Relationship between prostaglandin E₂ and vascular endothelial growth factor (VEGF) in angiogenesis in human vascular endothelial cells

Abstract

To address the role of prostaglandin E₂ (PGE₂) in tube formation of endothelial cells and the relationships between the action of PGE₂ and vascular endothelial growth factor (VEGF), cultured human umbilical vein endothelial cells (HUVECs) were used to evaluate tube formation on Matrigel and the expression of angiogenesis-related genes. PGE₂ treatment stimulated the tube-like formation of HUVECs. Whereas VEGF-induced tube formation was significantly suppressed by ETYA, an inhibitor of arachidonic acid metabolism, or SU5614, an inhibitor of VEGF-receptor tyrosine kinase, the stimulatory effect of PGE₂ was observed in the presence of ETYA or SU5614. Thus, PGE₂ counteracted both ETYA- and SU5614-induced blockage of angiogenesis in the presence of VEGF. VEGF induced cyclooxygenase (COX) -2 mRNA expression in HUVECs and increased the PGE₂ concentration in the medium. PGE₂ treatment enhanced the expression of VEGF mRNA. These findings suggest that PGE₂ directly stimulates angiogenesis, apart from VEGF signaling, and further induces VEGF expression in HUVECs. In addition, the effect of VEGF on angiogenesis may be mediated, in part, by PGE₂ secretion.

Introduction

Angiogenesis is the development of new microvessels from preexisting blood vessels (Folkman and D'Amore, 1996). Physiological angiogenesis occurs during fetal growth and development. It also occurs in the endometrium and ovarian corpus luteum of the female reproductive tract, which is reconstituted regularly during the menstrual cycle. Angiogenesis is seen in pathological conditions such as malignancy and chorionic inflammatory disorders, including rheumatoid arthritis and diabetic retinopathy. Vascular endothelial growth factor (VEGF) is a major regulator of endothelial proliferation and migration (Neufeld et al., 1999). VEGF has five isoforms composed of 121, 145, 165, 189, or 206 amino acids (Ferrara and Davis-Smyth, 1997). The secreted VEGF-121, VEGF-145, and VEGF-165 have mitogenic action on endothelial cells (Park et al., 1993). Although fibroblast growth factors (FGFs) as well as VEGF and their receptors are primarily responsible for the establishment of angiogenesis, numerous molecules have been reported to have angiogenic or anti-angiogenic actions (Carmeliet, 2003).

Cyclooxygenase (COX) is a rate-limiting enzyme in the biosynthetic pathway of prostaglandins (PGs). COX consists of two isoforms: COX-1, that is expressed constitutively in most cells, and COX-2, which is inducible by mitogens and proinflammatory cytokines during pathological processes including inflammation (Hla et al., 1993). COX-2 is also induced by hormones (Akarasereenont et al., 2000) and growth factors, including VEGF (Wheeler-Jones et al., 1997, He et al., 1999, Hernandez et al., 2001, Tamura et al., 2002) and basic FGF (Kage et al., 1999). Isomerization of PGH₂ to PGE₂ is catalyzed by PGE synthase (PGES). PGES, which is located downstream of COX in the arachidonate cascade, has multiple forms in mammalian cells (Murakami et al., 2002). Three isoforms of PGES have been identified. Cytosolic PGES (cPGES) is ubiquitous and constitutively expressed in cells. It converts COX-1-derived PGH₂ to PGE₂ in cooperation with heat shock protein 90. Microsomal PGES-1 (mPGES-1) is functionally coupled with COX-2 and up-regulated by proinflammatory stimuli. mPGES-2 is a unique PGES that couples with both COXs and is constitutively expressed in carious cells (Murakami et al., 2003). The functional coupling of COX-2 and mPGES-1 has been reported and may cause abundant PGE₂ production (Murakami et al., 2002). The co-localization of these two enzymes also has been described (Murakami et al., 2002). Endothelial cells produce PGE₂, PGI₂ and PGF_2α in response to various stimuli (Marcus et al., 1978, Camacho et al., 1998). PGs, especially PGE₂, have been implicated in angiogenesis (Leahy et al., 2000, Salcedo et al., 2003). It has been reported that PGE₂ stimulates VEGF expression in fibroblasts (Shweiki et al., 1992) and that COX-2-dependent VEGF induction enhances bFGF-induced angiogenesis in rat sponge implants (Amano et al., 2002).

We recently reported that the activity of cyclooxygenase (COX) -2 is associated with the formation of the ovarian corpus luteum via its stimulation of angiogenesis (Sakurai et al., 2003), and that PGE₂ overcomes the blockage of angiogenesis in the corpus luteum by COX-2 inhibitors (Sakurai et al., 2005). Further, VEGF enhances PGE₂ production by stimulating COX-2 and mPGES-1 expression and that PGE₂ induces VEGF expression in rat ovarian luteal cells (Sakurai et al., 2004). These findings suggest that luteal PGE₂ may be involved in angiogenesis in the corpus luteum. However, there are no reports examining a direct interaction between VEGF and eicosanoids, in particular PGE₂ in endothelial tube formation, and there is no evidence as to whether VEGF stimulates the pathway of PGE₂ formation in endothelial cells. In this study, we, therefore, evaluated the interaction of both angiogenic factors, VEGF and PGE₂, on in vitro angiogenesis using the Matrigel assay in human umbilical vein endothelial cells (HUVECs).