Our study confirmed that ZD6474 reduced cell proliferation and migration in TGF-β1-stimulated HTFs. Trans-differentiation to myofibroblasts and EMT were also attenuated in presence with ZD6474 in TGF-β1-stimulated HTFs. In contrast with the obvious toxicity of MMC [20, 21], ZD6474 showed a selective toxicity effect on the normal HTFs. Hence, we identified the anti-fibrosis effect of ZD6474 in preventing scar formation following GFS with better safety than MMC.
GFS is to deliberately open an extra aqueous outflow pathway in trabecular meshwork to decrease IOP effectively, but there are still numerous potential complications post-operatively. After GFS, the re-blockage of the pathway due to scar formation (severe fibroblasts proliferation), resulting in upregulating IOP again is very common . During the early stage of wound healing post-operatively, excessive TGF-β promotes fibrosis perhaps via recruiting fibroblasts and increase fibroblast proliferation . It has been reported that failure of GSF is mainly due to scar formation, particular in the presence of TGF‑β1 secreted by activated fibroblasts . We found that stimulated with TGF‑β1 significantly promoted HTFs proliferation and migration, but these effects were largely diminished in presence with ZD6474, suggesting that ZD6474 is capable of reducing scar formation following GFS. Thus, ZD6474 (a multi-kinase inhibitor) can also target to both VEGF and EGF pathways, which are able to achieve similar therapeutic effects as bevacizumab and 5-FU. Hence, such finding suggests that ZD6474 may also exert equal therapeutic effect, but substantially reduce adverse effect following GFS. Our explanation is in agreement with previous clinical study, showing that application of bevacizumab (a single VEGF inhibitor) and 5-FU (anti-metabolic drug) together during GFS significantly decreases failure rate after glaucoma surgery .
Trans-differentiation of HTFs to myofibroblasts and subsequent deposition of EMT are key steps during the scar formation following GFS . In the process of EMT and fibrosis [27, 28], various cytokines and inflammatory factors (TGF, EGF, CTGF) act as molecular switches to start a series of downstream biological functions. EGFR, one of the tyrosine kinases (HER1-4) receptors, has a wide range of physiological functions in many activities of life . Once EGFR binding with EGF, activated EGFR can trigger downstream signal pathways and ultimately elicit uncontrolled cell proliferation and fibrosis [29, 30]. ZD6474 can precisely target on EGFR to achieve its therapeutic effect. In the current study, we observed that higher expression of snail and α-SMA in TGF-β1-induced HTFs, but ZD6474 reversed the transformation from fibroblasts into myofibroblasts and EMT of TGF-β1-stimulated HTFs perhaps by inhibiting the activation of EGF to achieve the curative effect.
Conventional anti-metabolic drugs (MMC and 5‑FU) are routinely applied locally during and/or post operation (GFS) for inhibiting proliferation of fibroblasts in the wounded micro-environment. However, the major downside of MMC and 5‑FU is cytotoxicity, including bleb leakage, hypotony maculopathy, corneal toxicity and sclera melting [31, 32], compromising the application of MMC and 5‑FU significantly. In our current study, a transient 5-minutes exposure of MMC on HTFs could lead to mass cell death and apoptosis. By contrast, ZD6474 at the proper concentration was proved to be less toxic in HTFs. Therefore, ZD6474 had been confirmed with great curative effect and less side effects rather than MMC. Safety provides a strong guarantee for the follow-up research on ZD6474 to explore the curative effects.
Neovascularization, a major contributing factor for scaring formation, has been proved to be closely associated with fundus and glaucoma diseases [33, 34]. It is well documented that VEGF is secreted in both autocrine and paracrine fashions in the glaucomatous eyes at baseline, particularly over-secreted [5, 35, 36]. During the proliferative stage of wound healing, the overgrowth of angiogenesis results in new blood vessels formation with release of inflammatory molecular and cytokines that lead to scar formation and bleb disfunction [35, 36]. As for the anti-neovascularization in our study, the proliferation and migration of HUVECs and tube formation were notably inhibited in the presence of ZD6474, suggesting that ZD6474 can robustly suppress neovascularization. Our finding is consistent with others, showing that ZD6474 significantly inhibits neovascularization to achieve anti-tumor effect in thyroid and breast cancers [37, 38]. Thus, our finding further suggests that ZD6474 seems to be a good candidate in reducing re-closure of pathway in trabecular meshwork by interfering formation of neovascularization post-GFS, i.e. anti-proliferation, anti-fibrotic and anti-angiogenesis, but substantially reduced adverse effect following GFS effectively.
Our western blot data showed that AKT-mTOR signaling pathway was downregulated in HTFs in response to ZD6474 treatment, suggesting that ZD6474 prevents scarring after GFS via reducing aqueous outflow resistance, using AKT-mTOR signaling pathway. This is in line with the finding that, mTOR inhibitor abates TGF‑β2‑induced fibrotic changes in trabecular meshwork cells and increases aqueous outflow in glaucoma treatment . ZD6474 can accurately downregulate the downstream EGFR signaling pathway, which is at the heart of cell proliferation and migration , so targeting on EGFR to suppress its downstream network is widely exploited in fibrosis and EMT process [41, 42]. EGFR can recruit the PI3K lipid kinase via GAB1 and GAB2, and then PI3K catalyzes PIP2 into PIP3, which recruits AKT, leading to the activation of the PI3K -AKT-mTOR signaling pathway for various physiological functions . Phosphorylation of AKT eventually leads to the inhibition of antagonists and cell proliferation and division . Herein, EGF is closely related to the activation of AKT-mTOR signaling pathway. In present study, EGF-induced AKT-mTOR activation were considerably inhibited by ZD6474 treatment, as was EGF-induced EGFR phosphorylation.
VEGF, the most important angiogenesis factor, can be activated by binding with VEGF, and then many downstream signal-transduction networks associated with neovascularization can be successively activated, such as PI3K/AKT/mTOR, ERK1/2, and many others, so VEGF is crucial for angiogenesis-related processes . In several previous studies, VEGF activation is confirmed to be tightly associated with the activation of PI3K-AKT-mTOR signaling pathway [46, 47]. To explore the possible mechanism of ZD6474 on HUVECs, p-AKT and p-mTOR expression were detected and found were obviously diminished. Hence, it is suggested that ZD6474 attenuates AKT/mTOR phosphorylation probably through a reduction in VEGF-receptor activation in HUVECs, resulting to the reduce of angiogenesis to prevent surgery failure after GFS.
Integrated with anti-fibrosis and anti-angiogenesis effects of ZD6474, we found that ZD6474 can play a positive role in preventing bleb dysfunction after GFS. Considering the low toxicity and good efficacy, ZD6474 can undoubtedly obtain great outcomes and applications in clinical. Hence, the present study implicates that multi-kinase inhibitors may improve outcomes with good safety during the period of postoperative recurrence after GFS.
It should be stated that there are some limitations in the present study. In vitro assays are not ample to assess the anti-fibrotic and anti-angiogenesis effect of ZD6474. Thus, it cannot be extrapolated directly in clinical situations. Additionally, the mechanism underlying the anti-fibrosis and anti-angiogenesis effects of ZD6474 warrants further investigation in animal model and in human glaucoma patients.