Differentiation of vascular cells in vivo

Endothelial cells (EC) cover the inside of all blood vessels as a monolayer. In the adult mature vascular system EC contact pericytes (PC) or (sometimes in arteries/veins) smooth muscle cells (SMC) through breaks in the underlying basal membrane (BM). Under homeostasis, EC located in an intact monolayer as well as SMC do not proliferate and are referred to as “quiescent”. In this state SMC maintain a contractile apparatus able to respond to vasoactive agonists by contraction or relaxation. 

Stimulatory events such as wounding of blood vessels and the following healing process activate EC to degrade their underlying basal membrane, to migrate into the surrounding matrix, to proliferate and to establish new anastomosing  networks, which were again covered  by PC or SMC. Activated EC are referred to as “angiogenic”. SMC may temporarily acquire a “synthetic” phenotype to support the remodeling of a vessel wall. 

Differentiation of vascular cells in vitro

In vitro primary EC were usually cultured as two-dimensional flat monolayers reflecting some properties of the endothelial in vivo phenotype. Nevertheless, EC cultured in that way are not completely quiescent. Even in a confluent monolayer up to 10% of the EC proliferate and tend to lose their differentiation over time (e.g. downregulation of CD34). This also applies to cultured SMC, which preferentially acquire an activated synthetic rather than a quiescent contractile phenotype.

 

In order to preserve differentiation and quiescence of EC in vitro, we developed a novel method to culture EC as three-dimensional spheroids. Single suspended EC are seeded under non-adhesive conditions in round bottom 96-well plates or hanging drops. After 18-24h, all EC seeded in one well/drop contribute to the formation of a single spheroid. The spheroids organize over time (24h) to establish a quiescent, non-proliferating surface monolayer of ECs enclosing a core of (quiescent) unorganized cells. We exploit this cell culture technique to analyze angiogenesis and endothelial differentiation. 

 

Just like the spheroids generated from EC, corresponding  size- and cell number-defined aggregates of quiescent vascular smooth muscle cells (VSMC) are also utilized in various in vitro assays.  In fact, under 3D culture conditions VSMC cease to proliferate as evidenced by tracking the proliferation marker Ki67. Furthermore, they increase the protein level of contractile elements (e.g. alpha smooth muscle actin) and decrease the overall activity of MAP kinases (e.g. ERK1/2). 

 

Contact:

Prof. Thomas Korff, Abteilung Herz- und Kreislaufphysiologie 

E-Mail: korff(at)physiologie.uni-heidelberg.de