Department of Ophthalmology, Huadong Hospital, Fudan University, No. 221 East Yan'an Road, Shanghai, 200031, China.
Department of Ophthalmology, Tongren Hospital, Shanghai Jiaotong University, Shanghai, China.
Int Ophthalmol. 2023 May;43(5):1523-1536. doi: 10.1007/s10792-022-02548-3. Epub 2022 Oct 13.
To investigate the anti-fibrotic effect of ZD6474 (a novel inhibitor of VEGF and EGF) in TGF-β1 stimulated human Tenon's capsule fibroblasts (HTFs) and the anti-angiogenetic role in HUVECs, compared to that of mitomycin C (MMC).
The effects of ZD6474 on cell proliferation or migration in TGF-β1-stimulated HTFs and HUVECs were determined, using CCK8 or wound healing assay, respectively. The typical markers of fibrosis in TGF-β1-stimuated HTFs were detected, vimentin by immunofluorescence, α-SMA and snail by western blot. Tube formation was applied to validate the anti-angiogenesis effect in HUVECs following ZD6474 treatment. Furthermore, phosphorylated AKT and mTOR (p-AKT and p-mTOR) were evaluated, compared to the standardized total AKT and mTOR, using western blot.
There was almost no decreased cell viability in HTFs following ZD6474 (≤ 1 μM/mL) treatment, but MMC (> 50 μg/mL) significantly impaired cell viability. ZD6474 significantly inhibited TGF-β1-stimulated proliferation and migration in HTFs, compared to control group (**P < 0.01). ZD6474 also significantly attenuated the TGF-β1-stimulated expression of vimentin, α-SMA and snail in HTFs. Tube formation was notably interrupted in HUVECs following ZD6474 treatment (**P < 0.01). P-AKT and p-mTOR were significantly decreased in response to ZD6474 treatment in TGF-β1- induced HTFs and HUVECs.
ZD6474 exerts anti-proliferation and anti-fibrotic effects in TGF-β1-stimulated HTFs perhaps via regulating AKT-mTOR signaling pathway. ZD6474 also inhibited proliferation, migration and tube formation in HUVECs via the same signaling pathway. We concluded that ZD6474 may be potentially a novel agent in preventing bleb dysfunction following glaucoma filtration surgery (GFS).
研究新型血管内皮生长因子（VEGF）和表皮生长因子（EGF）抑制剂 ZD6474 对转化生长因子-β1（TGF-β1）刺激的人眼Tenon 囊成纤维细胞（HTFs）的抗纤维化作用，以及其在人脐静脉内皮细胞（HUVECs）中的抗血管生成作用，并与丝裂霉素 C（MMC）进行比较。
通过 CCK8 或划痕愈合实验分别测定 ZD6474 对 TGF-β1 刺激的 HTFs 和 HUVECs 中细胞增殖或迁移的影响。通过免疫荧光法检测 vimentin，通过 Western blot 检测α-SMA 和 snail，检测 TGF-β1 刺激的 HTFs 中的纤维化典型标志物。应用管形成实验验证 ZD6474 处理后对 HUVECs 的抗血管生成作用。此外，通过 Western blot 比较标准化总 AKT 和 mTOR（p-AKT 和 p-mTOR）来评估磷酸化 AKT 和 mTOR（p-AKT 和 p-mTOR）。
ZD6474（≤1 μM/mL）处理后 HTFs 中的细胞活力几乎没有降低，但 MMC（>50 μg/mL）显著损害细胞活力。与对照组相比，ZD6474 显著抑制 TGF-β1 刺激的 HTFs 增殖和迁移（**P<0.01）。ZD6474 还显著减弱了 TGF-β1 刺激的 HTFs 中 vimentin、α-SMA 和 snail 的表达。ZD6474 处理后，HUVECs 的管形成明显中断（**P<0.01）。TGF-β1 诱导的 HTFs 和 HUVECs 中，ZD6474 处理后 p-AKT 和 p-mTOR 明显减少。
ZD6474 通过调节 AKT-mTOR 信号通路，在 TGF-β1 刺激的 HTFs 中发挥抗增殖和抗纤维化作用。ZD6474 还通过相同的信号通路抑制 HUVECs 的增殖、迁移和管形成。我们得出结论，ZD6474 可能是预防青光眼滤过手术（GFS）后小梁泡功能障碍的一种潜在新型药物。
