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在成年内皮细胞（endothelial cells, ECs）中呈低水平表达，但其功能意义尚不明确。本研究旨在利用基因工程小鼠模型系统，阐明Nanog在成年内皮细胞中的作用。生化分析显示，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）可上调内皮细胞中Nanog与人端粒酶逆转录酶（human telomerase reverse transcriptase, hTERT）的表达，并增强这些细胞的端粒酶活性。染色质免疫沉淀实验表明，Nanog可直接结合至hTERT与eNOS的启动子/增强子DNA元件，从而调控二者的转录。向ROSAmT/mG::Nanogfl/+::Cdh5CreERT2小鼠给予低剂量他莫昔芬，可诱导单个Nanog等位基因的敲除，同时用绿色荧光蛋白标记内皮细胞，并导致Tert与eNos的表达水平下调。对这些小鼠的心脏组织切片进行组织学与形态计量学分析后发现，其心脏血管出现细胞死亡、微血管稀疏以及纤维化程度升高的现象。内皮细胞特异性Nanog单倍剂量不足可导致内皮细胞稳态与血管生成受损。反之，在Nanog敲低的内皮细胞中重新表达编码hTERT的互补DNA（complementary DNA, cDNA），可部分逆转Nanog缺失所带来的效应。本研究证实，成年个体中正常的内皮细胞稳态与血管生成依赖于Nanog的低水平表达。