Introduction
Pathogenesis of diabetic retinopathy (DR) is associated with deregulated expression of various growth factors of which elevated levels were found in the vitreous fluid of DR patients [
1‐
4]. Appearance of diabetic macular edema (DME), observed at any stage of DR, correlates with vascular endothelial growth factor-A (VEGF) induced permeability of retinal endothelial cells (REC) [
5]. DME can be treated with the recently approved VEGF-binding Fab-fragment ranibizumab or the recombinant protein VEGF-trap/aflibercept binding also to placenta growth factor (P
lGF) [
6‐
10]. VEGF inhibition might also be beneficial to patients with proliferative DR (PDR), which can be concluded from evidence indicating that proliferation and migration of REC associated with neovascularization is driven by angiogenic growth factors: Both isoforms VEGF
165 and VEGF
121, as well as P
lGF-1 and basic fibroblast growth factor (bFGF) or insulin-like growth factor-1 (IGF-1), stimulate proliferation of primary and immortalized REC of bovine or human origin over a wide range of concentrations including physiological conditions [
11‐
16]. Migration of immortalized and primary bovine REC ((i)BREC) is induced by VEGF
165 but not by VEGF
121 [
11,
14]. VEGF is only very weakly expressed by immortalized or primary BREC, but its secretion can be slightly enhanced with bFGF together with IGF-1 [
17,
18]. BREC also express P
lGF-1 which can be stimulated by binding of VEGF to VEGF receptor (VEGFR) 2 [
19,
20]. In addition to VEGFR2, VEGFR1 and neuropilin 1 (NRP-1) are REC-expressed receptors that are activated by members of the VEGF family [
17,
21]. VEGF
165 and VEGF
121 efficiently trigger signalling through VEGFR1 and VEGFR2 [
22]. NRP-1 is a potent receptor for VEGF
165, and is only weakly activated by VEGF
121 [
23,
24]. Both isoforms P
lGF-1 and P
lGF-2 bind to VEGFR1, but P
lGF-2 also to NRP-1 [
25‐
27].
Based on this experimental evidence and clinical observations, we studied the effects on proliferation and migration of iBREC (and their potential inhibition by ranibizumab) of combinations of involved growth factors (VEGF
121, P
lGF-1, P
lGF-2, bFGF, and IGF-1) in concert with VEGF
165. The well-established model of iBREC was used in view of its distinct advantages over primary cells or rodent models: compared to primary cells, it is a distinct advantage of iBREC that these are free of contaminating cells of other types. Although iBREC are of bovine origin, they behave like primary human retinal EC (HREC): their proliferation can be stimulated similarly with VEGF-A, IGF-1 or bFGF [
13‐
16]. Involved bovine proteins also show a high similarity to their human counterparts. Our systematic approach including all candidate factors was chosen to reveal their relative contributions to DR pathophysiology with immediate consequences for therapies targeting VEGF.
Discussion
We investigated the effects of a collection of growth factors potentially involved in the control of proliferation and migration in retinal endothelial cells, the key processes in PDR-associated neovascularization. To evaluate the potential of VEGF inhibition to counteract neovascularization even in the presence of other stimulating factors, VEGF-binding ranibizumab was included in the experiments.
All six growth factors tested stimulated proliferation of iBREC, but only VEGF
165, bFGF, and IGF-1 also enhanced migration (see also Table
2). That P
lGFs only stimulated proliferation was also observed in experiments with primary BREC [
11]. However, the proposed action of P
lGFs through mobilization of VEGF seems unlikely because P
lGF-stimulated proliferation of iBREC was not inhibited by ranibizumab [
24]. iBREC proliferation induced by co-stimulating growth factors including VEGF
165 was not completely inhibited by the Fab fragment, suggesting parallel activation of independent signalling pathways. This assumption was supported by previous studies also indicating that incomplete inhibition of VEGF
165-induced proliferation by the anti-VEGF antibody bevacizumab might be due to VEGF-upregulated P
lGF [
15,
19,
30]. Such possibly persistent induction of P
lGFs or other pro-proliferative factors might play a role in PDR that weakly respond to anti-VEGF therapies. However, proliferation stimulated by P
lGF in combination with VEGF can most likely be completely inhibited by VEGF trap because it also binds to P
lGF [
8,
9,
31]. Whether this is sufficient to block proliferation stimulated by a combination together with bFGF and IGF-1 remains to be shown.
Table 2
Receptor usage of VEGF family members and summary of their effects on iBREC proliferation and migration
VEGF-A |
VEGF121
| + | + | −/+ | Yes | No |
VEGF165
| + | + | + | Yes | Yes |
PlGF |
PlGF-1 | + | | | Yes | No |
PlGF-2 | + | | + | Yes | No |
VEGF-E | | + | + | Yes | Yes |
In contrast to its co-stimulation of REC proliferation, VEGF
165 seems so dominant in the regulation of migration that—despite some effects contributed by other growth factors observed in this study and by others—induced migration was almost completely suppressed by ranibizumab, even in experiments with the most complex combinations of factors. The dominant role of VEGF
165 was confirmed by our observation that the inhibitor of VEGFR KRN951 also blocked iBREC migration stimulated by a mixture of VEGF
165, bFGF, and IGF-1. At the concentration used in this study, KRN951 specifically inhibits the tyrosine kinase activity of VEGFR1 and -R2 without affecting other receptor tyrosine kinases [
29]. Interestingly, migration stimulated by VEGF
121 together with bFGF and IGF-1 was totally blocked by ranibizumab, although bFGF-induced migration was not affected, and VEGF
121 by itself did not even enhance iBREC migration. There is some evidence supporting the concept that bFGF facilitates binding of VEGF
121 by its induction of VEGFR2 expression [
32]. Then migration may be essentially driven by VEGF
121, and its removal with ranibizumab might be sufficient to normalize migration.
Migration of (i)BREC was not induced by P
lGF-1/-2, which is in contrast to the observation that these growth factors strongly stimulate migration of macrovascular endothelial cells of the human umbilical vein (HUVEC) [
31]. However, both isoforms stimulated proliferation of iBREC, thereby confirming that the used recombinant human polypeptides can activate the relevant bovine receptor VEGFR1. Another example for the obviously different behavior of macrovascular and microvascular EC is the observation that VEGF
121 induces migration of HUVEC, whereas iBREC do not respond [
14,
24]. Basic differences between HUVEC and HREC in their expression of genes important for angiogenic processes were also shown by expression profiling [
33].
Neovascularization requires migration and proliferation of REC, but therapeutic interference with only one of these processes might not be enough to prevent this hallmark of PDR. Results of several studies based on in-vitro or in-vivo models indeed suggest that more than inhibition of VEGF (which is not the only factor controling REC proliferation) is needed to suppress neovascularization. In an in-vitro model, sprouting of EC was stronger inhibited when bFGF was targeted in addition to VEGF [
34]. Efficient suppression of retinal neovascularization was also achieved with VEGF-trap, a chimeric recombinant protein consisting of the VEGF-binding domains of VEGFR1 and -R2 that inhibits VEGF as well as other members of the protein family such as P
lGFs [
8,
9]. This therapeutic effect is most likely due to inhibition of P
lGF-driven proliferation of REC.
Our results also suggest that different VEGF receptors (VEGFR1, -R2, and/or NRP-1) are involved in the processes leading to altered proliferation and migration of iBREC (Table
2): all members of the VEGF family tested—VEGF
165, VEGF
121, P
lGF-1, P
lGF-2, and VEGF-E—stimulated proliferation, suggesting that activation of either receptor is sufficient, which is in accordance with other studies using EC from various sources [
35]. In contrast, binding of a ligand to VEGFR2 and to NRP-1 seems to be necessary to enhance migration, because only VEGF
165 and VEGF-E, but not VEGF
121 were efficient stimulators. Because VEGF-E was most efficient in stimulating migration of iBREC, one could assume that VEGFR1 is a negative regulator. But combined activation of VEGFR1 and -R2 by VEGF
165/121 plus P
lGF-1/-2 did not result in a lower migration rate compared to stimulation with VEGF
165 alone. More likely, different receptors compete for the available factors, and VEGFR2-mediated signalling is the most efficient pathway leading to enhanced migration. The results of our experiments with immortalized BREC support the assumption that VEGFR2 is crucially involved in signal transduction in EC from various species and different tissues [
35]. Although VEGFR1 can play an important role in EC proliferation, its contribution to other cellular processes stimulated by VEGF is still unclear: potentiating the effect of VEGF by cross-activation of VEGFR2 through VEGFR1 in response to stimulation with P
lGF has been reported thereby [
35]. In contrast, activated VEGFR1 was also reported to be a negative regulator of VEGFR2 signaling in the event that VEGFR2 had been activated first [
21]. However, binding of both receptors by a combination of VEGF and P
lGF did not markedly affect VEGF-induced iBREC proliferation or migration.
Whereas stimulation of migration by the concerted action of growth factors can be at least partly prevented by ranibizumab, substantial inhibition of proliferation was not observed in such experimental setting. Both proliferation and migration of REC are required for neovascularization observed in PDR, and it will be interesting to learn from the ongoing clinical trials whether inhibition of one of these processes by ranibizumab can delay or slow down progression of the disease. The possible surplus benefit of inhibiting all VEGF family members by VEGF-trap remains to be shown, and in view of our results, additional targeting of bFGF and IGF-1 might be considered a more promising approach in the therapy of PDR [
9,
34]. However, these growth factors are also involved in retinal neuroprotection, and their complete inhibition might be detrimental to the function of retinal cells [
36‐
38]. Moderate instead of complete depletion of these growth factors might therefore be more promising, although complete inhibition or reversal of pathological processes may not be achieved under these conditions.