Dynamics of soluble vascular endothelial growth factor receptors and their ligands in aqueous humour during ranibizumab for age-related macular degeneration

Age-related macular degeneration (AMD) is an important cause of visual loss [1]. In neovascular AMD, choroidal neovascularization (CNV) leads to sub-retinal/intra-retinal macular edema, hemorrhage, and fibrosis, resulting in visual impairment. Development of agents targeting vascular endothelial growth factor (VEGF) has advanced considerably since an important role of VEGF in the pathogenesis of CNV was reported [2].

Four anti-VEGF agents are available for AMD, including an aptamer of VEGF (pegaptanib), two anti-VEGF antibodies (bevacizumab and ranibizumab) and a VEGF trapping agent (aflibercept) [3‐5]. However, recurrence of CNV after treatment or resistance to these agents is not uncommon; suggesting a role of other factors in CNV along with VEGF. Vitreous fluid levels of soluble VEGF receptor (sVEGFR)-1 and sVEGFR-2 are reported to be elevated in ischemic vitreoretinal diseases [6], and these receptors may have a role in the pathogenesis of macular edema [7]. Interactions between the VEGFRs and their ligands of these receptors (VEGF) may influence progression of AMD. While intravitreal ranibizumab injection (IRI) has been reported to reduce intraocular VEGF [8]. intraocular dynamics of the VEGF receptors after IRI remain unclear.

Accordingly, a prospective study was performed in which the concentrations of VEGFR-1, VEGFR-2, and their ligands (VEGF and placental growth factor (PlGF)) were measured in aqueous humors samples obtained from patients with wet AMD at initiation of IRI, and changes of these factors were evaluated during the first course of IRI treatment.

The AMD group (18 men and 6 women) was 76.0 ± 10.6 years old (mean ± SD) and the control group (7 men and 6 women) was 73.1 ± 5.9 years old. There were no significant differences of age (p = 0.369) or gender (p = 0.189) between two groups.

At the baseline examination (visit 0: just before the first IRI dose), the mean BCVA was 0.39 ± 0.34 in the AMD group and mean CMT was 401 ± 216 μm.

As shown in Table 1, the aqueous humor levels of VEGF, PlGF, sVEGFR-1, and sVEGFR-2 were significantly higher in the AMD group than in the control group (in descending order).

In the AMD group, there was a significant correlation between the levels of sVEGFR-1 and sVEGFR-2, as well as between VEGF and PlGF levels (Table 2), but there were no correlations between sVEGFR-1 and VEGF or PlGF (Table 2) or between sVEGFR-2 and VEGF or PlGF (Table 2).

In the AMD group, mean BCVA showed significantly imprvement from log MAR 0.39 ± 0.34 before IRI to log MAR 0.28 ± 0.33 after 1 month and log MAR 0.25 ± 0.36 after 2 months (Fig. 1a), while mean CMT decreased significantly from 401 ± 216 μm to 296 ± 199 μm after 1 month and 267 ± 161 μm after 2 months (Fig. 1b).

In the AMD group, the aqueous level of VEGF was significantly lower at 1 and at 2 months after starting IRI compared with baseline (Fig. 2a). The aqueous level of PlGF was significantly lower at 1 month and 2 months relative to baseline (Fig. 2b). The aqueous level of sVEGFR-1 was also significantly lower at 1 and at 2 months relative to baseline (Fig. 3a). However, there was no significant change of the aqueous sVEGFR-2 level between baseline and 1 or 2 months (Fig. 3b).

The main new findings were that aqueous levels of both sVEGFR-1 and sVEGFR-2 were significantly higher in the AMD group than the control group, with a significant correlation between sVEGFR-1 and sVEGFR-2 levels, suggesting that expression of these receptors shows concomitant upregulation in AMD. We recently reported [9, 10] that enhanced expression of both transmembrane VEGFR and sVEGFR is involved in regulating angiogenesis and vascular permeability based on the correlation between their expression in human umbilical vein endothelial cells stimulated with phorbol 12-myristate 13-acetate or basic fibroblast growth factor [11]. VEGF signaling may be tightly regulated by VEGFR-1 and VEGFR-2 [12]. VEGF binds to these receptors on vascular endothelial cells, and both receptors have tyrosine kinase activity [13]. VEGFR-1 is also expressed by monocytes/macrophages [14] and promotes VEGF-mediated recruitment of these cells to sites of angiogenesis and inflammation [14, 15]. On the other hand, VEGFR-2 is only expressed by endothelial cells, and VEGF signaling via its membrane-bound isoform influences physiological vascular permeability and angiogenesis [12, 16]. These actions of the VEGF receptors indicate the potential for an important role in the pathogenesis of AMD. Both sVEGFRs were increased in our AMD patients, suggesting that these receptors may be involved in development of CNV, and that the increase of sVEGFRs may be secondary to upregulation of transmembrane VEGFRs.

We also demonstrated that the aqueous humor level of VEGF was significantly suppressed in AMD patients by anti-VEGF therapy, possibly because ranibizumab binds and neutralize intraocular VEGF. These findings are also in agreement with previous reports [8]. IRI also significantly reduced the aqueous humor level of PlGF, which was an unexpected finding VEGF and PlGF exist as dimers in the eye, being either homodimers (VEGF/VEGF and PlGF/PlGF) or heterodimers (VEGF/PlGF) [17, 18]. Therefore, it is possible that the aqueous humor level of PlGF was reduced significantly because ranibizumab bound to and neutralized intraocular VEGF/PlGF heterodimers. Because VEGF acts as a chemoattractant for monocytes after binding to VEGFR-1 and promotes inflammation [19], it is also possible that inflammation was suppressed by ranibizumab binding and neutralizing VEGF. Accordingly, IRI may have reduced inflammation in AMD patients to indirectly down-regulate PlGF expression.

Interestingly, we noted a significant decrease in the aqueous level of sVEGFR-1 after IRI treatment. VEGFR-1 is expressed by monocytes/macrophages [14], and signaling via this receptor promotes VEGF-mediated recruitment of these cells to sites of inflammation [14, 20]. It has been reported that sVEGFR-1 has a pro-inflammatory effect [14, 21], suggesting that elevation of its expression may aggravate inflammation. This idea is supported by the report that sVEGFR-1 expression is correlated with the C-reactive protein level and the leukocyte count [22], which shows a possible link between sVEGFR-1 and inflammation. Chronic inflammation may have a role in the pathogenesis of AMD [23], and VEGF is known to be a chemoattractant for monocytes in primates [24]. Thus, inflammation may be suppressed by ranibizumab binding and neutralizing VEGF and this suppression of inflammation by IRI could be associated with down-regulation of sVEGFR-1 in AMD eyes. On the other hand, there was no significant decrease of sVEGFR-2 expression during IRI, suggesting that its effects are associated with down-regulation of VEGFR-1 rather than VEGFR-2. Because VEGFR-2 signaling mainly influences vascular permeability and angiogenesis [12], the sVEGFR-2 level might not decrease significantly when IRI suppresses inflammation. However, this study had a small sample size and further investigation will be required to clarify the kinetics of sVEGFRs in AMD patients receiving IRI.

According to our findings, improvement of BCVA and CMT occurred during IRI along with reduction of the aqueous humor levels of VEGF, PlGF, and sVEGFR-1. Therefore, IRI may achieve functional and morphological improvement in AMD patients by suppressing intraocular production of VEGF, PlGF, and sVEGFR-1.

In conclusion, the levels of sVEGFR-1, sVEGFR-2, VEGF, and PlGF in the aqueous humor were significantly higher in patients with AMD than in controls. Aqueous levels of sVEGFR-1 and sVEGFR-2 showed a significant correlation with each other, as did the levels of VEGF and PlGF. IRI significantly improved both BCVA and CMT, while aqueous levels of VEGF, PlGF, and sVEGFR-1 decreased significantly. These findings suggest that VEGFRs may have a role in AMD and that suppression of intraocular VEGF, PlGF, and sVEGFR-1 production by IRI may improve clinical parameters.