Isolation and Culture of Microvascular Endothelial Cells from the Primate Corpus Luteum1

Abstract

Endothelial cells have common as well as specialized roles in different tissues and organs; as such, the abundant endothelial cells in the corpus luteum (> 50% of total cell population) could have unique activities necessary for luteal function. Our objective was to establish a method for isolating a pure population of endothelial cells from the primate corpus luteum and to determine the basic conditions for in vitro culture. Corpora lutea collected from rhesus monkeys throughout the luteal phase of the menstrual cycle were minced and enzymatically dispersed into single cell suspensions. Endothelial cells were isolated from the remaining cells (i.e., steroidogenic, fibroblastic, etc.) utilizing magnetic beads labeled with a lectin, Ulex europaeus agglutinin-1 (UEA-1), which binds a sugar found only on primate endothelial cells. After exposure to a magnetic field, UEA-1-negative (-) cells were decanted from the pelleted UEA-1-positive (+) cells; to remove beads from the UEA-1 (+) cells, excess sugar was applied. After optimization of the bead-to-cell ratio, the UEA-1 (+) group contained a population of cells 8-12 microns in diameter (typical endothelial cell size) and < 1% steroidogenic cells. UEA-1 (-) cells were larger (15-35 microns) and stained histochemically for the steroidogenic marker, 3 beta-hydroxysteroid dehydrogenase. Immunocytochemical analysis demonstrated that > 93% of all cells in the UEA-1 (+) group stained positive for the specific endothelial cell marker, platelet/endothelial cell adhesion molecule-1. Cultured UEA-1 (+) cells produced low levels of progesterone and were unresponsive to hCG (100 ng/ml). In contrast, cultured UEA-1 (-) and mixed (unsorted) cells produced high basal levels of progesterone and exhibited a > 3-fold increase in response to hCG treatment. Preliminary experiments comparing different culture media and matrices demonstrated that 1) cell proliferation was unaffected by type of medium (i.e., Dulbecco's Modified Eagle Medium [DMEM]/ F12 or McCoy's 5A); 2) the presence of serum was essential in the absence of added growth factors, and 3) extracellular matrix had a profound effect on proliferation. UEA-1 (+) cells exhibited a dose-dependent increase in cell proliferation in response to vascular endothelial growth factor (VEGF) in the presence and absence of fetal calf serum. In the absence of serum, VEGF stimulated proliferation of UEA-1 (+) cells plated on fibronectin but not collagen I, whereas in the presence of 10% fetal calf serum, both matrices supported VEGF-induced mitogenesis. These studies provide for the first time an efficient, reliable method for isolating primate luteal endothelial cells and describe in vitro culture conditions for subsequent studies examining luteal endothelial cell function and regulation.

A novel physiological culture system that mimics luteal angiogenesis

“…This is in strong agreement with Christenson & Stouffer (1996) who demonstrated that fibronectin but not collagen I increased luteal endothelial cell proliferation in response to VEGFA. This is also supported by the in situ observations that fibronectin abundance is particularly high during the early growth phase of the CL while collagen I is relatively low, which again emphasizes its physiological relevance.…”

Vascular morphogenesis in the primate ovary

“…VEGF and EG-VEGF expression in these cells is regulated by hCG and not by progesterone [55]. In addition, endothelial cells from the macaque corpus luteum have also been isolated and studied in vitro [60] and human ovarian microvascular endothelium have been cultured from follicular aspirates [61]. Ovarian fibroblasts have also been isolated and co-culture models with steroidogenic cells have demonstrated paracrine regulation of CTGF by hCG [58].…”

Stimulation of tube formation mediated through the prostaglandin EP2 receptor in rat luteal endothelial cells

“…The multipotency of granulosa stem-like cells has been described by in vitro differentiation into other cell types (Kossowska-Tomaszczuk et al granulosa stem-like cells that show characteristics of endothelial progenitors capable of maturation into ECs under endothelium condition. This study is, to the best of our knowledge, the first to show in vitro tube formation in rat luteal ECs, although luteal ECs have already been isolated and characterized from other species including cows (Okuda et al 1999, Davis et al 2003, rhesus monkeys (Christenson & Stouffer 1996), and humans (Ratcliffe et al 1999). The PGE 2 -induced tube formation was observed either in the presence of COX inhibitors or in the presence of a blocker of the VEGF receptor-2, and without EC proliferation (data not shown).…”