Endogenous Cannabinoid-Like Arachidonoyl Serine Induces Angiogenesis through Novel Pathways.
Authors: Zhang Xuefeng , Wang Jian Feng , Maor Yehoshua , Kunos George , Groopman Jerome E. 
Published: April 2020

Abstract
Cannabinoid effects on the central nervous system have been well-described and include altered cognition, memory, motor function, and concentration. Studies have also shown that marijuana and its primary active constituent, THC (Tetrahydrocannabinol), can be salubrious in treating diverse medical conditions such as chemotherapy-induced nausea, HIV cachexia, glaucoma, and chronic pain. Recently, cannabinoids have been shown in animal models to inhibit the growth and metastasis of certain tumors through their effects on angiogenesis. There are also animal data showing that cannabinoids can alter circulatory responses by their effects on vascular tissues. However, the mechanisms involved in this modulation of angiogenesis and vascular function are not well elucidated. Δ9-THC binds with similar affinity to both CB1 and CB2, two well characterized G-protein-coupled receptors for cannabinoids. Both CB1 and CB2 are expressed to varying degrees by endothelium based on the origin of the tissue. Considerable evidence, including data from CB1/CB2 knockout mice, suggests that there is an additional endothelial cannabinoid receptor besides CB1 and CB2. This receptor has not yet been identified. We found that Δ9-THC inhibited in vitro angiogenesis in a dose dependent manner in the Matrigel assay. Δ9-THC reduced the expression of VEGFR-3 as well as the secretion of its cognate ligand VEGF-C. Blocking of the CB1 and CB2 receptors with their respective antagonists AM251 and AM630, individually or in combination, only partially reversed the THC-mediated inhibition of angiogenesis. However, pretreatment with N-Arachidonoyl Serine (ARA-S) or O-1918 [(−)-4-(3-3, 4-trans-p-menthadien-(1, 8)-yl)-orcinol], antagonists of the putative third endothelial cannabinoid receptor, significantly protected the endothelium from the inhibitory effects of Δ9-THC. In parallel, we found that abn-cbd [(−)-4-(3-3, 4-trans-p-menthadien-[1, 8]-yl)-olivetol], a selective agonist of this putative endothelial cannabinoid receptor, also inhibited in vitro angiogenesis. Pretreatment with ARA-S or O-1918 blocked the abn-cbd-mediated inhibition of angiogenesis, whereas pretreatment with AM251 or AM630 did not show significant protective effects following abn-cbd exposure. To clarify the mechanisms involved in this process, we studied the signaling pathways of cell growth and survival in endothelium following these treatments. We observed that both the ARA-S and O-1918 treatments activated ERK1/2 MAP kinase and Akt. Moreover, endothelial cells pretreated with ARA-S or O-1918 followed by THC treatment sustained significantly higher levels of ERK1/2 MAP kinase and Akt phosphorylation as compared to the THC-stimulated cells pretreated with vehicle control, the CB1 antagonist AM251, or the CB2 antagonist AM630. In addition, significantly more VEGF-C was detected in the culture supernatants of cells pretreated with the antagonists ARA-S and O-1918. ARA-S showed stronger effects than O-1918 in these studies. These results indicate that cannabinoids may regulate angiogenesis through novel pathways that are not mediated by the CB1 and CB2 receptors. Further understanding of such pathways may allow for therapeutic intervention with cannabinoids in disease states associated with neo-angiogenesis, like hematologic malignancies and solid tumors.