An important focus of the present study
An important focus of the present study has been the interaction of HUVECs with extracellular matrix in the regulation of angiogenesis. Matrigel is a gelatinous protein extract secreted by Engelbreth–Holm–Swarm mouse sarcoma cells, and is considered to be a good model of the extracellular matrix (Kleinman and Martin, 2005). Major constituents of matrigel are structural proteins such as laminin, entactin, collagen and heparan sulfate proteoglycans. It has emerged that complex substrates such as matrigel are essential for the Doxorubicin of classical endothelial genes and their response to prostacyclin (Doron et al., 1991). The importance of endothelial adhesion to the extracellular matrix through integrins and other factors in response to prostacyclin has been demonstrated (Aburakawa et al., 2013); this suggests that the effects of prostacyclin in HUVECs might be influenced by interactions with the extracellular matrix (Orpana et al., 1997). Sensitivity of HUVECs (as estimated by median effective concentration or EC50) appears to be higher for PGE2-related lipids compared to the prostacyclin-related lipid molecules. Considering that a variety of structurally unrelated lipids were used in this study it would be difficult to predict possible non-specific lipid-binding capabilities of matrigel that was used in all our experiments. Such possibility is supported by observations of non-specific binding of numerous lipids and lipoproteins to extracellular matrix (Pillarisetti et al., 1997). Non-specific binding may have resulted in decreased sensitivity to some of the lipids observed in our study in a relatively unpredictable manner. For these reasons, in our study, we did not assign specific importance to EC50, but rather compared their maximal biological responses (Emax).
Apart from activation of the IP receptor, prostacyclin was also reported to bind to peroxisome proliferator-activated receptors (PPARs), particularly PPARα and PPARγ. These nuclear receptors are transcription factors that exhibit broad specificity for ligands, including certain lipids and lipid metabolites (Forman et al., 1997). Activation of PPARs could contribute to prostacyclin-activated angiogenesis in vivo (Pola et al., 2004). Thus, iloprost, a stable prostacyclin analog, activated angiogenesis in vivo in control mice, but not in mice lacking PPARα (Biscetti et al., 2009). However, there is no evidence that PPARs regulate angiogenesis independently of the IP receptor and direct evidence of a role for PPARs in prostacyclin-driven angiogenesis in endothelium is lacking. For example, whether prostacyclin activates angiogenesis in IP-null mice has not been reported. Nevertheless, it is possible that PPARs could contribute to the regulation of angiogenesis by prostacyclin as an alternate pathway to the conventional IP receptor signaling.
Taken together, our findings suggest that prostacyclin and the IP receptor play a pivotal role in the activation and regulation of angiogenesis and that the role of PGE2 and EP4 receptors may be relatively minor. As suggested previously, prostacyclin synthase and the IP receptor may represent novel targets for the development of treatment strategies for the inhibition of pathological angiogenesis in disease states (Shiba and Ikeda, 2013).
Conflict of interest
Acknowledgments This work was supported in part by a Research Development Grant from Division of Health Sciences, University of South Australia (ID 20-2013) and by a Project Grant from the Australian National Health and Medical Research Council (ID 1051529).
Introduction Prostaglandins (PGs) are lipid mediators that are produced by nearly all cells in the body. They are synthesized from arachidonic acid by the cyclooxygenase (COX) enzymes (Smith, 1992). Among different types of PGs, PGE2 is the most abundant with diverse functions such as maintaining homeostasis, pro- and anti-inflammatory functions (Frolov et al, 2013, Nakanishi, Rosenberg, 2013, Yao et al, 2009).