Department of Pediatrics, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung 83301, Taiwan, R.O.C.
Department of Ophthalmology, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan, R.O.C.
Mol Med Rep. 2021 Nov;24(5). doi: 10.3892/mmr.2021.12442. Epub 2021 Sep 15.
Transforming growth factor‑β2 (TGF‑β2) has been implicated in the pathogenesis of proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR), due to its ability to stimulate the overproduction of pro‑angiogenic factors, such as vascular endothelial growth factor (VEGF), and remodeling of the extracellular matrix (ECM). Although intravitreal triamcinolone acetonide (TA) is clinically useful in the treatment of PVR and PDR, its molecular mechanism has yet to be fully elucidated. The present study investigated whether TA treatment altered TGF‑β2‑driven biological effects on the behavior of cultured human retinal pigment epithelial (RPE) cells, in order to determine which signaling pathway may be essential for the pharmacological action of TA. The R‑50 human RPE cell line was treated with TA in the presence of TGF‑β2, followed by analyses of cell viability and contraction using cell viability and collagen gel contraction assays. VEGF mRNA expression and protein production were measured using reverse transcription‑quantitative PCR and ELISA, respectively. The phosphorylation status of signaling mediators and the protein expression of type I collagen (COL1A1), α‑smooth muscle actin (α‑SMA), and ECM‑remodeling enzymes, including MMP‑2 and MMP‑9, were analyzed using western blotting. The gelatinolytic activity of MMPs was detected using gelatin zymography. TA treatment exhibited no prominent cytotoxicity but markedly antagonized TGF‑β2‑induced cytostatic effects on RPE cell viability and TGF‑β2‑enhanced contractility in collagen gels. In the context of TGF‑β2‑related signaling, TA significantly attenuated TGF‑β2‑elicited Smad2, extracellular‑regulated kinase (ERK)1/2 and p38 mitogen‑activated protein kinase (MAPK) phosphorylation. Moreover, TA markedly mitigated TGF‑β2‑induced VEGF upregulation through ablation of p38 signaling activity. TA also partially attenuated TGF‑β2‑elicted expression of COL1A1, α‑SMA, MMP‑2, and MMP‑9, but only suppressed TGF‑β2‑induced MMP‑9 gelatinolytic activity. Mechanistically, the MEK/ERK signaling pathway may have a critical role in the TGF‑β2‑induced upregulation of COL1A1, α‑SMA and MMP‑9. In conclusion, TA may be considered a useful therapeutic agent for treating TGF‑β2‑associated intraocular angiogenesis and tissue remodeling, the underlying mechanism of which may involve the ERK and p38 MAPK signaling pathways.
转化生长因子-β2(TGF-β2)能够刺激血管内皮生长因子(VEGF)等促血管生成因子的过度产生,并重塑细胞外基质(ECM),因此与增生性玻璃体视网膜病变(PVR)和增生性糖尿病视网膜病变(PDR)的发病机制有关。虽然玻璃体内曲安奈德(TA)在治疗 PVR 和 PDR 方面具有临床应用价值,但它的分子机制尚未完全阐明。本研究旨在探讨 TA 治疗是否改变了 TGF-β2 对培养的人视网膜色素上皮(RPE)细胞行为的驱动生物学效应,以确定哪种信号通路可能对 TA 的药理作用至关重要。采用 TA 处理 R-50 人 RPE 细胞系,并在存在 TGF-β2 的情况下,通过细胞活力和胶原凝胶收缩测定分析细胞活力和收缩。采用逆转录-定量 PCR 和 ELISA 分别测量 VEGF mRNA 表达和蛋白产生。采用 Western blot 分析信号转导介质的磷酸化状态以及Ⅰ型胶原(COL1A1)、α-平滑肌肌动蛋白(α-SMA)和 ECM 重塑酶(包括 MMP-2 和 MMP-9)的蛋白表达。采用明胶酶谱法检测 MMP 的胶溶活性。TA 治疗没有明显的细胞毒性,但显著拮抗 TGF-β2 诱导的 RPE 细胞活力的细胞静止作用和 TGF-β2 增强的胶原凝胶收缩性。在 TGF-β2 相关信号方面,TA 显著抑制 TGF-β2 诱导的 Smad2、细胞外调节激酶(ERK)1/2 和 p38 丝裂原激活蛋白激酶(MAPK)磷酸化。此外,TA 通过消除 p38 信号活性显著减轻 TGF-β2 诱导的 VEGF 上调。TA 还部分减轻了 TGF-β2 诱导的 COL1A1、α-SMA、MMP-2 和 MMP-9 的表达,但仅抑制了 TGF-β2 诱导的 MMP-9 胶溶活性。从机制上讲,MEK/ERK 信号通路可能在 TGF-β2 诱导的 COL1A1、α-SMA 和 MMP-9 上调中发挥关键作用。总之,TA 可被视为一种治疗 TGF-β2 相关眼内血管生成和组织重塑的有效治疗药物,其潜在机制可能涉及 ERK 和 p38 MAPK 信号通路。