Anandamide/LPI signaling & function in endothelial cells

Anandamide (ANA), also known as N-arachidonoylethanolamine, is a prominent substance of endocannabinoids, and exhibits multiple physiological functions in the brain, the immune system and the cardiovasculature. Although ANA is well known to contribute to human behavior, like eating and sleeping, and pain relief, evidence accumulated that ANA is an important vascular mediator that is released during tissue stress. Though in most tissues the physiological activity of anandamide can be well attributed to its binding to the classical cannabinoid receptors (CBR) 1 and 2, vascular reactivity to anandamide involves further, until recently or even still unknown binding sites that trigger e.g. the formation of nitric oxide and, thus, vascular relaxation in response to this endocannabinoids. Recently, by the discovery of GPR55 as putative receptor for anandamide and the characterization of its existence and functionality in endothelial cells, an intriguing candidate for an endothelial receptor that contributes to the vascular function of ANA was introduced. GPR55 does not share classical CBR properties but binds lysophosphatidylinositol (LPI), another putatively important lipid mediator in the vessel wall. In our previous work, we described that in human endothelial cells ANA but not LPI binds on both, the classical CB 1 R and the GPR55 that mediate divers signaling pathways. Intriguingly, GPR55-induced signaling is under the repression of the CB 1 R and becomes relieved once integrins clustering is initiated. Because integrin clustering plays an important role in cellular growth, migration, endothelial barrier function and inflammation, we speculate that ANA serves as an important vascular mediator of which its signaling properties changes pending the status of the endothelium: under resting conditions ANA is a rather calming signal while under cellularissue stress ANA stimulates endothelial cells in order to initiate counteraction against potentially harmful conditions. In contrast to ANA, LPI was found to be rather selective for GPR55 but also to exhibit numerous, most likely receptor independent effects on endothelial cells via yet unknown mechanisms. In view of the rather fragmentary understanding of the molecular mechanisms of ANA / LPI signaling and the vascular consequences thereof this proposal intends to investigate: the signal transduction pathways that are activated by ANA / LPI and to correlate them with their sites of action (WORK PACKAGE A), the consequences of the respective signaling pathways activated by ANA / LPI on endothelial cell functions (WORK PACKAGE B), and the role of ANA / LPI in the recruitment of hematopoietic stem cells to the endothelium as potential mechanisms of vascularissue repair (WORK PACKAGE C) The overall aim of this project is to explore ANA / LPI as important vascular mediators, to characterize their respective signal transduction, their interrelation and consequences to endothelial cell functions. In the end, our proposal aims to provide the scientific basis of consideration of the ANA / LPI system as therapeutic target for vascular-related medicine.

Anandamide (ANA), also known as N-arachidonoylethanolamine, is the most prominent substance of endocannabinoids, and exhibits multiple physiological functions in the brain, the immune system and the cardiovasculature. ANA is well known to contribute to human behavior, like eating and sleeping, and pain relief. Recently, evidence accumulated that ANA is an important vascular mediator under conditions of tissue stress. Though in most tissues the physiological activity of ANA correlates well with its binding to the classical cannabinoid receptors (CBR) 1 and 2, vascular reactivity to anandamide involves further, until recently or even still unknown binding sites that mediates vascular effects in response to these endocannabinoids. One of these putative new orphan endocannabinoid receptors binds lysophosphatidylinositol (LPI), another putatively important lipid mediator in the vessel wall. In view of the rather fragmentary understanding of the molecular mechanisms of ANA / LPI signaling and the vascular consequences thereof this proposal intended to investigate (A), the signal transduction pathways that are activated by ANA / LPI, (B) the consequences of the respective signaling pathways activated by ANA / LPI on cell functions, and (C) the role of ANA / LPI in the recruitment of hematopoietic stem cells. In course of this project, endocannabinoids and LPI were not only confirmed to act on their specific receptors on endothelial cells but also found to exhibit direct, non-receptor-mediated effects on various plasma membrane ion channels and carriers. Hence, N-arachidonoyl glycine, another member of the continuously growing family of endocannabinoids, was discovered to selectively prevent the molecular interaction between STIM1 and Orai1, thus, inhibiting the activity of store-operated Ca2+ channels in various cell types including endothelial cells. Accordingly, these data point to a considerable potential of endocannabinoids and LPI to act directly to plasma membrane ion channels. In line with these findings, we described for the very first time that anandamide stimulates the main mitochondrial Ca2+ channel i-MCC. This exciting finding provides a new characteristic of the physiology of endocannabinoids on mitochondrial function and energy metabolism and points to a role of endocannabinoids in energy sensing. Finally, endocannabinoids were found to distinctively activate endothelial progenitor cells (endothelial colony-forming cells, ECFC). In particular, AEA stimulates ECFC to perform angiogenesis via CB2R and TRPV1, a mechanism possibly important under hypoxic conditions and cancer angiogenesis. In contrast, LPI engages ECFCs to carry out wound healing via GPR55 and TRPV1. In course of this project several new, potentially important aspects on the physiological role of endocannabinoids, the underlying molecular mechanisms and therapeutic potential have been elaborated. These findings further underline the importance of endocannabinoids in vascular biology, distinct functions in cancer angiogenesis and wound healing, and highlight their messenger function in the cellular energy metabolism.