Endocannabinoids, cannabinoid receptors and inflammatory stress: an interview with Dr. PÃ¡l PacherHelene F. Rosenberg
December 1, 2007 - Journal of Leukocyte Biology
The manuscript "Cannabinoid-2 receptor agonist HU-308 protects against hepatic ischemia/reperfusion injury by attenuating oxidative stress, inflammatory response, and apoptosis" was selected as a Pivotal Advance because it considers the role of endocannabinoids, generally understood for their psychoactive features alone, as biochemical modulators of systemic inflammation. In this manuscript, Dr. PÃ¡l Pacher and his colleagues explore the role of the cannabinoid receptor 2 (CB2) agonist, HU-308, in preventing tissue damage in an experimental model of tissue ischemia/reperfusion injury.
Dr. Pacher, to begin, can you tell the readers a bit more about endocannabinoids and cannabinoid receptors? How were these receptors and their endogenous ligands discovered?
PP: Endocannabinoids are endogenous lipid mediators generated by virtually all cell types both in the brain and peripheral tissues, which exert broad range of biological effects (cardiovascular, psychoactive, antiinflammatory) similar to those of cannabis [1 ]. There are two major G protein-coupled cannabinoid receptors, CB1 and CB2 [2 , 3 ], although there is evidence suggesting that there are additional receptors remaining to be characterized [4 ]. Arachidonoyl ethanolamide or anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are the two most widely studied endocannabinoids which were isolated and characterized by the group of Raphael Mechoulam [5 , 6 ], who had earlier discovered the main psychoactive constituent of cannabis, delta-9-tetrahydrocannabinol [7 ]. Mechoulam hypothesized the existence of an endogenous brain ligand for this receptor from the pioneering work of Allyn Howlett, who with her then-graduate student Bill Devane, provided the first evidence for cannabinoid receptors in the brain [8 ]. His team (Bill Devane, Lumir Hanus, Aviva Breuer, and others) initially had to prepare an in-house radiolabeled probe (such probes were not commercially available that time), and they faced various difficulties in isolating the putative ligand from porcine brain. After two years of hard work, the endogenous ligand, named anandamide (from "ananda," meaning "supreme joy" in Sanskrit), was finally isolated [5 ], and was biologically characterized by Roger Pertwee in Scotland. Shortly after this, Sean Munro in Cambridge discovered a second cannabinoid receptor in the spleen, and Mechoulam’s group started to work on the identification of an endogenous ligand that activates this receptor. Mechoulam teamed up with a group of 15 brilliant scientists (Zvi Vogel, Roger Pertwee, Norb Kaminski, and Billy Martin, to name just a few) and in about two years they were able to isolate and characterize the second endocannabinoid 2-AG from canine gut [6 ]. Since these major groundbreaking discoveries, which were nicely described in a recent interview with Raphael Mechoulam in Addiction [9 ], it turned out that dysregulation of the endocannabinoid system may be implicated in virtually all diseases, and its pharmacological modulation holds tremendous promise in the treatment of various inflammatory, metabolic, and cardiovascular disorders, as well as pain and cancer [10 ].
What provided you and your group with the impetus to study these ligand-receptor interactions specifically in acute ischemia/reperfusion injury?
PP: Ischemia/reperfusion (I/R) is a pivotal mechanism of organ injury during myocardial infarction, stroke, organ transplantation and vascular surgery. Importantly, we wanted to understand the role of CB2 receptors in endothelial cell activation and in the endothelial/inflammatory cell interactions, which are critical steps not only in reperfusion injury, but also in atherosclerosis and various other inflammatory disorders. This issue indeed turned out to be very important, because CB2 cannabinoid agonists protected against hepatic I/R injury by decreasing the endothelial cell activation/inflammatory response, the expression of adhesion molecules, the modulation of inflammatory cytokines/chemokines levels, recruitment, adhesion and activation of inflammatory cells, and interrelated oxidative/nitrosative stress [11 , 12 ]. CB2 receptor activation also attenuates TNF-α-induced NF-κB and RhoA activation, up-regulation of adhesion molecules ICAM-1 and VCAM-1, increased expression of monocyte chemoattractant protein in human coronary endothelial cells, enhanced transendothelial migration of monocytes, and augmented monocyte-endothelial adhesion [13 ]. These findings, coupled with recent results from myocardial [14 ] and cerebral I/R models [15 ], and the known anti-fibrotic effects of CB2 receptor in the liver [16 ], suggest that selective CB2 receptor agonists may represent a novel protective strategy against hepatic and other forms of I/R injury via several interdependent mechanisms.
In your manuscript, you describe HU-308 as a highly selective CB2 agonist. Can you tell the readers of JLB a bit more about this agonist? How was HU-308 generated? Was it discovered accidentally or created via rational synthesis? What is the evidence indicating that it is highly specific?
PP: The bicyclic compound, HU-308, was created via rational synthesis by Dr. Raphael Mechoulam’s group at Hebrew University. They were looking for a specific CB2 receptor agonist and tested a whole variety of cannabinoid analogs. When they recognized that the phenolic side groups were not required for the activity they synthesized instead a series of ether compounds, among which the best turned out to be HU-308. The evidence for the high specificity, including the remarkable differential binding affinity, is described in their 1999 PNAS paper [17 ].
In this work, you clearly demonstrate diminished responses to ischemia/reperfusion (I/R) when HU-308 is administered either prior to injury or immediately thereafter. However, even in response to prior administration, the diminished responses are still substantially above baseline levels (such as in Fig. 1 ). Is this a limitation of HU-308 or of the experimental system? Can you envision agents that might function in an additive or even synergistic fashion with HU-308 to reduce the injury even further?
PP: I would not say that this is the limitation of HU-308, since we don’t see complete reversal of I/R injury with other CB2 agonists, such as JWH-133 [12 ]. The partial reversal of I/R injury by CB2 agonists most likely means that CB2 receptor activation can protect only against the second wave of injury, that mediated by infiltrating inflammatory cells during reperfusion, and is somewhat less effective against the early ischemic damage. There is also the possibility that CB2 agonists cannot reach sufficient concentrations that would permit complete elimination of tissue damage. In theory, any pharmacological intervention aimed at reducing oxidative/nitrosative stress, which has the crucial role of limiting reperfusion-related injury [18 ], may be combined with CB2 agonists to limit the I/R-induced damage. Some examples of these agents might be broad spectrum antioxidants, peroxynitrite scavengers, cannabinoids with antioxidant properties such as cannabidiol, and the like.
Figure 1. Dr. PÃ¡l Pacher received his M.D. degree in 1993 from Semmelweis University of Medicine and the Hungarian Academy of Sciences and was Assistant Professor of Pharmacology at Semmelweis University from then until 1999, while he was also working on his Ph.D. degree in Cardiovascular Physiology (received in 2000). After a post-doctoral fellowship in the laboratory of GyÃ¶rgy Hajnoczky at the Department of Pathology, Anatomy and Cell Biology of Thomas Jefferson University, he joined the group of Csaba SzabÃ³ at Inotek Pharmaceuticals, where he directed numerous in-house projects aimed at novel pathways for therapeutic strategies for diabetes, shock, and myocardial infarction. In 2003, he joined the NIAAA/National Institutes of Health, and was promoted to position of Chief of the Section on Oxidative Stress and Tissue Injury in 2005. He is pictured here with his wife, Sylvia Bercsenyi.
On a more personal note—what made you decide on a career in science? Can you identify a primary mentor or an event that sent you in this direction?
PP: During my school years, my father, who is a physicist, worked at the Joint Institute for Nuclear Research in Dubna, Russia, where numerous major breakthrough discoveries have been made in physics and chemistry, including the identification of several new elements in the periodic table, for example. This was a very special town and environment to grow up in, which most certainly had a major impact on my personal development and career choice. Almost all of my schoolmates’ parents were physicists, chemists, or mathematicians from various countries, and science appeared to be a very natural life for everybody. During my early childhood, my friend Oleg and I fell in love with chemistry and by the age of 14 we had set up a very sophisticated chemistry lab in our home, even better than the one we had at school! Since we lived in a forest-swamp area, it was not really too much of a problem when some of our experiments ended up in big explosions!
The memory of my grandfather, who was a prominent physician, and my strong desire to help people were probably the most important factors in my decision to attend medical school. Unfortunately, my grandfather died very early, but I heard lots of great stories about his character and personal integrity from my grandmother, with whom I lived during my university years. During my medical school years, I was fortunate to be surrounded with many great teachers and mentors. Among these I would have to identify my pharmacology teacher, Dr. TamÃ¡s Friedmann, as the person who provided me with such an attraction to pharmacology, and also my first mentor, Dr. Valeria Kecskemeti with whom I worked during my years at Semmelweis University until 1999, as the most important influences in my early career. Also, Dr. Csaba Szabo and Dr. George Kunos have both had considerable impact on my scientific thinking and on the progress of my career in biomedical sciences, and both have become recent collaborators and friends.
Dr. Pacher, you are the first NIH intramural scientist that we’ve had the pleasure of interviewing for the Pivotal Advance series. What do you see as the advantages and disadvantages of the intramural program? Can you comment on some changes you’d like to see in the coming years?
PP: Well, this is a bit of a provocative question given the number of recent critical remarks against NIH from the extramural scientific community. Clearly, these are very difficult times because of the decreasing amounts of research funding experienced by both the extramural and intramural research community. Despite these difficulties, NIH is a great place to learn and to develop as a researcher, because of its resources and network of excellent scientists. There are also countless opportunities for in-house collaborations so long as one can identify the right person with whom to interact. However, in my personal opinion, NIH is becoming more and more bureaucratic which slows down the research,. The intramural budgets for young investigators are often very low and there are virtually no other real opportunities to get money from outside sources, as most private foundations do not consider applications from intramural NIH investigators. In the coming years I would like to see an increase in the intramural research budget of the NIH and I would also like the NIH to explore additional granting mechanisms that allow talented young intramural investigators to compete for funding. I would also like to see more performance-based distribution of the research budgets and promotions.
Is there anything further that you’d like to add to this interview that has not been touched upon?
PP: I would like to thank NIAAA and our directors Dr. Ting-Kai Li and Dr. George Kunos for the support of our research studies and all the collaborators and authors of this paper for their dedicated hard work. I am personally grateful to Professor Raphael Mechoulam for assisting our studies with valuable tools and suggestions. I would also like to thank my wife Sylvia Bercsenyi for her love, support, and understanding over the course of my research career, and the Editorial Board of the Journal of Leukocyte Biology and the reviewers of our paper for selecting it as a Pivotal Advance.
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