Low-Level Nanog Expression in the Regulation of Quiescent Endothelium

Abstract

Objective: 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. Approach and Results: 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 ROSA mT/mG ::Nanog fl/+ ::Cdh5 CreERT2 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. Conclusions: We showed that low-level Nanog expression is required for normal EC homeostasis and angiogenesis in adulthood.

A requirement for Krüppel Like Factor‐4 in the maintenance of endothelial cell quiescence

“…These methods have been previously described ( Baruah et al, 2020 ; Chatterjee et al, 2016 ; Humtsoe et al, 2003 ; Kohler et al, 2011 ; Kohler et al, 2013 ). All primary endothelial cells (ECs) were cultured up to 3-5 passages in EndoGro TM Basal Medium with EndoGro TM VEGF Supplement Kit (SCME-BM; SCME002-S; millipore) on 0.2% gelatin (G-2625, Sigma-Aldrich) in PBS-coated plates.…”

A requirement for Krüppel-Like Factor-4 in the maintenance of endothelial cell quiescence

“…All experimental procedures and husbandry have been approved by the University of Illinois at Chicago (UIC) Institutional Animal Care and Use Committee (IACUC). We procured Klf4 fl/wt mice on a C57BL background (Dr. Klaus Kaestner, University of Pennsylvania, Philadelphia, PA) (57), obtained from Mutant Mouse Resource & Research Centers (MMRRC) through an institutional Material Transfer Agreement (MTA). Introns 1 and 3 of the Klf4 gene have loxP sites to create the floxed allele.…”

“…Following TAM (2.0mg/kg body weight, BW) administration for each of five consecutive days, all ECs (expressing EC-specific Cdh5 CreERT2 ) turn green due to mG expression, while non-ECs fluoresce tomato (red) due to baseline expression of mT. The transgenic Cdh5 CreERT2 driver line served as a proxy for EC specificity (53,57). After a series of breeding and backcrossing regimens, Klf4 fl/fl ::Cdh5 CreERT2 , Rosa mT/mG ::Cdh5 CreERT2 and Rosa mT/mG ::Klf4 fl/fl ::Cdh5 CreERT2 GEMM were produced.…”

“…Mice were anesthetized and sacrificed humanely as previously described (53)(54)(55)(56)(57). For cryosections, lungs were perfused with PBS, collected, rinsed in PBS, and placed in increasing concentrations of PBS/sucrose solution overnight at 4ºC (3%, 10%, 30%) for cryoprotection and in sucrose:OCT (TissueTek 4583) (30% in PBS) until the specimen reached the bottom of the mold.…”

“…Microbeads coated with anti-mouse CD31 (Miltenyi Biotech., 130-097-418) and antimouse CD45 antibodies (Miltenyi Biotech., 130-052-301) were used to isolate ECs from mouse tissue as previously described (53)(54)(55)(56)(57). For EC isolation, mice were humanely sacrificed.…”