Low level of Nanog in endothelial cells

Nanog is expressed in adult endothelial cells (ECs) at a low-level, however, its functional significance is not known. The goal of our study was to elucidate the role of Nanog in adult ECs using a genetically engineered mouse model system. Biochemical analyses showed that Nanog is expressed in both adult human and mouse tissues. Primary ECs isolated from adult mice showed detectable levels of Nanog, Tert (telomerase reverse transcriptase), and eNos (endothelial nitric oxide synthase). Wnt3a (Wnt family member 3A) increased the expression of Nanog and hTERT (human telomerase reverse transcriptase) in ECs and increased telomerase activity in these cells. In a chromatin immunoprecipitation experiment, Nanog directly bound to the hTERT and eNOS promoter/enhancer DNA elements, thereby regulating their transcription. Administration of low-dose tamoxifen to ROSAmT/mG::Nanogfl/+::Cdh5CreERT2 mice induced deletion of a single Nanog allele, simultaneously labeling ECs with green fluorescent protein and resulting in decreased Tert and eNos levels. Histological and morphometric analyses of heart tissue sections prepared from these mice revealed cell death, microvascular rarefaction, and increased fibrosis in cardiac vessels. Accordingly, EC-specific Nanog-haploinsufficiency resulted in impaired EC homeostasis and angiogenesis. Conversely, re-expression of cDNA encoding the hTERT in Nanog-depleted ECs, in part, restored the effect of loss of Nanog. We showed that low-level Nanog expression is required for normal EC homeostasis and angiogenesis in adulthood.

Nanog在成体内皮细胞（endothelial cells，ECs）中呈低水平表达，但其功能意义尚未明确。本研究旨在借助基因工程小鼠模型系统，阐明Nanog在成体内皮细胞中的作用。
生化分析结果显示，Nanog在成人及小鼠组织中均有表达。从成年小鼠体内分离的原代内皮细胞中，可检测到Nanog、端粒酶逆转录酶（telomerase reverse transcriptase，Tert）以及内皮型一氧化氮合酶（endothelial nitric oxide synthase，eNos）的表达水平。
Wnt3a（Wnt家族成员3A，Wnt family member 3A）可上调内皮细胞中Nanog与人类端粒酶逆转录酶（human telomerase reverse transcriptase，hTERT）的表达，并增强此类细胞的端粒酶活性。
染色质免疫沉淀（chromatin immunoprecipitation）实验结果显示，Nanog可直接结合至hTERT与eNOS的启动子/增强子DNA元件，进而调控二者的转录。
对ROSAmT/mG::Nanogfl/+::Cdh5CreERT2小鼠给予低剂量他莫昔芬（tamoxifen），可诱导单个Nanog等位基因发生缺失，同时以绿色荧光蛋白（green fluorescent protein）标记内皮细胞，并导致Tert与eNos的表达水平降低。
对上述小鼠的心脏组织切片开展组织学与形态计量分析后发现，心血管系统出现细胞死亡、微血管稀疏及纤维化程度升高的现象。
相应地，内皮细胞特异性Nanog单倍体不足（haploinsufficiency）会引发内皮细胞稳态受损与血管生成能力下降。
反之，在Nanog敲低的内皮细胞中重新导入编码hTERT的互补DNA（complementary DNA，cDNA），可部分恢复Nanog缺失所导致的表型缺陷。
本研究证实，低水平的Nanog表达对于成年个体正常内皮细胞稳态及血管生成是必需的。