Background
Surgery must be performed in cases where maximal medical therapy cannot control intraocular pressure (IOP) in patients with glaucoma. Glaucoma filtration surgery (GFS) is currently one of the most effective methods for treating glaucoma [
1,
2]. The goal of GFS is to create an incision to bypass the trabecular meshwork and drain the aqueous humor outward through the subconjunctival filtering bleb to relieve the elevated IOP [
3]. Unlike with most surgeries, the success of GFS is achieved by inhibiting wound healing [
4]. Postoperative conjunctival scarring at the site of the filtering bleb, however, promotes adhesion to the episcleral tissue, which leads to the resealing of the bleb inhibiting the aqueous flow and poor control of IOP [
5]. Human Tenon’s fibroblast (HTF) is regarded as the major cell type contributing to the formation of subconjunctival scar after GFS [
6].
Safely inhibiting the formation of scars in GFS and improving the success rate of surgery has noticeably attracted the attention of glaucoma specialists. Antimetabolites, such as 5-fluorouracil (5-Fu) and mitomycin-C (MMC), are used to modulate the healing process and improve the success rate of surgery. However, despite their effectiveness, these drugs can lead to thin-walled filtering bleb, which is related to the high-risk of leakage, hypotony, and endophthalmitis [
7,
8]. When vascular endothelium growth factor (VEGF) expression is upregulated in the early stage of GFS [
9,
10], the treated eyes receiving subconjunctival injection of bevacizumab (BVZ) can develop larger filtration blebs than the non-treated eyes [
11], or the IOP is reduced with a better safety profile compared with the MMC-treated group [
12]. However, the scar formation after GFS involves complex processes of angiogenesis and fibrosis, and hence it is inadequate to aim only at anti-VEGF or other single targets [
13‐
15].
Placental growth factor (PIGF) is primarily a pro-angiogenic growth factor only upregulated under pathological conditions [
16]. Previous studies [
17] showed that the inhibition of PIGF could effectively reduce angiogenesis, vascular leakage, and inflammation, besides affecting reactive gliosis in the retina. Conbercept can bind to dual targets (VEGF and PIGF) for antiangiogenic therapy [
18‐
20]. Zhang et al. [
21] used the subconjunctival injection of conbercept as an adjuvant to GFS for open-angle glaucoma and compared its efficacy with that of 5-Fu. Less vascularity of filtration blebs, lower IOP, and lower incidence of corneal epithelial stripping were achieved after the surgery in the conbercept treatment group. However, evidence showing the direct effects of conbercept on HTFs [
22,
23] and its safety profile is still lacking. Also, the underlying mechanisms of conbercept in inhibiting scar formation in GFS are still unclear.
In the present study, HTFs and HUVECs were cultured in vitro and then treated with conbercept, BVZ, 5-Fu, or MMC. The results revealed that conbercept significantly inhibited HTF cell migration and collagen type I alpha1 (Col1A1) mRNA expression level [
24] in HTFs with a significant anti-PIGF effect and an inferior anti-VEGF effect compared with BVZ; also, the low cytotoxicity of conbercept was observed. Our research might assist in better understanding the role of conbercept during the GFS wound healing process.
Discussion
After GFS, increased angiogenesis in conjunctiva and fibroblast migration at the site of the filtering bleb, leading to fibroblast proliferation with collagen deposition, are the direct causes of filtering bleb failure [
5]. Various anti-scarring treatments are adjunctively used for GFS to improve the success rate of surgery. Conbercept has been used as an adjunct in GFS for treating open-angle glaucoma and has been effective in improving the surgical outcome [
21]. However, its direct effect on HTF is still unknown, and its mechanism for improving the prognosis of GFS has not been clearly explained. In this study, HTFs were incubated with conbercept, and the direct inhibitory effects of conbercept on HTF cell migration, Col1A1 mRNA expression of HTFs, and VEGF(R) mRNA expression of HTFs were detected. Also, the low cytotoxicity of conbercept was assessed, while the inhibitory effect of concepcept on the expression of PIGF and VEGF in HUVECs was examined.
Clinically, increased bleb vascularity is associated with a poorer prognosis for GFS [
30]. VEGF expression increased in the Tenon tissue of patients who experienced failed GFS compared with patients in whom the surgery was successful and patients without glaucoma [
31]. VEGF [
9,
32] is a key mediator of angiogenesis; inhibiting the VEGF pathway inhibits the angiogenic process [
10,
32,
33]. These findings suggest the potential usefulness of anti-VEGF therapy in promoting the success of GFS. Vandewalle et al. [
34] and Grewal et al. [
35] reported that using BVZ as an adjuvant for GFS could help control IOP after the surgery. However, several anti-VEGF compounds lack efficacy in preventing fibrosis, possibly because PIGF is simultaneously upregulated following the use of anti-VEGF(R) antibodies, leading to a profibrotic effect of PIGF via binding to VEGFR-1 [
36]. This leads to an overall profibrotic effect [
37].
PIGF is another member of the VEGF family, which shows no effect under physiological conditions, while it is important for pathological angiogenesis, plasma extravasation, and compensatory growth in response to hypoxia, inflammation, wound healing, and cancer [
38‐
40]. Additionally, PIGF is considered as a profibrotic growth factor [
41]. Anti-PIGF agents have a direct inhibitory effect on reactive gliosis in the retina [
37]. Van Bergen et al. [
42] found that the expression level of PIGF in the aqueous humor of patients with glaucoma after anti-VEGF treatment significantly increased, indicating an important contribution of this growth factor to wound healing after trabeculectomy. PIGF can be a possible target for improving the outcome of GFS. Anti-PIGF agents can significantly reduce postoperative proliferation, inflammation, and angiogenesis, as well as collagen deposition in later stage of GFS in animal models [
42].
However, treatment with a single antiangiogenic drug may lead to the upregulation of other growth factors. This is based on escape mechanisms via induction of an angiogenic rescue program [
42]. Therefore, the combination of anti-VEGF and anti-PIGF agents may attenuate the escape mechanism and affect the three most important wound healing phases: inflammation, angiogenesis, and collagen deposition [
42]. In the clinical treatment of vitreoretinal diseases, aflibercept exhibits ambivalent profibrotic effects because it possesses both anti-fibrotic (via PIGF inhibition) and profibrotic properties. After the treatment, the decreased VEGF expression level increases the connective tissue growth factor (CTGF)/VEGF ratio [
43,
44], resulting in an overall profibrotic effect [
45]. In the choroidal neovascularization model and the mouse streptozotocin model [
37], whether the reduction of scar formation after treatment with anti-PIGF antibody is associated with the absence of an angiofibrotic switch (i.e., CTGF release) remains unclear. Nevertheless, it is concluded that the inhibition of PlGF can reduce the process of fibrosis, known as a common side effect of VEGF inhibition [
37]. For the anti-scarring effect of GFS, it has been suggested that the optimal dose of anti-PIGF agent combined with the suboptimal dose of anti-VEGF agent (which has no side effects) may better inhibit scarring compared with monotherapy of either [
42].
Conbercept has a dual effect on binding to PIGF and VEGF [
18‐
20]. In the present study, conbercept showed a significant inhibitory effect on the PIGF expression with a weaker anti-VEGF effect than BVZ in vascular endothelial cells. It could directly inhibit HTF migration and Col1A1 mRNA expression level in HTFs. Besides, it was found that the inhibitory effect of conbercept on the expression level of VEGFR-1 mRNA in HTFs was more noticeable than that of BVZ. In contrast, the inhibitory effect of conbercept on the expression level of VEGFR-2 mRNA was lower than that of BVZ. It was suggested that conbercept could inhibit the upregulation of PIGF while inhibiting VEGF and also inhibit the signaling pathway of the binding of PIGF to VEGFR-1. Therefore, our study initially indicated that concepcept might be a valuable anti-scarring therapy for GFS. It provided an experimental basis for the clinical application of conbercept as an adjunct in GFS [
21].
The results of this study revealed that conbercept + 5-Fu and conbercept + MMC had a remarkable anti-PIGF effect and inferior anti-VEGF effect. Conbercept combined with 5-Fu or MMC could also significantly inhibit HTF migration and the expression of Col1A1 mRNA in HTFs. Conbercept + 5-Fu was superior to 5-Fu in inhibiting HTF migration and expression of VEGF-R1 mRNA and VEGF-R2 mRNA. The cytotoxicity of conbercept combined with 5-Fu or MMC was not higher than that of 5-Fu or MMC, while the cytotoxicity of 2.5 mg/mL BVZ on HTFs was obvious [
46]. As a result, the experimental results suggested that the combined use of conbercept with 5-Fu or MMC, especially the combination of conbercept and 5-Fu, might also be effective in delaying the wound healing of GFS.
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