Published On: Thu, Apr 18th, 2019

Prostaglandin E2 promotes embryonic vascular development and maturation in zebrafish [RESEARCH ARTICLE]

INTRODUCTION

Prostaglandin (PG) E-2 is a prostanoid, which is endogenously synthesized from the arachidonic acid of vertebrate cell membranes by an enzyme, cyclooxygenase (COX)-2. PGE2 binds to four different G-coupled receptors (EPs), EP 1–4, which have various cellular functions (Sugimoto and Narumiya, 2007). EP2 and EP4 receptors share the cAMP/PKA pathway; however, EP4 additionally signals through the phosphatidylinositol 3-kinase (PI3K)/AKT pathway (Fujino et al., 2003). In vertebrates, PGE2 plays major physiological roles in embryonic development by regulating the homeostatic balance of hematopoietic stem cells (HSC) during early embryonic growth. The role of PGE synthase (Ptges) in embryonic differentiation and growth in zebrafish was well investigated by Cha et al. (2006). They showed that knockdown of Ptges in the zebrafish embryo completely abrogated early cell differentiation and cell polarization, which was retrieved with PGE2 addition, as embryos could recover all phenotypes. They have also shown that PGE2 regulates zebrafish growth via EP4/PI3K/Akt pathways (Cha et al., 2006). Supporting this, North et al. (2007) showed that PGE2 regulates the differentiation of embryonic stem cells. This study showed that treatment with chemicals that enhance PGE2 synthesis induced HSC and in contrast, chemicals, which block prostaglandin synthesis, decreased stem cell numbers (North et al., 2007).

PGE2 induces breast cancer stem-like cells (SLCs) via the upregulation of stem cells marker (NANOG and SOX2) in tumors and by stimulation of NOTCH and Wnt genes expression (Majumder et al., 2016). With a COX-2 inhibitor (COX2-I) and specific EP4 antagonist (EP4A) treatment, we could abrogate COX-2/PGE2 induced SLCs in breast cancer. In addition, PI3K/Akt inhibitors also abrogated PGE2 induced NOTCH and WNT genes expression in human breast cancer. Therefore, we have established that COX-2 and PGE2 induce human breast SLCs and were regulated by EP4/PI3K/Akt/NOTCH/WNT pathways (Majumder et al., 2016). However, we never tested the roles of PGE2 in vertebrate vascular development.

Zebrafish are widely used as vertebrate models for pathophysiological studies (North et al., 2007; Zon and Peterson, 2005). The transparency of the zebrafish identifies it as a good model for the investigation of vascular development. Moreover, zebrafish mutually share many structural, functional and molecular features with other vertebrates and are perfect models for xenotransplantation due to their inability to reject graft within 48 hpf (Benyumov et al., 2012; Jung et al., 2017; Mulligan and Weinstein, 2014). In vertebrates, lymphangiogenesis begins from HSC fate determination, through several differentiation steps to develop arterial and venous progenitors (Nicenboim et al., 2015). The genetic interactions involving HSC homeostasis and replenishment in zebrafish is regulated by PGE2 through the Wnt pathway (Goessling et al., 2009). Similarly, cardiac muscle development and pigmentation in zebrafish was shown to be regulated through Wnt signaling (Dohn and Waxman, 2012; Vibert et al., 2017). In animal models, cell lineage and vascular differentiation stages could be monitored with Texas Red low-molecular-weight dextran, a widely used fluorescent dye for tracing vascular lineages in vertebrate development (Zhao et al., 2011). There is no comprehensive report on the roles of PGE2 in zebrafish embryonic angiogenesis and lymphangiogenesis. Therefore in this study, we investigated the effects of PGE2 on zebrafish embryonic vascular development and maturation.

The process of vascular development and maturation principally involves the interaction of vascular endothelial growth factors (vegf)s with their cognate receptors (vegfr)s in zebrafish. VEGF/VEGFR interaction plays a critical role in the formation and modification of vascular network during embryonic development in vertebrates. In zebrafish, primarily vegfa and vegfd directly interact with kdrl (equivalent to VEGFR2 in human) to regulate angiogenesis and lymphangiogenesis during early embryonic growth (Covassin et al., 2006; Bahary et al., 2007; Bower et al., 2017). Alternatively, angiogenesis is partly regulated by vegfa receptor flt1 (equivalent to VEGFR1 in humans), tested in mouse endothelial cell models (Nesmith et al., 2017). In zebrafish, angiogenesis involves a coordinated regulation of kdrl and flt4 (equivalent to VEGFR3 in humans) receptors, partly controlled by Erk and Notch signaling (Phng and Gerhardt, 2009). Expression of NOTCH transmembrane ligand efnb2a and its cognate receptor ephb4a on vascular endothelial cells and blood vessels selectively promotes cardiovascular development and angiogenesis in mouse embryos (Gerety, et al., 1999; Chen et al., 2015). However, Krueger et al. (2011) showed that flt1 negatively regulates efnb2a during early angiogenic sprouting and erythropoiesis in zebrafish (Krueger et al., 2011). Furthermore, the roles of another vascular transcription factor etv2 and its receptor G protein gamma-2 in VEGF-mediated angiogenesis and lymphangiogenesis in vertebrates remains unclear (Leung et al., 2006; Gomez et al., 2009; Davis et al., 2018). Roles of PGE2 in vascular marker expression have never been tested in zebrafish.

Previously, we have shown that in mouse breast cancer cell lines COX-2 induces PGE2 synthesis, which in turn induces tumor-associated angiogenesis and lymphangiogenesis via overproduction of VEGF-C and VEGF-D. This was regulated by EP4/PI3K/Akt pathway, and selective COX-2I and EP4A could inhibit tumor associated angiogenesis and metastasis in a mouse model (Xin et al., 2012; Majumder et al., 2014; Lala et al., 2018). Furthermore, using rat lymphatic mesenteric lymphatic endothelial cells (RMLEC)s we showed that PGE2 induced lymphangiogenesis in vitro could be abrogated with COX2-I and EP4A (Nandi et al., 2017). In human breast cancer we have also shown that PGE2 induces cancer cell migration, invasion and tumor-associated angiogenesis and lymphangiogenesis via upregulation of the EP4/PI3K/AKT pathways. Each of these phenotypes could be abrogated with a specific COX-2I and an EP4A (Majumder et al., 2016, 2018). Non-steroidal anti-inflammatory drugs (NSAIDs) have emerged as powerful COX-2 inhibitors, which inhibit the synthesis of prostaglandins, hence are commonly used as pain medication (Ricciotti and FitzGerald, 2011). However, chronic consumption of NSAIDs by North Americans resulted in severe side effects like gastrointestinal ulcers, perforation, bleeding, cardiac strokes, myocardial infarction, hypertension and renal dysfunction (Harirforoosh et al., 2013; Pai et al., 2018). Most of these effects might be due to blockage of protective physiological functions of PGE2 in humans (Guo et al., 2012; Przygodzki et al., 2015). So, in this article, we tested the physiological roles of PGE2 during early embryonic vascular growth and maturation using zebrafish as an in vivo model.

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