Department of Ophthalmology, Boston Children's Hospital, Harvard Medical School, 300 Longwood Ave, Boston, MA 02115, USA.
Manton Center for Orphan Disease, Boston Children's Hospital, 300 Longwood Ave, Boston, MA 02115, USA.
Int J Mol Sci. 2020 Feb 11;21(4):1188. doi: 10.3390/ijms21041188.
The aim of the current study was to investigate the impact of long-acting fibroblast growth factor 21 (FGF21) on retinal vascular leakage utilizing machine learning and to clarify the mechanism underlying the protection. To assess the effect on retinal vascular leakage, C57BL/6J mice were pre-treated with long-acting FGF21 analog or vehicle (Phosphate Buffered Saline; PBS) intraperitoneally (i.p.) before induction of retinal vascular leakage with intravitreal injection of mouse (m) vascular endothelial growth factor 164 (VEGF164) or PBS control. Five hours after mVEGF164 injection, we retro-orbitally injected Fluorescein isothiocyanate (FITC) -dextran and quantified fluorescence intensity as a readout of vascular leakage, using the Image Analysis Module with a machine learning algorithm. In FGF21- or vehicle-treated primary human retinal microvascular endothelial cells (HRMECs), cell permeability was induced with human (h) VEGF165 and evaluated using FITC-dextran and trans-endothelial electrical resistance (TEER). Western blots for tight junction markers were performed. Retinal vascular leakage in vivo was reduced in the FGF21 versus vehicle- treated mice. In HRMECs in vitro, FGF21 versus vehicle prevented hVEGF-induced increase in cell permeability, identified with FITC-dextran. FGF21 significantly preserved TEER compared to hVEGF. Taken together, FGF21 regulates permeability through tight junctions; in particular, FGF21 increases Claudin-1 protein levels in hVEGF-induced HRMECs. Long-acting FGF21 may help reduce retinal vascular leakage in retinal disorders and machine learning assessment can help to standardize vascular leakage quantification.
本研究旨在利用机器学习研究长效成纤维细胞生长因子 21（FGF21）对视网膜血管渗漏的影响，并阐明其保护机制。为评估对视网膜血管渗漏的影响，将 C57BL/6J 小鼠预先用长效 FGF21 类似物或载体（磷酸盐缓冲盐水；PBS）腹腔内（i.p.）预处理，然后用玻璃体内注射小鼠（m）血管内皮生长因子 164（VEGF164）或 PBS 对照诱导视网膜血管渗漏。在 mVEGF164 注射后 5 小时，我们经眼眶后注射荧光素异硫氰酸酯（FITC）-葡聚糖，并使用带有机器学习算法的图像分析模块量化荧光强度作为血管渗漏的读数。在 FGF21 或载体处理的原代人视网膜微血管内皮细胞（HRMEC）中，用人（h）VEGF165 诱导细胞通透性，并使用 FITC-葡聚糖和跨内皮电阻（TEER）进行评估。进行了紧密连接标志物的 Western blot 分析。与载体处理的小鼠相比，FGF21 处理的小鼠体内视网膜血管渗漏减少。在体外 HRMEC 中，与载体相比，FGF21 可防止 hVEGF 诱导的细胞通透性增加，这通过 FITC-葡聚糖鉴定。与 hVEGF 相比，FGF21 显著保留了 TEER。总之，FGF21 通过紧密连接调节通透性；特别是，FGF21 增加了 hVEGF 诱导的 HRMEC 中 Claudin-1 蛋白水平。长效 FGF21 可能有助于减少视网膜疾病中的视网膜血管渗漏，机器学习评估有助于标准化血管渗漏的定量。
