Endogenous Cannabinoid-Like Arachidonoyl Serine Induces Angiogenesis through Novel Pathways.
Xuefeng Zhang, MD, PhD1,
Jian Feng Wang, MD, PhD1,
Yehoshua Maor, PhD3,*,
George Kunos, MD, PhD4,* and
Jerome E. Groopman, MD1,2
1 Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; 2 Department of Surgery, BIDMC, Harvard Medical School, Boston, MA, USA; 3 Department of Medicinal Chemistry and Natural Products, Hebrew University, Jerusalem, Israel and 4 NIAAA, NIH, Bethesda, MD, USA.
Cannabinoid effects on the central nervous system have beenwell-described and include altered cognition, memory, motorfunction, and concentration. Studies have also shown that marijuanaand its primary active constituent, THC (Tetrahydrocannabinol),can be salubrious in treating diverse medical conditions suchas chemotherapy-induced nausea, HIV cachexia, glaucoma, andchronic pain. Recently, cannabinoids have been shown in animalmodels to inhibit the growth and metastasis of certain tumorsthrough their effects on angiogenesis. There are also animaldata showing that cannabinoids can alter circulatory responsesby their effects on vascular tissues. However, the mechanismsinvolved in this modulation of angiogenesis and vascular functionare not well elucidated. 9-THC binds with similar affinity toboth CB1 and CB2, two well characterized G-protein-coupled receptorsfor cannabinoids. Both CB1 and CB2 are expressed to varyingdegrees by endothelium based on the origin of the tissue. Considerableevidence, including data from CB1/CB2 knockout mice, suggeststhat there is an additional endothelial cannabinoid receptorbesides CB1 and CB2. This receptor has not yet been identified.We found that 9-THC inhibited in vitro angiogenesis in a dosedependent manner in the Matrigel assay. 9-THC reduced the expressionof VEGFR-3 as well as the secretion of its cognate ligand VEGF-C.Blocking of the CB1 and CB2 receptors with their respectiveantagonists AM251 and AM630, individually or in combination,only partially reversed the THC-mediated inhibition of angiogenesis.However, pretreatment with N-Arachidonoyl Serine (ARA-S) orO-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 inhibitoryeffects of 9-THC. In parallel, we found that abn-cbd [()-4-(3-3,4-trans-p-menthadien-[1, 8]-yl)-olivetol], a selective agonistof this putative endothelial cannabinoid receptor, also inhibitedin vitro angiogenesis. Pretreatment with ARA-S or O-1918 blockedthe abn-cbd-mediated inhibition of angiogenesis, whereas pretreatmentwith AM251 or AM630 did not show significant protective effectsfollowing abn-cbd exposure. To clarify the mechanisms involvedin this process, we studied the signaling pathways of cell growthand survival in endothelium following these treatments. We observedthat both the ARA-S and O-1918 treatments activated ERK1/2 MAPkinase and Akt. Moreover, endothelial cells pretreated withARA-S or O-1918 followed by THC treatment sustained significantlyhigher levels of ERK1/2 MAP kinase and Akt phosphorylation ascompared to the THC-stimulated cells pretreated with vehiclecontrol, the CB1 antagonist AM251, or the CB2 antagonist AM630.In addition, significantly more VEGF-C was detected in the culturesupernatants of cells pretreated with the antagonists ARA-Sand O-1918. ARA-S showed stronger effects than O-1918 in thesestudies. These results indicate that cannabinoids may regulateangiogenesis through novel pathways that are not mediated bythe CB1 and CB2 receptors. Further understanding of such pathwaysmay allow for therapeutic intervention with cannabinoids indisease states associated with neo-angiogenesis, like hematologicmalignancies and solid tumors.