Blood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness.

Paracrine interactions between endothelial cells (EC) and mural cells act as critical regulators of vessel wall assembly, vessel maturation and define a plasticity window for vascular remodeling. The present study was aimed at studying blood vessel maturation processes in a novel 3-dimensional spheroidal coculture system of EC and smooth muscle cells (SMC). Coculture spheroids differentiate spontaneously in a calcium-dependent manner to organize into a core of SMC and a surface layer of EC, thus mimicking the physiological assembly of blood vessels with surface lining EC and underlying mural cells. Coculture of EC with SMC induces a mature, quiescent EC phenotype as evidenced by 1) a significant increase in the number of junctional complexes of the EC surface layer, 2) a down-regulation of PDGF-B expression by cocultured EC, and 3) an increased resistance of EC to undergo apoptosis. Furthermore, EC cocultured with SMC become refractory to stimulation with VEGF (lack of CD34 expression on VEGF stimulation; inability to form capillary-like sprouts in a VEGF-dependent manner in a 3-dimensional in gel angiogenesis assay). In contrast, costimulation with VEGF and Ang-2 induced sprouting angiogenesis originating from coculture spheroids consistent with a model of Ang-2-mediated vessel destabilization resulting in VEGF responsiveness. Ang-2 on its own was able to stimulate endothelial cells in the absence of Ang-1 producing SMC, inducing lateral sheet migration as well as in gel sprouting angiogenesis. Taken together, the data establish the spheroidal EC/SMC system as a powerful cell culture model to study paracrine interactions in the vessel wall and provide functional evidence for smooth muscle cell-mediated quiescence effects on endothelial cells.

Analysis of blood vessel maturation processes during cyclic ovarian angiogenesis.

Cyclic angiogenic processes in the ovarian corpus luteum (CL) of monovulatory species are characterized by distinct phases of blood vessel growth, vessel maturation, and vessel regression. To characterize molecular and cellular systems that may play a role in regulating blood vessel maturation, we have (a) analyzed the spatiotemporal expression of vascular endothelial growth factor (VEGF) and its receptors VEGF-R1 (Flt-1) and VEGF-R2 (Flk-1) throughout the ovarian cycle, (b) examined the recruitment of pericytes during vessel maturation, and (c) quantitatively measured the ratio of angiopoietin-2 (Ang-2) to angiopoietin-1 (Ang-1) throughout the ovarian cycle. The data indicate that the VEGF/VEGF-receptor system is expressed not only during ovarian angiogenesis, but also with similar intensity in the nonangiogenic midstage CL. In fact, VEGF is expressed through most of the ovarian cycle, only being down-regulated during luteolysis, which leads to regression of the CL neovasculature. Pericytes are recruited soon after the induction of CL angiogenesis following the front of invading endothelial cells. Based on a double-staining immunohistochemistry technique, we developed a microvessel maturation index (MMI) that reflects the percentage of the capillary neovasculature that is associated with pericytes. The MMI in the angiogenic corpus rubrum is approximately 0.60. This value is not significantly higher in the nonangiogenic midstage CL but increases to close to 0.90 during CL regression. Lastly, an RT-PCR analysis of Ang-1 and Ang-2 expression revealed that both molecules are expressed throughout the ovarian cycle. The quantitative Ang-2/Ang-1 ratio does, however, change from 1.34 in the angiogenic CL and 1.07 in the midstage CL to 7.59 during CL regression, reflecting the strong overexpression of Ang-2 over Ang-1 during blood vessel regression. Taken together, the data support a model of a transiently maturated vasculature in the midstage CL, which is characterized by VEGF and pericyte contact-mediated endothelial cell survival and an induction of blood vessel regression during luteolysis that is characterized by the down-regulation of VEGF and the up-regulation of Ang-2.