In mice, VEGF is primarily secreted as VEGF₁₂₀, VEGF₁₆₄, and VEGF₁₈₈ (human VEGF is one amino acid longer: VEGF₁₂₁, VEGF₁₆₅, VEGF₁₈₉) [3]; longer isoforms include C-terminal motifs that increase binding to heparin and HSPGs in the ECM [21,22]. This increased matrix affinity can reduce the effective diffusivity of the isoform, altering the spatial gradient. For example, transgenic mice expressing only VEGF₁₂₀, an isoform lacking HSPG affinity [23], show a shallow VEGF gradient in the developing hindbrain [13] and retina [12], whereas wildtype mice, which predominantly express the heparin-binding VEGF₁₆₄, have a VEGF spatial distribution that is markedly more localized (Figure ​(Figure1A).1A). Systems secreting VEGF₁₈₈ show the greatest levels of ECM and basement membrane VEGF deposition [6,24].

As noted above, VEGF sensed by endothelial VEGF receptors has two isoform-specific components: the spatial differences between VEGF isoforms; and the isoform specificity of binding to the receptors themselves as discussed below. These give rise to a spectrum of isoform-dependent vascular phenotypes (Figure ​(Figure1B).1B). VEGF regulates sprouting angiogenesis at the level of tip cell filopodial guidance and migration and stalk cell proliferation [12]. Systems (mice or tumor xenografts) secreting only the diffusible VEGF₁₂₀ usually develop wide, tortuous, malformed vessels with infrequent branching (Figure ​(Figure1B)1B) [6,12,13]. This phenotype suggests excessive proliferation and an insufficiency in sprout guidance. On the other hand, VEGF₁₈₈ alone gives rise to vessels that are thin and numerous and have high branching density [3,6]. VEGF₁₆₄, similar to wild type mice, displays intermediate behaviour [4,6,13,25]. While these behaviours are thought to be primarily dependent upon the receptor tyrosine kinases VEGFR2 (KDR/Flk-1) [12], VEGFR1 (Flt-1) also seems to play an important but not yet fully understood role, in part by modulating VEGFR2 signaling [26]. VEGF isoforms also differentially bind to neuropilin-1 and -2 (NRP1 and NRP2), a class of semaphorin coreceptors that function as coreceptors for VEGFR2. Neuropilin-1, for example, can greatly enhance the signaling of VEGF₁₆₅ relative to VEGF₁₂₁ [23,27].

Proteolytic release of VEGF from the matrix seems to biologically mimic secretion of shorter isoforms (Figure ​(Figure1B).1B). For example, VEGF₁₂₀ elicits vascular effects at greater distances away from the source of secretion than VEGF₁₆₄ [6,12]; similarly, VEGF release by the protease MMP9 enhances VEGFR2 binding in quiescent vasculature by increasing soluble (diffusible) VEGF levels [16], activating tumorigenesis through the angiogenic switch [16,18,19]. Tumor xenografts expressing only the cleaved isoform VEGF₁₁₃ have large dilated vessels [7]. Inhibiting MMP9 results in markedly localized VEGF distributions reminiscent of VEGF₁₆₄ or VEGF₁₈₈, with higher levels of matrix-bound VEGF [6,13,19]. Secretion of a synthetic VEGF₁₆₄ that is resistant to proteolysis (VEGF_164Δ108-118) induces vascular patterning phenotypes similar to that of VEGF₁₈₈ alone, specifically high vascular densities [7] (Figure ​(Figure1B).1B). While the direct cleavage of VEGF's C-terminal heparin-binding and NRP1-binding domain is the most accepted mechanism of release (plasmin produces VEGF₁₁₀ from VEGF₁₆₅ [23]; MMP3 produces VEGF₁₁₃ from VEGF₁₆₄ [7]), cleavage of HSPGs and ECM by proteases or heparinases is also a potent release mechanism [15,28-30], but one that liberates intact VEGF. Recently, degradation of soluble VEGF inhibitors such as connective tissue growth factor and soluble VEGFR1 (sVEGFR1) has also been suggested to regulate VEGF [31-33].

The effects of protease-mediated VEGF release are not fully understood. VEGF_164Δ108-118- and VEGF₁₈₈-expressing tumors show similar intratumoral vessel architectures; however, paradoxically, their ability to enhance tumor growth are vastly different: VEGF₁₈₈ either significantly delays tumor growth [25] or cannot support it at all [6], while VEGF_164Δ108-118 results in tumor hyperproliferation [7]. Similarly, while most studies implicate MMPs, e.g. MMP9, in pro-angiogenic and carcinogenic behaviors through VEGF [15,16,20,29,32,33], in others, VEGF proteolysis results in an angiogenic response that is uncoordinated and ineffective [17], leading to lower vessel density and decreased tumor growth [7].

It is not fully understood how the spatial patterning of different VEGF isoforms and the processing of VEGF by proteases control endothelial behavior, however several possibilities have been suggested by experiments [7,12,34]. For example, increased matrix-bound VEGF in tissues (due to expression of longer isoforms or inhibition of proteolysis) may increase branching behavior and decrease vessel diameters by increasing p38/MAPK signaling [7,35]. Alternatively, low levels of soluble VEGF may independently increase sprouting behavior [36]. On the other hand, vascular patterning might arise due to neuropilin-1-dependent activation of p38/MAPK signaling [34], supported by the high affinity of VEGF₁₆₅ and VEGF₁₈₉ for NRP1 [37]. Finally, steep VEGF isoform gradients may improve tip cell filopodial stability while mitigating stalk cell proliferation [12].

Whatever the dominant signaling modality that guides vascular patterning, it appears from experimental data to have two particular properties. First, the seemingly monotonic increase in vascular density and decrease in vessel diameter with VEGF isoform length (Figure ​(Figure1B)1B) suggests that the critical feature of the VEGF distribution that controls vascular behavior also varies in a monotonic fashion. Monotonicity is also evident in the similar vascular phenotypes arising from mice dually expressing VEGF₁₂₀ and VEGF₁₈₈, and those only expressing VEGF₁₆₄, at the same total rate [6,13]. Second, increased VEGF cleavage has the opposite effect to increased HSPG binding [6,7] (Figure ​(Figure1A).1A). All of the above modalities seem to support both conditions; for example, among the isoforms, VEGF₁₈₈ would be expected to have the highest levels of matrix-bound VEGF [24], lowest levels of soluble VEGF [21,22], most directional gradients [13], and possibly greatest NRP1 affinity [37]. Similarly, VEGF cleavage prevents VEGF binding to HSPGs and NRP1 [23], and by solubilizing VEGF should lead to greater diffusion.

To separate out the above effects, we seek to develop a precise understanding of the effects of HSPG binding and VEGF proteolysis on the VEGF distribution by creating a computational model. The model should replicate key experimental observations regarding the VEGF distribution (summarized in Table ​Table1):1): longer isoforms with stronger heparin binding affinity have higher degrees of localization (shorter propagation distances and higher local peaks) (Figure ​(Figure1A)1A) [13]; increased heparin binding affinity results in greater levels of matrix deposition and decreased levels of soluble-phase VEGF [7,21,22,24,38]; and VEGF-cleaving proteases should reverse these patterns [7,16,19]. The inverse relationship between soluble and matrix-bound VEGF gives rise to the question of whether total VEGF is conserved in vivo. While this is expected in vitro [22,39], and was also seen by certain studies in vivo [7,19], other studies found significantly higher total VEGF levels for VEGF₁₆₅-expressing systems [25,39].

The current study extends our work on understanding the nature of VEGF transport in angiogenesis [40-44]. Among the numerous mechanisms of proteases modulating VEGF, we focus on VEGF cleavage, which we have previously studied [44]. Our model describes a sprout situated in a VEGF concentration gradient, to represent a range of biological systems, e.g. the hypoxic front of the hindbrain, tumor, or retina.

We use the computational model (Figure ​(Figure2)2) to understand mechanisms that might allow isoform-dependent VEGF localization and proteolytic redistribution to arise. Using a VEGF-VEGFR kinetic binding model, we investigate how VEGF signals may be detected by endothelial cells, e.g. soluble or matrix-bound VEGF, and the impact of NRP1. The results suggest that the system must exhibit isoform specificity in VEGF clearance or degradation: along with longer isoforms binding to the ECM with greater affinity, the longer isoforms must also be cleared or degraded from the soluble fraction at faster rates.

In the developing hindbrain system, VEGF₁₂₀ distributes in a much more disperse manner (Figure ​(Figure1A)1A) than VEGF₁₆₄ [13]. We find that while VEGF₁₆₄ may diffuse slower in tissues than VEGF₁₂₀, differences in grading will only arise when VEGF₁₆₄ is also degraded more rapidly by the surrounding hindbrain parenchyma, possibly due to specific uptake by cell surface HSPGs or NRP1, preventing it from diffusing far (Figure ​(Figure9A).9A). We propose that this difference in degradation rates between isoforms (which we term isoform-specific degradation, and is here a result of degradation of matrix-bound VEGF) also explains why secretion of non-heparin binding isoforms such as VEGF₁₁₃ or VEGF₁₂₀ leads to greater VEGF levels in the solution phase of tissues than secretion of heavier isoforms, given identical secretion rates [7,21]. Note that assuming a simpler mechanism of reversible HSPG binding in an in vivo system is not able to explain this behavior at steady state (Figure ​(Figure4A).4A). Importantly, our mechanism of isoform-specific degradation also provides a general explanation for the phenomena of protease-mediated redistribution of VEGF activity: proteases, by either cleaving VEGF [7], cleaving HSPGs [15], or by cleaving VEGF inhibitors [32], inhibit the degradation of VEGF (Figure ​(Figure9A),9A), thereby leading to the accumulation of VEGF in tissues and an increased range of receptor activation on the vasculature. This hypothesis is distinct from what is commonly noted in the literature as VEGF release, in which VEGF is focally released from the matrix by the action of proteases and diffuses to activate vasculature.

The second thrust of our current work is the elucidation of how VEGF isoforms and VEGF-releasing proteases might regulate vascular morphology (Figure ​(Figure1B).1B). We originally asked the question: which VEGF metrics may be responsible for guiding vascular patterning, to recapitulate both isoform monotonicity [6,12,13,25] and the protease dependence in vascular patterning [6,7]? We identified several possibilities including the levels of matrix-bound VEGF, soluble VEGF levels, NRP1 dependent signaling, and even VEGF gradient directionality. Using the results of our model, we can now provide a more specific answer to this question.

For example, the model predicts as expected that VEGF₁₈₈-secreting tumors would have the greatest levels of peritumoral matrix-bound VEGF [6], however, farther away at the vascular front, matrix-bound VEGF levels may actually be lower than if VEGF₁₆₅ were being secreted (Figure ​(Figure9B).9B). Thus, how a sprout experiences the ordering of the isoforms by sensing matrix-bound VEGF may not always be monotonic with respect to the heparin binding affinity of the VEGF isoform; it will be dependent on the distance between the sprout and the VEGF source (Figure ​(Figure9B).9B). (Note that the specific ordering depends on HSPG concentrations, protease levels, degradation rates, etc. Given their intrinsic variability in biological systems, the conclusion that a sufficiently strong matrix-binding isoform will have less matrix-bound VEGF is robust.) Instead, the possibility that VEGF₁₈₉-secreting systems also have the lowest levels of soluble VEGF of the different isoform-expressing systems seems to be consistent, even in the presence of proteases (Figure ​(Figure9B9B).

An important alternative to the concentration of matrix-bound VEGF levels may however be its directionality, especially as VEGF₁₈₉ seems to have the sharpest distributions in the absence of proteases (Figure 4Diii). While this holds true in the absence of proteases and can possibly explain observations in the mouse hindbrain [13], this metric does not account for the effect of VEGF-cleaving proteases, which further sharpen the distribution, instead of behaving in a HSPG-antagonistic fashion (Figure S4.3A). VEGF₁₈₉ also may have stronger NRP1 binding than VEGF₁₆₅ [37], however our results show that soluble uncleaved VEGF₁₈₉ levels may be so low (the majority will be cleaved or degraded), that total NRP1-potentiated VEGFR2 signaling may be weaker than that in VEGF₁₆₅-secreting systems. In fact, our model shows a surprising result: VEGFR1 binding of total soluble VEGF seems to better reconcile both isoform monotonicity and the antagonistic relationship between HSPG affinity and MMP activity than VEGFR2 binding does (Figure ​(Figure99).

Our results may provide insight into the nature of several experimental observations regarding vascular patterning. A comparison of our results to those of Ruhrberg et al's hindbrain data suggest that while the overall VEGF distribution is spatially non-monotonic (Figure ​(Figure1A)1A) [13], the underlying soluble fraction of VEGF is monotonic and is the basis for endothelial behavior. We note, however, that several important biological effects need to first be taken into account, for example the roles of filopodia [12,13,74] and the direct receptor signaling of matrix-bound VEGF [35].

The loss of monotonicity for matrix-bound VEGF (Figure ​(Figure7B,7B, Figure ​Figure9B)9B) is a result of the isoform-dependent decrease of the soluble uncleaved VEGF fraction in space. We now discuss how the tip cell filopodia can contribute to the process of isoform sensing. By projecting out in front of the tip cell, filopodia may be able to detect a region of space where matrix-bound VEGF may in fact operate in an isoform-monotonic manner (Figure ​(Figure9B),9B), effectively increasing the spatial range of the matrix-bound VEGF fraction's isoform monotonicity. We conceptualize this to sprouting angiogenesis. In the initial stages of sprouting where the sprout is far away from the VEGF source, only soluble VEGF will exhibit isoform monotonicity. However, as a sprout continues to invade closer into the VEGF secreting tissue, matrix-bound VEGF signaling and VEGF gradients will also exhibit the correct isoform monotonicity (refer to Additional file 1, section S3.2, Figure S4.4). These effects would be relevant in vivo as VEGF gradients never seem to be more than 50 μm in front of the vascular front [12,13], a range that can easily be sensed by filopodia [12]. Thus, while total soluble VEGF is theoretically the most isoform-monotonic signal, there may be no practical differences between it and matrix-bound VEGF. In fact, the information provided in matrix-bound VEGF may be even more relevant than that of soluble VEGF as it can result in differential VEGFR2 signaling that favors activation of p38/MAPK compared to soluble VEGF [35] and may mediate increased interaction with cell-surface NRP1 [34], both of which may play a direct role in branching and migration behaviors leading to increased vascular density. Unlike the behavior of matrix-bound VEGF, an isoform monotonic signal in NRP1-mediated VEGFR2 signaling does not necessarily emerge as a sprout invades into the VEGF secreting tissue (a result of reaction limitations in VEGF/VEGFR2/NRP1 coupling) (Figure S4.4C), indicating that NRP1-mediated VEGFR2 signaling may be less capable of giving rise to isoform-specific increases in vascular branching complexity. However, this conclusion is based on assuming a model where receptors only bind soluble VEGF; how the signaling of matrix-bound VEGF changes these conclusions is not known.

Our results indicated that VEGFR1 signaling of soluble VEGF may be able to also mediate isoform-specific differences in vascular patterning. However, this conclusion is at odds with the VEGFR2-dependent nature of angiogenesis in the retina [12], in in vitro patterning of porcine aortic endothelial cells [7], and in MMP9 induced carcinogenesis [16]. In addition, VEGFR1 signaling supports migratory behavior through p38/MAPK [26], however our results suggest an inhibitory role of VEGFR1 signaling on vascular density (Figure 8Bi). While our model cannot elucidate the importance of VEGFR1 signaling, it is interesting to note that when VEGFR2 signaling is monotonically increasing with the isoform length, VEGFR1 signaling is decreasing, suggesting that a balance between the two receptors may be important.

The unique role of matrix-bound VEGF in mediating the branching phenotype through filopodia may offer an interesting solution to the paradoxes of VEGF-cleaving MMPs in tumor growth. For example, both VEGF₁₈₈ tumors and VEGF_164Δ108-118 tumors are similar in that they have higher intratumoral vascular density and lower-diameter vessels compared to VEGF₁₆₄ tumors; however, whereas VEGF_164Δ108-118 tumors are markedly more proliferative than VEGF₁₆₄ tumors [7], VEGF₁₈₈ tumors show negligible growth [6]. We may conclude that this discrepancy indicates that the resulting vascular beds in the two tumors are different in ways that the intratumoral vessel density cannot measure, for example, in their connectivity to peritumoral vasculature [6], which may be a limiting factor for nutrient delivery; we propose that a separate aspect of VEGF signaling controls this latter behavior. Our model suggests a crucial difference between the two tumors: while VEGF₁₈₈ tumors show minimal VEGFR2 activation, even less than that of VEGF₁₂₀ tumors, VEGF_164Δ108-118 tumors instead show even higher VEGFR2 activation than VEGF₁₆₄ tumors (Figure ​(Figure8A).8A). As a result, it may be possible that the ability of the vasculature to support tissue growth may be dictated by VEGFR2 activation at the vessel itself, due to the receptor binding behavior of soluble VEGF, as suggested in numerous studies [16,17]. Furthermore, notice that peak VEGFR2 activation occurs between the VEGF₁₆₅ and VEGF₁₂₁ isoforms (Figure 8Ai). This may support the observation that VEGF₁₂₀ is at least as tumorigenic as VEGF₁₆₄ in some systems due to intrinsic differences between systems [25,39]. Overall, we hypothesize that intratumoral vessel density and branching is determined by matrix-bound VEGF detected by filopodia removed from the vessel surface, while vessel efficacy is dictated by VEGFR2 activation at the vessel body. As a result, we thus posit that the tumorigenic behavior ultimately depends on the ability of VEGF to keep its receptor and NRP1 binding domains intact. The inability of VEGF₁₈₈ tumors to elicit VEGFR2 activation, despite it potentially having higher affinity to NRP1 than VEGF₁₆₄, is a result of its degradation and cleavage (Figure ​(Figure7C7C).

This hypothesis may also explain the paradoxical roles of specific proteases in tumorigenesis. Our mechanism of MMP-mediated VEGF redistribution (Figure ​(Figure9A)9A) shows that proteases will increase the functional soluble VEGF concentration by inhibiting the process of VEGF degradation. However, while several modes of VEGF redistribution, such as through MMP9, heparinases, or VEGF inhibitor cleavage are implicated as pro-tumorigenic, mediating an angiogenic switch [15,16,19,20,29,30,32], there are several notable exceptions where protease activity instead leads to diminished tumor growth [7], an effect similar to the plasmin-mediated loss of wound healing due to a loss of angiogenesis [75]. We note an important trend: in studies where pro-tumorigenic behavior occurs from VEGF release, VEGF has not been shown to be directly cleaved. Bergers et al have shown that MMP9 mediates VEGF-release induced carcinogenesis in pancreatic islets [16] without determining the mechanism of release. However, subsequent studies have shown that MMP9 does not necessarily cleave VEGF [15,31-33], as suggested by [7], but instead acts to cleave HSPGs directly [15]. In fact, Joyce et al [30] show that MMPs and heparinases have similar effects in the pancreatic islet system, which strengthens the argument that the MMP9 induced angiogenic switch in [16] may have been mediated by HSPG cleavage. In contrast, studies where proteases reduce the angiogenic potential of VEGF show direct evidence of VEGF being cleaved and/or degraded [7,75,76]. We propose that VEGF needs to maintain coreceptor domains for effective tumorigenesis. Cleavage of VEGF, while increasing the total soluble VEGF concentration, may decrease overall VEGFR2 activation due to a loss of NRP1 binding (recapitulated in Figure 8Aii); heparinases, MMP9, and VEGF inhibitor proteases also prevent VEGF degradation but redistribute intact, coreceptor-binding VEGF.

Our results suggest that a central facet of VEGF patterning in vivo is its degradation, which we show necessarily occurs in an isoform-specific manner. Several mechanisms may underlie VEGF's isoform-specific degradation, and it is not currently known what, if any, mechanisms operate in the different in vivo experimental systems used. Interstitial cells and endothelial cells from nearby vessels may uptake VEGF isoform in an HSPG- or NRP1- dependent manner. This is supported by observations that NRP1 and VEGF receptors are typically present in many types of parenchyma, e.g. hindbrain [77], astrocytes [78], tumor [27,37], and skeletal muscle [79]. On the other hand, degradation may be due to the action of VEGF-degrading proteases, possibly the same proteases that also initially cleave VEGF; however, this possibility does not seem to be consistent with developmental systems where VEGF cleavage has not been detected [17]. An interesting possibility that has recently been raised is that of VEGF inhibition by soluble VEGF inhibitors, e.g. sVEGFR1 [11,33] or connective tissue growth factor [32], operating in an isoform-dependent manner through either HSPG complexation or through NRP1 complexation. Interestingly, each of these mechanisms also supports the ability of proteases to allow VEGF to escape degradation, through either HSPG binding or NRP1 binding, mediating VEGF redistribution. An important uncertainty in VEGF catabolism is whether endothelial cells represent the primary source of VEGF receptors and degradation in vivo or not. For example, while the background neural progenitor cells in the hindbrain may express NRP1 [78], endothelial expression is typically thought to be very strong [80]. Furthermore, immunochemical staining usually shows that VEGF concentrations diminish precisely at the vascular front [12,13].

Besides being able to reproduce experimental observations of VEGF patterning in vivo, is there evidence for isoform-specific degradation in tissues? VEGF (specifically, VEGF bioactivity) has been shown to degrade in vitro under cell culture conditions [53,81] and in fact, proteases that cleave VEGF into shorter isoforms also seem to degrade it further [51,82]. In addition, while VEGF degradation has not typically been studied as a cause of VEGF patterning, this view is commonly accepted in numerous developmental systems (e.g. Decapentaplegic, Wingless in Drosophilia) [52,83,84]. Evidence for isoform-specific degradation however may come from an indirect source. Perlecan knockdown in zebrafish establishes a diffusible VEGF phenotype [85]. Surprisingly, it also increased total tissue VEGF levels. Relevant to the present study is the fact that total VEGF levels did not decrease, supporting the view that HSPGs may be important mediators of VEGF degradation. However, there are other pieces of evidence that seem to contradict isoform-specific degradation: intravenously-injected bevacizumab shows significantly greater tumoral deposition in VEGF₁₈₉-expressing tumors compared to VEGF₁₆₅ tumors and VEGF₁₂₁ tumors [86]. Preliminary computational results suggest that this observation supports the HSPG-binding-only model on the basis of the total number of bevacizumab binding sites, i.e. VEGF, increases proportionally to the VEGF isoform matrix affinity.

An interesting prediction of the isoform-specific degradation model is that the extracellular residence times of different VEGF isoforms are roughly equal. Thus, a test of the model can be made by injecting labeled VEGF isoforms into tissues and measuring their half-lives in the interstitial fluid or lymph. If this test shows that the longer isoforms have significantly greater residence times in tissues, it would disprove the isoform-specific degradation model. The residence time of VEGF in tissues is a direct measure of the overall degradation and clearance rate, and its constancy is identical to the statement that the total levels of VEGF in tissue are roughly constant against differences in patterns of isoform secretion and VEGF proteolysis, a finding that is suggested by results in [7,19]. Note that this statement, however, does not contradict isoform-specific degradation. In this model, the rate of degradation (or clearance) of the soluble fraction of VEGF is isoform specific, with heavier isoforms showing more rapid degradation; however, accounting for all phases of VEGF (e.g. matrix bound, receptor bound), the average rate of degradation of any VEGF isoform is nearly identical (refer to Additional file 1, section S2).