Low-Level Nanog Expression in the Regulation of Quiescent Endothelium.

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 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. CONCLUSIONS: We showed that low-level Nanog expression is required for normal EC homeostasis and angiogenesis in adulthood.

The allosteric glycogen synthase kinase-3 inhibitor NP12 limits myocardial remodeling and promotes angiogenesis in an acute myocardial infarction model.

A key feature of acute myocardial infarction (AMI) is an alteration in cardiac architecture. Signaling events that result in the inhibition of glycogen synthase kinase-3 (GSK-3)ß represent an adaptive response that might limit the extent of adverse remodeling in the aftermath of AMI. Here, we report that an allosteric inhibitor of GSK-3ß, 4-benzyl-2-(naphthalene-1-yl)-1,2,4-thiadiazolidine-3,5-dione (NP12), lessens the magnitude of adverse myocardial remodeling and promotes angiogenesis. Male and female mice 8-10 weeks old were grouped (six animals in each group) into sham surgery (sham group), left anterior descending (LAD) ligation of the coronary artery followed by intramyocardial PBS injections (control group), and LAD ligation followed by NP12 administration (NP12 group). After 7 and 14 days, the extents of fibrosis and integrity of blood vessels were determined. Intramyocardial administration of NP12 increased phosphorylation of GSK-3ß, reduced fibrosis, and restored diastolic function in the mice that had experienced an AMI. Morphometric analyses revealed increased CD31+ and Ki67+ vascular structures and decreased apoptosis in these mice. NP12 administration mediated proliferation of reparative cells in the AMI hearts. In a time-course analysis, Wnt3a and NP12 stabilized ß-catenin and increased expression of both Nanog and VEGFR2. Moreover, NP12 increased the expression of ß-catenin and Nanog in myocardium from AMI mice. Finally, loss- and gain-of-function experiments indicated that the NP12-mediated benefit is, in part, Nanog-specific. These findings indicate that NP12 reduces fibrosis, reestablishes coronary blood flow, and improves ventricular function following an AMI. We conclude that NP12 might be useful for limiting ventricular remodeling after an AMI.

MicroRNA Regulation of Endothelial Junction Proteins and Clinical Consequence.

Cellular junctions play a critical role in structural connection and signal communication between cells in various tissues. Although there are structural and functional varieties, cellular junctions include tight junctions, adherens junctions, focal adhesion junctions, and tissue specific junctions such as PECAM-1 junctions in endothelial cells (EC), desmosomes in epithelial cells, and hemidesmosomes in EC. Cellular junction dysfunction and deterioration are indicative of clinical diseases. MicroRNAs (miRNA) are ~20 nucleotide, noncoding RNAs that play an important role in posttranscriptional regulation for almost all genes. Unsurprisingly, miRNAs regulate junction protein gene expression and control junction structure integrity. In contrast, abnormal miRNA regulation of junction protein gene expression results in abnormal junction structure, causing related diseases. The major components of tight junctions include zonula occluden-1 (ZO-1), claudin-1, claudin-5, and occludin. The miRNA regulation of ZO-1 has been intensively investigated. ZO-1 and other tight junction proteins such as claudin-5 and occludin were positively regulated by miR-126, miR-107, and miR21 in different models. In contrast, ZO-1, claudin-5, and occludin were negatively regulated by miR-181a, miR-98, and miR150. Abnormal tight junction miRNA regulation accompanies cerebral middle artery ischemia, brain trauma, glioma metastasis, and so forth. The major components of adherens junctions include VE-cadherin, ß-catenin, plakoglobin, P120, and vinculin. VE-cadherin and ß-catenin were regulated by miR-9, miR-99b, miR-181a, and so forth. These regulations directly affect VE-cadherin-ß-catenin complex stability and further affect embryo and tumor angiogenesis, vascular development, and so forth. miR-155 and miR-126 have been shown to regulate PECAM-1 and affect neutrophil rolling and EC junction integrity. In focal adhesion junctions, the major components are integrin ß4, paxillin, and focal adhesion kinase (FAK). Integrin ß4 has been regulated by miR-184, miR-205, and miR-9. Paxillin has been regulated by miR-137, miR-145, and miR-218 in different models. FAK has been regulated by miR-7, miR-138, and miR-135. Deregulation of miRNAs is caused by viral infections, tumorigenesis, and so forth. By regulation of posttranscription, miRNAs manipulate junction protein expression in all cellular processes and further determine cellular fate and development. Elucidation of these regulatory mechanisms will become a new alternative therapy for many diseases, such as cancers and inflammatory diseases.

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

Rationale and Goal: Endothelial cells (ECs) are quiescent and critical for maintaining homeostatic functions of the mature vascular system, while disruption of quiescence is at the heart of endothelial to mesenchymal transition (EndMT) and tumor angiogenesis. Here, we addressed the hypothesis that KLF4 maintains the EC quiescence. Methods and Results: In ECs, KLF4 bound to KLF2, and the KLF4-transctivation domain (TAD) interacted directly with KLF2. KLF4-depletion increased KLF2 expression, accompanied by phosphorylation of SMAD3, increased expression of alpha-smooth muscle actin (αSMA), VCAM-1, TGF-ß1, and ACE2, but decreased VE-cadherin expression. In the absence of Klf4, Klf2 bound to the Klf2-promoter/enhancer region and autoregulated its own expression. Loss of EC-Klf4 in Rosa mT/mG ::Klf4 fl/fl ::Cdh5 CreERT2 engineered mice, increased Klf2 levels and these cells underwent EndMT. Importantly, these mice harboring EndMT was also accompanied by lung inflammation, disruption of lung alveolar architecture, and pulmonary fibrosis. Conclusion: In quiescent ECs, KLF2 and KLF4 partnered to regulate a combinatorial mechanism. The loss of KLF4 disrupted this combinatorial mechanism, thereby upregulating KLF2 as an adaptive response. However, increased KLF2 expression overdrives for the loss of KLF4, giving rise to an EndMT phenotype.

Chromatin-modifying agents convert fibroblasts to OCT4+ and VEGFR-2+ capillary tube-forming cells.

RATIONALE: The human epigenome is plastic. The goal of this study was to address if fibroblast cells can be epigenetically modified to promote neovessel formation. METHODS AND RESULTS: Here, we used highly abundant human adult dermal fibroblast cells (hADFCs) that were treated with the chromatin-modifying agents 5-aza-2'-deoxycytidine and trichostatin A, and subsequently subjected to differentiation by activating Wnt signaling. Our results show that these epigenetically modified hADFCs increasingly expressed ß-catenin, pluripotency factor octamer-binding transcription factor-4 (OCT4, also known as POU5F1), and endothelial cell (EC) marker called vascular endothelial growth factor receptor-2 (VEGFR-2, also known as Fetal Liver Kinase-1). In microscopic analysis, ß-catenin localized to cell-cell contact points, while OCT4 was found to be localized primarily to the nucleus of these cells. Furthermore, in a chromatin immunoprecipitation experiment, OCT4 bound to the VEGFR-2/FLK1 promoter. Finally, these modified hADFCs also transduced Wnt signaling. Importantly, on a two-dimensional (2D) gel substrate, a subset of the converted cells formed vascular network-like structures in the presence of VEGF. CONCLUSION: Chromatin-modifying agents converted hADFCs to OCT4+ and VEGFR-2+ capillary tube-forming cells in a 2D matrix in VEGF-dependent manner.

PAR1 Scaffolds TGFßRII to Downregulate TGF-ß Signaling and Activate ESC Differentiation to Endothelial Cells.

We studied the function of the G-protein-coupled receptor PAR1 in mediating the differentiation of mouse embryonic stem cells (mESCs) to endothelial cells (ECs) that are capable of inducing neovascularization. We observed that either deletion or activation of PAR1 suppressed mouse embryonic stem cell (mESC) differentiation to ECs and neovascularization in mice. This was mediated by induction of TGFßRII/TGFßRI interaction, forming an active complex, which in turn induced SMAD2 phosphorylation. Inhibition of TGF-ß signaling in PAR1-deficient mESCs restored the EC differentiation potential of mESCs. Thus, PAR1 in its inactive unligated state functions as a scaffold for TGFßRII to downregulate TGF-ß signaling, and thereby promote ESC transition to functional ECs. The PAR1 scaffold function in ESCs is an essential mechanism for dampening TGF-ß signaling and regulating ESC differentiation.