Department of Ophthalmology, National Defense Medical College, Namiki 3-2, Tokorozawa, Saitama, 359-8513, Japan.
Department of Developmental Anatomy and Regenerative Biology, National Defense Medical College, Namiki 3-2, Tokorozawa, Saitama, 359-8513, Japan.
Exp Eye Res. 2022 Jul;220:109094. doi: 10.1016/j.exer.2022.109094. Epub 2022 Apr 28.
Diabetic retinopathy is a major cause of blindness in developed countries, and is characterized by deterioration of barrier function causing vascular hyperpermeability and retinal edema. Vascular endothelial growth factor (VEGF) is a major mediator of diabetic macular edema. Although anti-VEGF drugs are the first-line treatment for diabetic macular edema, some cases are refractory to anti-VEGF therapy. Osteopontin (OPN) is a phosphoglycoprotein with diverse functions and expressed in various cells and tissues. Elevated OPN level has been implicated in diabetic retinopathy, but whether OPN is involved in hyperpermeability remains unclear. Using streptozotocin-induced diabetic mice (STZ mice) and human retinal endothelial cells (HRECs), we tested the hypothesis that up-regulated OPN causes tight junction disruption, leading to vascular hyperpermeability. The serum and retinal OPN concentrations were elevated in STZ mice compared to controls. Intravitreal injection of anti-OPN neutralizing antibody (anti-OPN Ab) suppressed vascular hyperpermeability and prevented decreases in claudin-5 and ZO-1 gene expression levels in the retina of STZ mice. Immunohistochemical staining of retinal vessels in STZ mice revealed claudin-5 immunoreactivity with punctate distribution and attenuated ZO-1 immunoreactivity, and these changes were prevented by anti-OPN Ab. Intravitreal injection of anti-OPN Ab did not change VEGF gene expression or protein concentration in retina of STZ mice. In an in vitro study, HRECs were exposed to normal glucose or high glucose with or without OPN for 48 h, and barrier function was evaluated by transendothelial electrical resistance and Evans blue permeation. Barrier function deteriorated under high glucose condition, and was further exacerbated by the addition of OPN. Immunofluorescence localization of claudin-5 and ZO-1 demonstrated punctate appearance with discontinuous junction in HRECs exposed to high glucose and OPN. There were no changes in VEGF and VEGF receptor-2 expression levels in HRECs by exposure to OPN. Our results suggest that OPN induces tight junction disruption and vascular hyperpermeability under diabetic conditions. Targeting OPN may be an effective approach to manage diabetic retinopathy.
糖尿病性视网膜病变是发达国家失明的主要原因，其特征是屏障功能恶化导致血管通透性增加和视网膜水肿。血管内皮生长因子（VEGF）是糖尿病性黄斑水肿的主要介质。虽然抗 VEGF 药物是糖尿病性黄斑水肿的一线治疗方法，但有些病例对抗 VEGF 治疗有抗性。骨桥蛋白（OPN）是一种具有多种功能的磷糖蛋白，在各种细胞和组织中表达。OPN 水平升高与糖尿病性视网膜病变有关，但 OPN 是否与通透性增加有关尚不清楚。我们使用链脲佐菌素诱导的糖尿病小鼠（STZ 小鼠）和人视网膜内皮细胞（HRECs），验证了上调的 OPN 导致紧密连接破坏，从而导致血管通透性增加的假设。与对照组相比，STZ 小鼠的血清和视网膜 OPN 浓度升高。STZ 小鼠玻璃体腔内注射抗 OPN 中和抗体（抗 OPN Ab）可抑制血管通透性增加，并防止 STZ 小鼠视网膜中 Claudin-5 和 ZO-1 基因表达水平降低。STZ 小鼠视网膜血管免疫组织化学染色显示 Claudin-5 免疫反应呈点状分布，ZO-1 免疫反应减弱，抗 OPN Ab 可预防这些变化。抗 OPN Ab 玻璃体腔内注射不会改变 STZ 小鼠视网膜中 VEGF 基因表达或蛋白浓度。在一项体外研究中，将 HRECs 暴露于正常葡萄糖或高葡萄糖中，或在高葡萄糖中加入或不加入 OPN 48 小时，通过跨内皮电阻和 Evans 蓝渗透评估屏障功能。在高葡萄糖条件下，屏障功能恶化，加入 OPN 后进一步恶化。在暴露于高葡萄糖和 OPN 的 HRECs 中，Claudin-5 和 ZO-1 的免疫荧光定位显示点状外观，连接不连续。HRECs 暴露于 OPN 时，VEGF 和 VEGF 受体-2 的表达水平没有变化。我们的结果表明，OPN 在糖尿病条件下诱导紧密连接破坏和血管通透性增加。靶向 OPN 可能是治疗糖尿病性视网膜病变的有效方法。
