Lauren C. Goldie

Regulation of Endothelial Cell Differentiation and Specification

The circulatory system is the first organ system to develop in the vertebrate embryo and is critical throughout gestation for the delivery of oxygen and nutrients to, as well as removal of metabolic waste products from, growing tissues. Endothelial cells, which constitute the luminal layer of all blood and lymphatic vessels, emerge de novo from the mesoderm in a process known as vasculogenesis. The vascular plexus that is initially formed is then remodeled and refined via proliferation, migration, and sprouting of endothelial cells to form new vessels from preexisting ones during angiogenesis. Mural cells are also recruited by endothelial cells to form the surrounding vessel wall. During this vascular remodeling process, primordial endothelial cells are specialized to acquire arterial, venous, and blood-forming hemogenic phenotypes and functions. A subset of venous endothelium is also specialized to become lymphatic endothelium later in development. The specialization of all endothelial cell subtypes requires extrinsic signals and intrinsic regulatory events, which will be discussed in this review.

Cell signaling directing the formation and function of hemogenic endothelium during murine embryogenesis

During developmental hematopoiesis, multilineage hematopoietic progenitors are thought to derive from a subset of vascular endothelium. Herein, we define the phenotype of such hemogenic endothelial cells and demonstrate, on a clonal level, that they exhibit multilineage hematopoietic potential. Furthermore, we have begun to define the molecular signals that regulate their development. We found that the formation of yolk sac hemogenic endothelium and its hematopoietic potential were significantly impaired in the absence of retinoic acid (RA) signaling, and could be restored in RA-deficient (Raldh2(-/-)) embryos by provision of exogenous RA in utero. Thus, we identify a novel, critical role for RA signaling in the development of hemogenic endothelium that contributes to definitive hematopoiesis.

Hemogenic Endothelial Cell Specification Requires c-Kit, Notch Signaling, and p27-Mediated Cell-Cycle Control

Delineating the mechanism or mechanisms that regulate the specification of hemogenic endothelial cells from primordial endothelium is critical for optimizing their derivation from human stem cells for clinical therapies. We previously determined that retinoic acid (RA) is required for hemogenic specification, as well as cell-cycle control, of endothelium during embryogenesis. Herein, we define the molecular signals downstream of RA that regulate hemogenic endothelial cell development and demonstrate that cell-cycle control is required for this process. We found that re-expression of c-Kit in RA-deficient (Raldh2(-/-)) primordial endothelium induced Notch signaling and p27 expression, which restored cell-cycle control and rescued hemogenic endothelial cell specification and function. Re-expression of p27 in RA-deficient and Notch-inactivated primordial endothelial cells was sufficient to correct their defects in cell-cycle regulation and hemogenic endothelial cell development. Thus, RA regulation of hemogenic endothelial cell specification requires c-Kit, notch signaling, and p27-mediated cell-cycle control.

A critical assessment of the factors affecting reporter gene assays for promoter SNP function : a reassessment of -308 TNF polymorphism function using a novel integrated reporter system

One of the greatest challenges facing genetics is the development of strategies to identify functionally relevant genetic variation. The most common test of function is the reporter gene assay, in which allelic regulatory regions are used to drive the expression of a reporter gene, and differences in expression in a cell line after transient transfection are taken to be a reflection of the polymorphism. Many studies have reported small differences in single nucleotide polymorphism (SNP)-specific reporter activity, including the tumor necrosis factor (TNF) G−308A polymorphism. However, we have established that many variables inherent in the reporter gene approach can account for the reported allelic differences. Variables, such as the amount of DNA used in transfection, the amount of transfection control vector used, the method of transfection, the growth history of the host cells, and the quality and purity of DNA used, all influence TNF −308 SNP-specific transient reporter gene assays and serve as a caution for those researchers who apply this method to the functional assessment of polymorphic promoter sequences. We have developed an integrated reporter system that obviates some of these problems and shows that the TNF G−308A polymorphism is functionally relevant in this improved assay, thus confirming that the −308A allele expresses at a higher level compared with the −308G allele.

Embryonic vasculogenesis and hematopoietic specification.

Vasculogenesis is the process by which blood vessels are formed de novo. In mammals, vasculogenesis occurs in parallel with hematopoiesis, the formation of blood cells. Thus, it is debated whether vascular endothelial cells and blood cells are derived from a common progenitor. Whether or not this is the case, there certainly is commonality among regulatory factors that control the differentiation and differentiated function of both cell lineages. VEGF is a major regulator of both cell types and plays a critical role, in coordination with other signaling pathways and transcriptional regulators, in controlling the differentiation and behavior of endothelial and blood cells during early embryonic development, as further discussed herein.

Integration site-specific transcriptional reporter gene analysis using Flp recombinase targeted cell lines

While high-throughput genome-wide approaches are useful to identify important regulatory regions, traditional reporter gene methodologies still represent the ultimate steps in fine structure analysis of transcriptional control elements. However, there are still several inherent limitations in the currently available transient and stable transfection systems often leading to aberrant function of specific cis elements. In this study we overcome these problems and have developed a novel and widely applicable system that permits the comparison of transcriptional reporter gene activities following site-specific genomic integration. By using Flp recombinase-mediated integration, the system allows the integration and expression of a series of reporter gene constructs at exactly the same genomic location and orientation in all cells of any one culture. The resulting reporter gene lines carry a single reporter gene, which is incorporated within a measurably active chromatinized setting, thus more closely reflecting the endogenous gene environment.

Stem Cell-Derived Vascular Endothelial Cells and Their Potential Application in Regenerative Medicine

Although a ‘vascular stem cell’ population has not been identified or generated, vascular endothelial and mural cells (smooth muscle cells and pericytes) can be derived from currently known pluripotent stem cell sources including human embryonic stem cells and induced pluripotent stem cells. We review the vascular potential of these human pluripotent stem cells, the mechanisms by which they are induced to differentiate toward a vascular endothelial cell fate, and their applications in regenerative medicine.