Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis.

In sprouting angiogenesis, specialized endothelial tip cells lead the outgrowth of blood-vessel sprouts towards gradients of vascular endothelial growth factor (VEGF)-A. VEGF-A is also essential for the induction of endothelial tip cells, but it is not known how single tip cells are selected to lead each vessel sprout, and how tip-cell numbers are determined. Here we present evidence that delta-like 4 (Dll4)-Notch1 signalling regulates the formation of appropriate numbers of tip cells to control vessel sprouting and branching in the mouse retina. We show that inhibition of Notch signalling using gamma-secretase inhibitors, genetic inactivation of one allele of the endothelial Notch ligand Dll4, or endothelial-specific genetic deletion of Notch1, all promote increased numbers of tip cells. Conversely, activation of Notch by a soluble jagged1 peptide leads to fewer tip cells and vessel branches. Dll4 and reporters of Notch signalling are distributed in a mosaic pattern among endothelial cells of actively sprouting retinal vessels. At this location, Notch1-deleted endothelial cells preferentially assume tip-cell characteristics. Together, our results suggest that Dll4-Notch1 signalling between the endothelial cells within the angiogenic sprout serves to restrict tip-cell formation in response to VEGF, thereby establishing the adequate ratio between tip and stalk cells required for correct sprouting and branching patterns. This model offers an explanation for the dose-dependency and haploinsufficiency of the Dll4 gene, and indicates that modulators of Dll4 or Notch signalling, such as gamma-secretase inhibitors developed for Alzheimer's disease, might find usage as pharmacological regulators of angiogenesis.

Phosphatase and tensin homolog regulates the pluripotent state and lineage fate choice in human embryonic stem cells.

Understanding the intrinsic and extrinsic signals that regulate the molecular basis of the pluripotent state may improve our understanding of mammalian embryogenesis, different states of pluripotency, and our ability to tailor lineage differentiation. Although the role of the PI3K/Akt pathway in the self-renewal and maintenance of mESCs is well-established, the specific contribution of the pathway or of its negative regulator, PTEN, in the maintenance of the human pluripotent state is less understood. To explore the PI3K/AKT pathway in human embryonic stem cell (hESC) pluripotency and differentiation, we generated stable PTEN knockdown (KD) hESCs using short hairpin RNA. Similar to mESCs, we found that PTEN KD hESCs have increased self-renewal, cell survival, and proliferation over multiple passages compared to control cells. However, in contrast to mESCs, in vitro, PTEN KD hESCs differentiated inefficiently in directed differentiation assays, in part due to the continued maintenance of OCT4 and NANOG expression. In teratoma assays, PTEN KD hESCs generated tissues from the three germ layers, although with a bias toward neuroectoderm differentiation. These results demonstrate that PTEN is a key regulator of hESC growth and differentiation, and manipulation of this pathway may improve our ability to regulate and understand the pluripotent state.

Integrated chemical genomics reveals modifiers of survival in human embryonic stem cells.

Understanding how survival is regulated in human embryonic stem cells (hESCs) could improve expansion of stem cells for production of cells for regenerative therapy. There is great variability in comparing the differentiation potential of multiple hESC lines. One reason for this is poor survival upon dissociation, which limits selection of homogeneous populations of cells. Understanding the complexity of survival signals has been hindered by the lack of a reproducible system to identify modulators of survival in pluripotent cells. We therefore developed a high-content screening approach with small molecules to examine hESC survival. We have identified novel small molecules that improve survival by inhibiting either Rho-kinase or protein kinase C. Importantly, small molecule targets were verified using short hairpin RNA. Rescreening with stable hESCs that were genetically altered to have increased survival enabled us to identify groups of pathway targets that are important for modifying survival. Understanding how survival is regulated in hESCs could overcome severe technical difficulties in the field, namely expansion of stem cells to improve production of cells and tissues for regenerative therapy.

Combinatorial regulation of the murine RAG-2 promoter by Sp1 and distinct lymphocyte-specific transcription factors.

The recombination activation genes, RAG-1 and RAG-2, encode the critical components of the recombinase complex responsible for the generation of functional antigen receptor genes. In order to gain an insight into the transcription factors and cis-acting elements that regulate the lymphocyte-specific expression of RAG-2, the promoter-region of this gene was isolated and characterized. This analysis demonstrated that a relatively small promoter fragment could confer lymphocyte-restricted expression to a reporter construct. Our work and that of others subsequently revealed that RAG-2 promoter expression is positively regulated by BSAP (PAX-5) and c-Myb transcription factors in B- and T-lineage cells, respectively. Although BSAP and c-Myb were deemed necessary for lymphocyte-specific expression, our analysis also uncovered a G-rich region at the 5'-end of the core promoter that was essential for full activity in lymphocyte cell lines. Site-directed mutagenesis revealed that a GA-box within the G-rich region was required for full promoter activity and subsequent DNA binding assays demonstrated that this element was specifically recognized by Sp1. Apart from showing that Sp1 interacts within the RAG-2 promoter, we also demonstrate that the Sp1-binding site is necessary for the high-level activation of this promoter.

Selective binding of lectins to embryonic chicken vasculature.

Chicken embryos are an excellent model system for studies related to vascular morphogenesis. Development in ovo allows manipulations otherwise difficult in mammals, and the use of chicken-quail chimeras offers an additional advantage to this experimental system. Furthermore, the chicken chorioallantoic membrane has been extensively used for in vivo assays of angiogenesis. Surprisingly, few markers are available for a comprehensive visualization of the vasculature. Here we report the use of lectins for identification of embryonic chicken blood vessels. Nine lectins were evaluated using intravascular perfusion and directly on sections. Our results indicate that Lens culinaris agglutinin, concanavalin A, and wheat germ agglutinin can be used effectively for visualization of vessels of early chicken embryos (E2.5-E4). At later developmental stages, Lens culinaris agglutinin is a better choice because it displays equal affinity for the endothelia of arteries, veins, and capillaries. The findings presented here expand our understanding of lectin specificity in the endothelium of avian species and provide information as to the use of these reagents to obtain comprehensive labeling of the embryonic and chorioallantoic membrane vasculature.

A spatially and chemically defined platform for the uniform growth of human pluripotent stem cells.

We report the design of a chemically defined platform engineered for the culture of human pluripotent stem cells (hPSCs) that supports the long-term maintenance of self-renewing hPSC populations in a more uniform manner than standard culture systems. Microcontact printing (µCP) of alkanethiol self-assembled monolayers (SAMs) was used to spatially direct hPSC adherence. This technique not only establishes control over hPSC colony size and shape but also preserves genetic stability and provides unprecedented uniformity in the pluripotency of hPSC populations that is quantitatively assessed in the present study.

Human pluripotent stem cells: the development of high-content screening strategies.

High-content screening (HCS) permits simultaneous observation and analysis of multiple cellular variables including cell morphology, survival, and differentiation in live cells at the single-cell level, at the level of the culture well, and across the entire culture. By combining high-throughput technologies such as robotics, chemical libraries, and automated high-resolution microscopy, scientists are able to evaluate a much broader array of experimental conditions than can be studied using conventional cell biological techniques that study fewer parameters at any one time. Thus, HCS assays provide a means to vastly improve our basic understanding of stem cell biology. We have developed a HCS assay that allows the study of the effects of hundreds of small molecules in parallel. The protocol described in this chapter was developed to assess the effects of small molecules on the survival, proliferation, and expression of pluripotent markers following single-cell dissociation of human embryonic stem cells, but can be applied to the study of other types of stem cells including induced pluripotent stem cells. A detailed protocol for the setup of HCS assays and the parameters used to identify chemical modifiers of survival in human pluripotent stem cells, as well as secondary assays used to validate the small-molecule "hits" obtained during the high-content screen, are described.

Increased lysis of stem cells but not their differentiated cells by natural killer cells; de-differentiation or reprogramming activates NK cells.

The aims of this study are to demonstrate the increased lysis of stem cells but not their differentiated counterparts by the NK cells and to determine whether disturbance in cell differentiation is a cause for increased sensitivity to NK cell mediated cytotoxicity. Increased cytotoxicity and augmented secretion of IFN-gamma were both observed when PBMCs or NK cells were co-incubated with primary UCLA oral squamous carcinoma stem cells (UCLA-OSCSCs) when compared to differentiated UCLA oral squamous carcinoma cells (UCLA-OSCCs). In addition, human embryonic stem cells (hESCs) were also lysed greatly by the NK cells. Moreover, NK cells were found to lyse human Mesenchymal Stem Cells (hMSCs), human dental pulp stem cells (hDPSCs) and human induced pluripotent stem cells (hiPSCs) significantly more than their differentiated counterparts or parental lines from which they were derived. It was also found that inhibition of differentiation or reversion of cells to a less-differentiated phenotype by blocking NFkappaB or targeted knock down of COX2 in monocytes significantly augmented NK cell cytotoxicity and secretion of IFN-gamma. Taken together, these results suggest that stem cells are significant targets of the NK cell cytotoxicity. However, to support differentiation of a subset of tumor or healthy untransformed primary stem cells, NK cells may be required to lyse a number of stem cells and/or those which are either defective or incapable of full differentiation in order to lose their cytotoxic function and gain the ability to secrete cytokines (split anergy). Therefore, patients with cancer may benefit from repeated allogeneic NK cell transplantation for specific elimination of cancer stem cells.

Role of the Jak/STAT pathway in the regulation of interleukin-8 transcription by oxidized phospholipids in vitro and in atherosclerosis in vivo.

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) and its component phospholipid, 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine, induce endothelial cells (EC) to synthesize chemotactic factors, such as interleukin 8 (IL-8). Previously, we demonstrated a role for c-Src kinase activation in Ox-PAPC-induced IL-8 transcription. In this study, we have examined the mechanism regulating IL-8 transcription by Ox-PAPC downstream of c-Src. Our findings demonstrate an important role for JAK2 in the regulation of IL-8 transcription by Ox-PAPC. Treatment of human aortic EC with Ox-PAPC and 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine induced a rapid yet sustained activation of JAK2; activation of JAK2 by Ox-PAPC was dependent on c-Src kinase activity. Furthermore, pretreatment with selective JAK2 inhibitors significantly reduced Ox-PAPC-induced IL-8 transcription. In previous studies, we also demonstrated activation of STAT3 by Ox-PAPC. Here we provide evidence that STAT3 activation by Ox-PAPC is dependent on JAK2 activation and that STAT3 activation regulates IL-8 transcription by Ox-PAPC in human EC. Transfection with small interfering RNA against STAT3 significantly reduced Ox-PAPC-induced IL-8 transcription. Using chromatin immunoprecipitation assays, we demonstrated binding of activated STAT3 to the sequence flanking the consensus gamma-interferon activation sequence (GAS) in the IL-8 promoter; site-directed mutagenesis of GAS inhibited IL-8 transcription by Ox-PAPC. Finally, these studies demonstrate a role for STAT3 activation in atherosclerosis in vivo. We found increased staining for activated STAT3 in the inflammatory regions of human atherosclerotic lesions and reduced fatty streak formation in EC-specific STAT3 knock-out mice on the atherogenic diet. Taken together, these data demonstrate an important role for the JAK2/STAT3 pathway in Ox-PAPC-induced IL-8 transcription in vitro and in atherosclerosis in vivo.

VE-cadherin-CreERT2 transgenic mouse: a model for inducible recombination in the endothelium.

To introduce temporal control in genetic experiments targeting the endothelium, we established a mouse line expressing tamoxifen-inducible Cre-recombinase (Cre-ERT2) under the regulation of the vascular endothelial cadherin promoter (VECad). Specificity and efficiency of Cre activity was documented by crossing VECad-Cre-ERT2 with the ROSA26R reporter mouse, in which a floxed-stop cassette has been placed upstream of the beta-galactosidase gene. We found that tamoxifen specifically induced widespread recombination in the endothelium of embryonic, neonatal, and adult tissues. Recombination was also documented in tumor-associated vascular beds and in postnatal angiogenesis assays. Furthermore, injection of tamoxifen in adult animals resulted in negligible excision (lower than 0.4%) in the hematopoietic lineage. The VECad-Cre-ERT2 mouse is likely to be a valuable tool to study the function of genes involved in vascular development, homeostasis, and in complex processes involving neoangiogenesis, such as tumor growth.

VE-Cadherin-Cre-recombinase transgenic mouse: a tool for lineage analysis and gene deletion in endothelial cells.

The ability to target gene deletion to a specific cellular compartment via the Cre/loxP system has been a powerful tool in the analysis of broadly expressed genes. Here, we report the generation of a transgenic mouse line in which expression of Cre-recombinase is under the regulatory control of the VE-Cadherin promoter. Temporal distribution and activity of the enzyme was evaluated with two independent Cre reporter lines. Histological analysis was performed throughout development and in the adult. Recombination of lox P sites with subsequent expression of beta-galactosidase or GFP was detected as early as E7.5 in endothelial cells of the yolk sac. Progressive staining of the embryonic vasculature was noted from E8.5-13.5; however, more contiguous reporter expression was only seen by E14.5 onward in all endothelial compartments including arteries, veins, and capillaries. In addition, we found Cre activity in lymphatic endothelial cells. Unlike other endothelial-specific Cre mice, this model showed expression in the adult quiescent vasculature. Furthermore, the constitutive nature of the VE-Cadherin promoter in the adult can be advantageous for analysis of gene deletion in pathological settings.

Notch signaling in vascular morphogenesis.

PURPOSE OF REVIEW: This review highlights recent developments in the role of the Notch signaling pathway during vascular morphogenesis, angiogenesis, and vessel homeostasis. RECENT FINDINGS: Studies conducted over the past 4 years have significantly advanced the understanding of the effect of Notch signaling on vascular development. Major breakthroughs have elucidated the role of Notch in arterial versus venular specification and have placed this pathway downstream of vascular endothelial growth factor. SUMMARY: An emerging hallmark of the Notch signaling pathway is its nearly ubiquitous participation in cell fate decisions that affect several tissues, including epithelial, neuronal, hematopoietic, and muscle. The vascular compartment has been the latest addition to the list of tissues known to be regulated by Notch. Unraveling the contribution of Notch signaling to blood vessel formation has resulted principally from gain-of-function and loss-of-function experiments in mouse and zebrafish. During the past 4 years, these mechanistic studies have revealed that Notch is required for the successful completion of several steps during vascular morphogenesis and differentiation. In addition, the findings that Notch mutations are linked to some late-onset hereditary vascular pathologic conditions suggest the added contribution of this signaling pathway to vascular homeostasis.