependent bidirectionality. The paradoxical effect of VEGF-A resembles that of TGF-β, which is proangiogenic at low doses and antiangiogenic at high doses, an effect attributed to differential activation of the TGF-β receptors ALK1 and ALK5 (45
), akin to the differential routing via VEGFR-1 and VEGFR-2. This Janus-like effect reveals novel therapeutic strategies to modulate angiogenesis in the setting of inflammation and highlights the importance of developing assays for markers such as SPARC to target therapeutics more specifically.
In most systems, VEGFR-1 tyrosine kinase activity has been described as weak. Our findings provide a paradigm by which its activation is controlled in vivo by SPARC and illustrate the multifunctional nature of this receptor in promoting or curtailing angiogenesis. These data might also explain the divergent outcomes of experiments involving VEGFR-1 function in different angiogenesis models. The poor intrinsic kinase activity of VEGFR-1 in many systems may be due to repressive elements in its juxtamembrane domain (55
). It is also possible that VEGFR-1 signaling could occur without phosphorylation of consensus-positive regulatory tyrosine residues, i.e., VEGFR-1 could be a poor substrate for itself. Further investigation of the role of SPARC in maintaining repression of VEGFR-1 may improve our fragmentary understanding of its activation, particularly during development and in cancer, where this receptor appears functional. In addition, the propensity of the macula to develop CNV despite widespread subretinal disease in AMD may be related to the concentration of SPARC in this central region in monkeys (56
) (and presumably in humans), which may prevent downregulation of proangiogenic VEGFR-2 signaling by VEGFR-1.
The expression and activity of SPARC are segregated largely to tissues that are undergoing remodeling or turnover. As such, it is well placed to regulate the activity of potent growth factors such as VEGF-A, which often are the primary stimuli for remodeling of the local microenvironment. The present study provides a physiological example of how SPARC potentially regulates the angiogenic process. The initiation of angiogenesis by soluble factors such as VEGF-A is complex and involves the activation of individual endothelial cells that express both VEGFR-1 and VEGFR-2. Our data point to extracellular adaptor proteins as critical linchpins in the induction of the VEGFR-2–driven angiogenic program. Previous studies (17
) showed that SPARC can interfere with VEGF-A ligation of VEGFR-1 and that SPARC-mediated inhibition of VEGF-A–induced phosphorylation of VEGFR-1 was long lived (at least 24 hours). In the context of CNV, this time frame is relevant to the demonstrated activity of VEGF-A in that setting. Investigations using mice with a deficiency in a matricellular protein (e.g., thrombospondin-1 and/or -2, the SPARC ortholog hevin, or SPARC) support the hypothesis that the function of matricellular proteins is contextual and that regulated expression of these proteins is important for maintenance of tissue homeostasis and responses to injury (16
The basal CNV response of Sparc–/–
mice was greater than that of wild-type counterparts (Figure D), a result consistent with the heightened neovascular response in sponge implants reported in Sparc–/–
animals as well as the synergistic increase in vascularization in the foreign body response of SPARC/hevin double-null mice (59
). The inhibitory effect of SPARC on vessel growth resides in part in the C terminal, Ca+2
-binding EF hand, whereas other peptides released by plasmin or stromelysin-1 stimulate angiogenesis by affecting the cell cycle and/or migration of endothelial cells (27
). Our present study emphasizes the contextual dependence of SPARC and its activity as a regulator of angiogenesis.
Our data define a previously unrecognized autoregulatory potential of excess VEGF-A and are relevant to ongoing clinical trials examining anti–VEGF-A therapy in AMD and VEGF-A over-expression in ischemic limb and cardiac disease. Data from the pegaptanib clinical trials in patients with AMD showed an inverse therapeutic dose response (15
), indicating that partial inhibition of VEGF-A could be optimal. The inability of this drug to arrest the increasing size of the CNV lesion with time in patients with AMD indicates that VEGF-A might have dual actions in the human eye as well. However, that pegaptanib decreased the rate of vascular leakage points to a divergent response of VEGF-A in mediating growth versus hyperpermeability of new blood vessels. Whether similar bifurcation of VEGF-A signaling occurs in laser-induced CNV warrants investigation.
Our findings provide insight into the context and stage dependency of the role of VEGF-A in ocular neovascularization as well as interactions between a matricellular protein and a VEGF receptor that regulate and route signaling by VEGF-A. Our findings also demonstrate alternative pathways of VEGF-A signal transduction and emphasize the need to clarify the complex effects of interactions among VEGF-A, its receptors, SPARC, and the chemokine network in ocular neovascularization before VEGF-A alone can be considered an ideal therapeutic target.