Department of Biochemistry and Molecular Biology, Augusta, GA, 30912, USA.
Department of Ophthalmology, Augusta, GA, 30912, USA.
Exp Eye Res. 2022 Aug;221:109129. doi: 10.1016/j.exer.2022.109129. Epub 2022 May 29.
Preservation of retinal barrier function is critical to maintenance of retinal health. Therefore, it is not surprising that loss of barrier integrity is a pathologic feature common to degenerative retinal diseases such as diabetic retinopathy. Our prior studies demonstrate the importance of hydroxycarboxylic acid receptor 2/GPR109A (HCAR2/GPR109A) expression in the retinal pigment epithelium (RPE) to outer retinal barrier integrity. However, whether HCAR2/GPR109A is expressed in retinal endothelial cells and has a similar relationship to inner blood retinal barrier regulation is not known. In the current study, we examined relevance of receptor expression to endothelial cell dependent-blood retinal barrier integrity. siRNA technology was used to modulate HCAR2/GPR109A expression in human retinal endothelial cells (HRECs). Cells were cultured in the presence or absence of VEGF, a pro-inflammatory stimulus, and/or various concentrations of the HCAR2/GPR109A-specific agonist beta-hydyroxybutyrate (BHB). HCAR2/GPR109A expression was monitored by qPCR and electrical cell impedance sensing (ECIS) was used to evaluate barrier function. Complementary in vivo studies were conducted in wildtype and HCAR2/GPR109A knockout mice treated intraperitoneally with lipopolysaccharide and/or BHB. Vascular leakage was monitored using fluorescein angiography and Western blot analyses of albumin extravasation. Additionally, retinal function was evaluated by OptoMotry. Decreased (siRNA knockdown) or absent (gene knockout) HCAR2/GPR109A expression was associated with impaired barrier function both in vitro and in vivo. BHB treatment provided some protection, limiting disruptions in retinal barrier integrity and function; an effect that was found to be receptor (HCAR2/GPR109A)-dependent. Collectively, the present studies support a key role for HCAR2/GPR109A in regulating blood-retinal barrier integrity and highlight the therapeutic potential of the receptor toward preventing and treating retinal diseases such as diabetic retinopathy in which compromised barrier function is paramount.
视网膜屏障功能的维持对于维持视网膜健康至关重要。因此，毫不奇怪，屏障完整性的丧失是糖尿病性视网膜病变等退行性视网膜疾病的共同病理特征。我们之前的研究表明，羟基羧酸受体 2/GPR109A（HCAR2/GPR109A）在视网膜色素上皮（RPE）中的表达对于外视网膜屏障的完整性很重要。然而，HCAR2/GPR109A 是否在视网膜内皮细胞中表达，以及它与内血视网膜屏障调节是否存在类似的关系尚不清楚。在本研究中，我们研究了受体表达与内皮细胞依赖性血视网膜屏障完整性的相关性。siRNA 技术用于调节人视网膜内皮细胞（HRECs）中的 HCAR2/GPR109A 表达。细胞在存在或不存在血管内皮生长因子（VEGF）、促炎刺激物和/或各种浓度的 HCAR2/GPR109A 特异性激动剂β-羟基丁酸（BHB）的情况下进行培养。通过 qPCR 监测 HCAR2/GPR109A 表达，并用细胞阻抗传感（ECIS）评估屏障功能。在接受腹腔内注射脂多糖和/或 BHB 的野生型和 HCAR2/GPR109A 敲除小鼠中进行了补充的体内研究。通过荧光素血管造影术监测血管渗漏，并通过 Western blot 分析评估白蛋白外渗。此外，通过 OptoMotry 评估视网膜功能。体外和体内研究均表明，HCAR2/GPR109A 表达减少（siRNA 敲低）或缺失（基因敲除）与屏障功能受损有关。BHB 治疗提供了一定的保护作用，限制了视网膜屏障完整性和功能的破坏；这种作用被发现是受体（HCAR2/GPR109A）依赖性的。总之，这些研究支持 HCAR2/GPR109A 在调节血视网膜屏障完整性方面的关键作用，并强调了该受体在预防和治疗糖尿病性视网膜病变等视网膜疾病中的治疗潜力，在这些疾病中，屏障功能受损至关重要。
