Merrigan Stephanie L, Kennedy Breandán N
UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Dublin, Ireland.
Br J Pharmacol. 2017 Aug;174(16):2636-2651. doi: 10.1111/bph.13875. Epub 2017 Jul 7.
Pathological growth of ocular vasculature networks can underpin visual impairment in neovascular age-related macular degeneration, proliferative diabetic retinopathy and retinopathy of prematurity. Our aim was to uncover novel pharmacological regulators of ocular angiogenesis by phenotype-based screening in zebrafish.
A bioactive chemical library of 465 drugs was screened to identify small molecule inhibitors of ocular hyaloid vasculature (HV) angiogenesis in zebrafish larvae. Selectivity was assessed by evaluation of non-ocular intersegmental vasculature development. Safety pharmacology examined visual behaviour and retinal histology in larvae. Molecular mechanisms of action were scrutinized using expression profiling of target mRNAs and miRNAs in larval eyes.
Library screening identified 10 compounds which significantly inhibited HV developmental angiogenesis. The validated hit calcitriol selectively demonstrated dose-dependent attenuation of HV development. In agreement, vitamin D receptor (VDR) agonists paricalcitol, doxercalciferol, maxacalcitol, calcipotriol, seocalcitol, calcifediol and tacalcitol significantly and selectively attenuated HV development. VDR agonists induced minor ocular morphology abnormalities and affected normal visual function. Calcitriol induced a three to sevenfold increase in ocular dre-miR-21 expression. Consistently, all-trans-retinoic acid attenuated HV development and increased ocular dre-miR-21 expression. Interestingly, zebrafish ocular vegfaa and vegfab expression was significantly increased while, vegfc, flt1 and kdrl expression was unchanged by calcitriol.
These studies identified VDR agonists as significant and selective anti-angiogenics in the developing vertebrate eye and miR21 as a key downstream regulated miRNA. These targets should be further evaluated as molecular hallmarks of, and therapeutic targets for pathological ocular neovascularization.
眼部血管网络的病理性生长是新生血管性年龄相关性黄斑变性、增殖性糖尿病视网膜病变和早产儿视网膜病变导致视力损害的基础。我们的目的是通过在斑马鱼中进行基于表型的筛选来发现眼部血管生成的新型药理学调节剂。
对一个包含465种药物的生物活性化学文库进行筛选，以鉴定斑马鱼幼虫眼部玻璃体血管系统（HV）血管生成的小分子抑制剂。通过评估非眼部节间血管系统的发育来评估选择性。安全药理学研究了幼虫的视觉行为和视网膜组织学。使用幼虫眼睛中靶mRNA和miRNA的表达谱来仔细研究作用的分子机制。
文库筛选鉴定出10种可显著抑制HV发育性血管生成的化合物。经验证的活性成分骨化三醇选择性地表现出剂量依赖性的HV发育减弱。同样，维生素D受体（VDR）激动剂帕立骨化醇、度骨化醇、马沙骨化醇、钙泊三醇、司骨化醇、骨化二醇和他骨化醇均显著且选择性地减弱了HV发育。VDR激动剂诱导了轻微的眼部形态异常并影响了正常视觉功能。骨化三醇使眼部dre-miR-21表达增加了三到七倍。同样，全反式维甲酸减弱了HV发育并增加了眼部dre-miR-21表达。有趣的是，骨化三醇使斑马鱼眼部vegfaa和vegfab表达显著增加，而vegfc、flt1和kdrl表达未改变。
这些研究确定VDR激动剂是发育中的脊椎动物眼睛中重要且选择性的抗血管生成剂，miR21是关键的下游调节miRNA。这些靶点应作为病理性眼部新生血管形成的分子标志和治疗靶点进行进一步评估。
