Martin N. Nakatsu

Cell-autonomous notch signaling regulates endothelial cell branching and proliferation during vascular tubulogenesis

The requirement for notch signaling during vascular development is well‐documented but poorly understood. Embryonic and adult endothelial cells (EC) express notch and notch ligands; however, the necessity for cell‐autonomous notch signaling during angiogenesis has not been determined. During angiogenesis, EC display plasticity, whereby a subset of previously quiescent cells loses polarity and becomes migratory. To investigate the role of notch in EC, we have used a three‐dimensional in vitro system that models all of the early steps of angiogenesis. We find that newly forming sprouts are composed of specialized tip cells that guide the sprout and trunk cells that proliferate and rearrange to form intercellular lumens. Furthermore, we find that notch acts cell‐autonomously to suppress EC proliferation, thereby regulating tube diameter. In addition, when notch signaling is blocked, tip cells divide, and both daughter cells take on a tip cell phenotype, resulting in increased branching through vessel bifurcation. In contrast, notch signaling is not required for re‐establishment of EC polarity or for lumen formation. Thus, notch is used reiteratively and cell‐autonomously by EC to regulate vessel diameter, to limit branching at the tip of sprouts, and to establish a mature, quiescent phenotype.

The requirement for fibroblasts in angiogenesis: fibroblast-derived matrix proteins are essential for endothelial cell lumen formation

The combination of a candidate gene approach, column chromatography, and mass spectrometry identifies several fibroblast-derived proteins essential for endothelial cell sprouting and lumen formation. Furthermore, proteins responsible for EC lumen formation increase matrix stiffness, which correlates with EC lumenogenesis.

An optimized three-dimensional in vitro model for the analysis of angiogenesis.

Angiogenesis is the formation of new blood vessels from the existing vasculature. It is a multistage process in which activated endothelial cells (EC) degrade basement membrane, sprout from the parent vessel, migrate, proliferate, align, undergo tube formation, and eventually branch and anastomose with adjacent vessels. Here we describe a three-dimensional in vitro assay that reproduces each of these steps. Human umbilical vein endothelial cells (HUVEC) are cultured on microcarrier beads, which are then embedded in a fibrin gel. Fibroblasts cultured on top of the gel provide factors that synergize with bFGF and VEGF to promote optimal sprouting and tube formation. Sprouts appear around day 2, lumen formation begins at day 4, and at day 10 an extensive anastomosing network of capillary-like tubes is established. The EC express a similar complement of genes as angiogenic EC in vivo and undergo identical morphologic changes during tube formation. This model, therefore, recapitulates in vivo angiogenesis in several critical aspects and provides a system that is easy to manipulate genetically, can be visualized in real time, and allows for easy purification of angiogenic EC for downstream analysis.

VEGF 121 and VEGF 165 Regulate Blood Vessel Diameter Through Vascular Endothelial Growth Factor Receptor 2 in an in vitro Angiogenesis Model

Vascular endothelial growth factor (VEGF) is essential for the induction of angiogenesis and drives both endothelial cell (EC) proliferation and migration. It has been suggested that VEGF also regulates vessel diameter, although this has not been tested explicitly. The two most abundant isoforms, VEGF121 and VEGF165, both signal through VEGF receptor 2 (VEGFR-2). We recently optimized a three-dimensional in vitro angiogenesis assay using HUVECs growing on Cytodex beads and embedded in fibrin gels. Fibroblasts provide critical factors that promote sprouting, lumen formation, and vessel stability. Using this assay, we have examined the role of VEGF in setting vessel diameter. Low concentrations of both VEGF121 and VEGF165 promote growth of long, thin vessels, whereas higher concentrations of VEGF remarkably enhance vessel diameter. Placental growth factor, which binds to VEGFR-1 but not VEGFR-2, does not promote capillary sprouting. Moreover, specific inhibition of VEGFR-2 signaling results in a dramatic reduction of EC sprouting in response to VEGF, indicating the critical importance of this receptor. The increase in vessel diameter is the result of cell proliferation and migration, rather than cellular hypertrophy, and likely depends on MEK1-ERK1/2 signaling. Both phosphatidylinositol 3-kinase and p38 activity are required for cell survival. We conclude that the diameter of new capillary sprouts can be determined by the local concentration of VEGF and that the action of VEGF on angiogenic EC in this assay is critically dependent on signaling through VEGFR-2.

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo. Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel. Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads. After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.