VEGF has long been recognized as an endothelial cell survival factor and mitogen as well as having a role in endothelial cell migration and vessel repair. There are multiple VEGF isoforms with VEGF165, a heparin-binding variant, being the most widely expressed in vivo. We have recently observed that neutrophil elastase can cleave VEGF and generate VEGFf, a biologically-active fragment of VEGF with altered receptor binding and activity . Unlike several of the other proteolytically processed forms of VEGF, which generally involve truncation of the primary VEGF polypeptide chain [27–29], NE appears to cleave the VEGF chain within internal regions in addition to the termini. In this way, VEGFf is held together by intra- and inter-chain disulfide bonds and shows interesting features resulting from selective alterations in its binding properties. The present paper focuses on coupling computer simulations with experimental studies to delve further into the impact of NE on the VEGF network in order to gain insight on the larger question of how proteolytic damage to growth factor-laden tissues during injury and inflammation might modulate bioavailability and activity.
Enzymatic processing of the extracellular matrix, a depository site for important biological molecules, can result in the release of stored growth factors thereby increasing their availability for activity. In addition, the degradation of the ECM may alter its ability to capture additional growth factors. We show this to be the case with NE and VEGF. Treatment of both surface-deposited fibronectin and cell-derived ECM with NE resulted in a significant decrease in VEGF binding (Figure 2). We postulated that elastase damage to ECM might result in increased cellular VEGFR binding due to a reduction in competition from the ECM sites. Using simulations, however, we found that the impact was negligible unless the sites were of very high number or high affinity (comparable or better than the receptors for VEGF, data not shown) (Figure 3). While the effect of ECM competition was negligible under the in vitro cell culture conditions used in our study, it is likely that this process would be very relevant in matrix-rich tissues in vivo where VEGF is stored and binding sites are abundant such as in the lung and blood vessel wall. Under those conditions, our simulations suggest that degradation of sites would have a major impact on VEGF availability for receptor binding.
Our studies suggest that a more important consequence of NE treatment with regard to the VEGF system may be the conversion of VEGF to VEGFf. Simulations demonstrate that the presence of VEGFf, with its altered binding properties compared to VEGF, could have a significant impact on VEGF receptor binding. Previously we have shown that VEGFf does not bind to VEGFR2  and data included herein show a loss of binding to NP-1 as well as to fibronectin (Figure 4). We postulated that VEGFf addition would result in increased VEGF binding to VEGFR2 due to a reduction in VEGF-VEGFR1 complexes as a simple consequence of VEGFf-VEGFR1 binding. Using simulations, however, we find that VEGFf can impact VEGF receptor interactions in a more complex manner (Figure 5). When VEGFf binding to VEGFR1 did not prevent VEGFR1 interactions with NP-1 or VEGFf was able to bind to VEGFR1 bound to NP-1 (Model 1), VEGFf did not increase overall VEGF binding to VEGFR2 and had only a limited effect. In contrast, when VEGFf binding to VEGFR1 prevented VEGFR1 interactions with NP-1 (Model 2), VEGFf essentially acted as a release mechanism for the VEGF-VEGFR2 stabilizer, NP-1, and increased VEGF interactions with VEGFR2 were evident.
The role of VEGFf appears therefore to be more of an indirect regulator of VEGF through NP-1 than a direct regulator of VEGF-receptor interactions. This was illustrated further by looking at the impact of NP-1 density (Figure 6) and the coupling rate between NP-1 and VEGFR1 (Figure 7). This type of ternary regulation is not unique to the VEGF system. Many heparin-binding growth factors, such as fibroblast growth factor -2 (FGF-2), form stabilizing complexes with cellular receptors and HSPGs, resulting in significant enhancement of receptor activation. Certainly receptors such as gp130 have been shown to play an important role in cytokine signaling within a number of systems making it a potentially more important system regulator than any individual component. This ternary complex regulation may also have a role in heterodimerization and cross-talk within (i.e., VEGFR1-R2 heterodimers) and between growth factor systems such as what is suggested for the IGF-I and EGF families .
Our original hypothesis was that elastase conversion of VEGF to VEGFf would result in the generation of a non-stimulating form of the growth factor due to its inability to bind the major signaling receptor VEGFR2. Both our experimental and simulation results suggest that this model is an oversimplification of this complex ligand-receptor system. VEGF alone stimulates endothelial cell migration while VEGFf alone does not (Figure 9). Conversion of VEGF to VEGFf should result in increased VEGFf and decreased VEGF with the same overall concentration of total growth factor to be found. With cell migration studies designed to test this effect, we found an increase in migration when the VEGF concentration was reduced by 25% with a corresponding substitution of VEGFf, in contrast to what one might expect based on the results with 100% VEGF and 100% VEGFf. Further increases in VEGFf beyond 25% and corresponding decreases in VEGF resulted in reduced migration. Simulations showed that this biphasic response could not be explained simply by an increase and then decrease in VEGF binding to VEGFR2 caused by VEGFf addition (Figure 10) regardless of whether VEGFf bound to VEGFR1-NP-1 complexes or not.
There is however evidence that NP-1  and VEGFR1 are also involved in VEGF signaling  and may be involved in mediating the migration process. Previously we found that Akt but not ERK 1/2 was activated by VEGFf suggesting that this key signal pathway might be mediated through VEGFR1 . Using simulations, we find that under conditions where the total VEGF plus VEGFf remains constant, VEGF binding to VEGFR1 and VEGFR2 is reduced while VEGFf binding to VEGFR1 is increased (Figure 10). Further, when one focuses on NP-1 stabilized VEGF and VEGFf complexes, we find that, with Model 1, a biphasic binding situation exists with a peak sum of VEGF-VEGFR2-NP-1 and VEGF-VEGFR1-NP-1 at an intermediate combination of VEGF and VEGFf. Certainly this is not direct evidence but simply further illustrates how NP-1 might be critical to the regulation of VEGF-VEGFf activity. Further experimental studies are needed to determine exact mechanisms but our simulations suggest possibilities worth exploring with regard to NP-1. There are also recent reports indicating that VEGFR1 and VEGFR2 can form heterodimers . Thus, VEGFf also has the potential to influence the formation of these complexes directly and through NP-1 interactions. As experimental data become available it will be interesting to explore these additional aspects of the system to fully appreciate how its complexity can be used to produce sophisticated modes of regulation.