Schepens Eye Research Institute, Mass Eye and Ear Infirmary and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
Schepens Eye Research Institute, Mass Eye and Ear Infirmary and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States University of Exeter Medical School, Exeter, Devon, United Kingdom.
Invest Ophthalmol Vis Sci. 2014 Jul 11;55(8):5191-9. doi: 10.1167/iovs.14-14046.
Pericytes, the vascular cells that constitute the outer layer of capillaries, have been shown to have a crucial role in vascular development and stability. Loss of pericytes precedes endothelial cell dysfunction and vascular degeneration in small-vessel diseases, including diabetic retinopathy. Despite their clinical relevance, the cellular pathways controlling survival of retinal pericytes remain largely uncharacterized. Therefore, we investigated the role of Notch signaling, a master regulator of cell fate decisions, in retinal pericyte survival.
A coculture system of ligand-dependent Notch signaling was developed using primary cultured retinal pericytes and a mesenchymal cell line derived from an inducible mouse model expressing the Delta-like 1 Notch ligand. This model was used to examine the effect of Notch activity on pericyte survival using quantitative PCR (qPCR) and a light-induced cell death assay. The effect of Notch gain- and loss-of-function was analyzed in monocultures of retinal pericytes using antibody arrays to interrogate the expression of apoptosis-related proteins.
Primary cultured retinal pericytes differentially expressed key molecules of the Notch pathway and displayed strong expression of canonical Notch/RBPJK (recombination signal-binding protein 1 for J-kappa) downstream targets. A gene expression screen using gain- and loss-of-function approaches identified genes relevant to cell survival as downstream targets of Notch activity in retinal pericytes. Ligand-mediated Notch activity protected retinal pericytes from light-induced cell death.
Our results have identified signature genes downstream of Notch activity in retinal pericytes and suggest that tight regulation of Notch signaling is crucial for pericyte survival.
周细胞是构成毛细血管外层的血管细胞，已被证明在血管发育和稳定性中起着关键作用。在小血管疾病（包括糖尿病视网膜病变）中，周细胞的丧失先于内皮细胞功能障碍和血管退化。尽管它们具有临床相关性，但控制视网膜周细胞存活的细胞途径在很大程度上仍未被描述。因此，我们研究了 Notch 信号通路在视网膜周细胞存活中的作用，Notch 信号通路是细胞命运决定的主要调节剂。
使用原代培养的视网膜周细胞和一种间质细胞系（该细胞系源自可诱导表达 Delta-like 1 Notch 配体的小鼠模型），开发了一种依赖配体的 Notch 信号通路共培养系统。该模型用于使用定量 PCR（qPCR）和光诱导细胞死亡测定来研究 Notch 活性对周细胞存活的影响。使用抗体阵列分析 Notch 功能获得和功能丧失对视网膜周细胞的单培养物的影响，以研究凋亡相关蛋白的表达。
原代培养的视网膜周细胞差异表达 Notch 通路的关键分子，并显示出强烈的经典 Notch/RBPJK（J 激酶的重组信号结合蛋白 1）下游靶标表达。使用增益和功能丧失方法进行的基因表达筛选确定了与细胞存活相关的基因作为 Notch 活性在视网膜周细胞中的下游靶标。配体介导的 Notch 活性可保护视网膜周细胞免受光诱导的细胞死亡。
我们的研究结果确定了 Notch 活性在视网膜周细胞中的下游特征基因，并表明 Notch 信号通路的严格调节对于周细胞的存活至关重要。
