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

Abstract

To explore the role of prostaglandin E 2 (PGE 2 ) in angiogenesis in the developing corpus luteum, luteal microvascular endothelial-like cells (luteal ECs) were prepared from highly luteinizing ovaries of rats using the percoll density gradient method. The cells abundantly expressed the mRNAs of the endothelial markers CD31 (PECAM-1) and responded to the vascular endothelial growth factor (VEGF) to form in vitro tube structures on Matrigel. Cyclooxygenase (COX) inhibitors significantly suppressed tube formation in luteal ECs, whereas PGE 2 counteracted the COX inhibitorinduced blockage. PGE 2 -induced tube formation was blocked by a cyclic AMP-dependent protein kinase A (PKA) inhibitor, H89. The antagonist against the PGE receptor type 2 (EP2 receptor), AH6809, completely inhibited PGE 2 -induced tube formation and partly suppressed the VEGF-induced tube formation but did not attenuate PGE 2 -induced phosphorylation of both AKT kinase and extracellular signal-regulated kinase 1/2. VEGF significantly enhanced the expression of COX-2 mRNAs detected by realtime RT-PCR and PGE 2 secretion into the media measured by ELISA in luteal ECs. In turn, PGE 2 stimulated VEGF expression. In vitro co-culture of luteal ECs with steroidogenic luteal cells (SLCs) promoted tube formation. Pre-treatment of SLCs with VEGF further enhanced tube formation of ECs, and this effect was blocked by the COX-2 inhibitor. This stimulatory effect was inhibited by treatment with AH6809. These data indicate that PGE 2 exerts a direct stimulatory effect on tube formation mainly via the EP2 receptor/PKA signaling in luteal ECs. Our results suggest the possibility that the endogenous PGE 2 that is produced from luteinizing follicular cells as well as ECs may stimulate luteal angiogenesis.

Prostaglandin receptor EP2 in the crosshairs of anti-inflammation, anti-cancer, and neuroprotection

“…For example, aromatase-dependent estrogen synthesis is associated with hormone-dependent breast carcinogenesis, and EP2 can regulate cytochrome P450 aromatase via the cAMP/PKA/CREB pathway [71]. In addition, PGE 2 facilitates tube formation via EP2/PKA signaling in rat luteal endothelial cells, indicating the involvement of EP2 receptor in luteal angiogenesis and progression of ovarian cancer [72]. EP2 activation promotes growth of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs), by increasing β-catenin-mediated c-Myc and VEGF expression, in which both Epac and PKA pathways are engaged [73].…”

Inhibitory effect of insulin-like growth factor-binding protein-7 (IGFBP7) on in vitro angiogenesis of vascular endothelial cells in the rat corpus luteum

“…Highly luteinized ovaries were collected from immature female Wistar-Imamichi rats (3 weeks old, Imamichi Institute for Animal Reproduction, Ibaraki, Japan) treated with equine chorionic gonadotropin (50 IU, Asuka Pharmaceutical, Tokyo) and hCG (25 IU, Asuka Pharmaceutical). Whole dispersed cells obtained from the ovaries of 3 rats were subjected to the centrifugation procedure for endothelial cell enrichment with the gradient Percoll solution in each experiment [ 14 ]. Isolated LECs (Cell Systems, Kirkland, WA, USA) were grown on a 0.1% gelatin-coated dish in MCDB131 medium containing 10% fetal bovine serum (FBS; JRH Biosciences, ACSL, Lenexa, MD, USA), ECGS (50 μg/ml), and antibiotics.…”

Inflammation and angiogenesis in the corpus luteum

“…23 Under the effect of numerous proangiogenesis factors, the continually growing luteal vessels recruit the EC, and these cells proliferate and form EC networks. 24 Intensive luteal reorganization occurs after ovulation, and EC sprout to form tube-like capillaries during bovine luteal angiogenesis. 25 In fact, almost every steroidogenic cell or parenchymal cell is associated with one or more capillaries.…”

“…The follicular vessels are located in the thecal layer and invade into the granulosa cell layer following the breakdown of the basement membrane . Under the effect of numerous proangiogenesis factors, the continually growing luteal vessels recruit the EC, and these cells proliferate and form EC networks . Intensive luteal reorganization occurs after ovulation, and EC sprout to form tube‐like capillaries during bovine luteal angiogenesis .…”