Centre de Recherches des Cordeliers, UMR_S INSERM 1138, Équipe 17, Université Paris Cité, Université Paris Sorbonne Cité, Paris, France.
APHP, Hôpital Universitaire Necker-Enfants Malades, Paris, France.
Mol Vis. 2022 Sep 4;28:280-299. eCollection 2022.
Choroidal and retinal neovascularization plays an essential role in various ocular diseases. In this study, we examined the role of nestin in this process. Nestin is an intermediate filament protein known to play several roles, including as a marker of neural progenitor and proliferating endothelial cells.
We used Brown Norway rats, in which choroidal and retinal neovascularization was induced using intraocular laser impacts. The role of nestin was examined using angiography, western blot from the second to the 14th day after laser impacts, and intraocular injection of nestin siRNA. The localization of the protein was specified by co-immunoreactivity with glial fibrillary protein (GFAP), glutamine synthetase (GS), and von Willebrand factor (vWF).
In the control retina, nestin was found principally in glial structures in the ganglion cell layer, as confirmed by nestin/GFAP immunolabeling. Two days after the laser impacts, the nestin expression extended to numerous radial processes at the site of the impacts. With Bruch's membrane ruptured, these processes penetrated into the choroid. Nestin immunolabeling remained high from the third to the seventh day but appeared reduced on the 14th day. The nature of these processes was not clearly defined, but co-immunolabeling with GFAP suggested that they were principally in activated Müller cells from the third day after the laser impacts. However, the co-immunoreactivity of nestin and GS, a marker of mature functional Müller cells, could be observable only from the seventh day. Nestin was also observed in some vascular cells, as demonstrated by the co-immunoreactivity of the protein with vWF in the choroid and retina. As observed on angiography, the numbers of choroidal and retinal blood vessels were significantly increased (principally on the seventh day) after the laser impacts. An intraocular injection of nestin siRNAs led to a significant decrease in the number of blood vessels.
Our results confirmed the presence of nestin in glial (e.g., astrocytes), reactive Müller, and endothelial cells. They demonstrated their critical involvement in a rat model of retinal and choroidal neovascularization experimentally induced using ocular laser impacts.
脉络膜和视网膜新生血管在各种眼部疾病中起着重要作用。在这项研究中，我们研究了巢蛋白在这一过程中的作用。巢蛋白是一种中间丝蛋白，已知其具有多种功能，包括作为神经祖细胞和增殖内皮细胞的标志物。
我们使用棕色挪威大鼠，通过眼内激光冲击诱导脉络膜和视网膜新生血管。使用血管造影、激光冲击后第 2 天至第 14 天的 Western blot 以及眼内注射巢蛋白 siRNA 来研究巢蛋白的作用。通过与神经胶质纤维酸性蛋白 (GFAP)、谷氨酰胺合成酶 (GS) 和血管性血友病因子 (vWF) 的共免疫反应性来确定蛋白质的定位。
在对照视网膜中，巢蛋白主要存在于神经节细胞层的神经胶质结构中，巢蛋白/GFAP 免疫标记证实了这一点。激光冲击后两天，巢蛋白的表达延伸到冲击部位的许多放射状突起。当 Bruch 膜破裂时，这些突起穿透脉络膜。从第 3 天到第 7 天，巢蛋白免疫标记仍然很高，但在第 14 天减少。这些突起的性质尚不清楚，但与 GFAP 的共免疫标记表明，它们主要是从激光冲击后第 3 天开始激活的 Müller 细胞。然而，只有从第 7 天开始，巢蛋白与 GS（成熟功能 Müller 细胞的标志物）的共免疫反应才能观察到。巢蛋白也存在于一些血管细胞中，如在脉络膜和视网膜中，该蛋白与 vWF 的共免疫反应表明。如血管造影所示，激光冲击后脉络膜和视网膜的血管数量显著增加（主要在第 7 天）。眼内注射巢蛋白 siRNA 可导致血管数量显著减少。
我们的结果证实了巢蛋白存在于神经胶质（如星形胶质细胞）、反应性 Müller 和内皮细胞中。它们证明了它们在使用眼内激光冲击实验诱导的大鼠视网膜和脉络膜新生血管模型中具有重要作用。
