Chapters Transcript Adenosine Metabolism & Receptors in Aging Course: NYU Langone Seminar in Advanced Rheumatology So I, I think, uh, you know, for many of us, uh, investigators who have been at this for a long time, we suddenly wake up one day and realize that we're old and I'm not sure how that happened, but, uh, it happened to me too so. Um, so I, you know, it gives you an opportunity to think back on what you've been doing and whether that has anything to do with the fact that, uh, uh, that people age, and I thought, uh, what I do is, uh, um, is review some of the work that we've done and many others in the field, uh, have done on the role of, uh, purine metabolism, purine receptors in aging. And if you believe this, I've got a bridge to sell you, so. Um, that is the Brooklyn Bridge. Oh, these are my disclosures. Uh, the only relevant one is regenousing. Um, so, The aging population is growing in the United States and worldwide. Uh, uh, in the US in 2014, the, uh, population, um, uh, about 14.5% of the population was, uh, aged 65 or older. And uh by 2060, that number is projected to be 23.5% so I guess we've been uh reasonably successful as uh as physicians. Uh, by 2030, the aging population is going to comprise 77 million people and um this is the first time in history, at least in the United States, that the older population is projected to outnumber children. Uh, this is, this is a problem for those of us who are, uh, facing, uh, Social Security for the country as a whole, and the same is true for, uh, many other countries. So so far there uh there have been a number of mechanisms uh elucidated that uh contribute to aging. uh, first the genomic damage uh with uh telomere shortening, um, this is easily measurable in blood epigenetic alterations of uh uh that stick around and, uh, uh, ultimately disrupt function. This regulation of uh proteostasis, protein uh uh synthesis and degradation and nutrient sensing mitochondrial dysfunction has been described as uh as part of the molecular basis for aging, uh, the collapse of the stem cells pool, the the the longer we live, the fewer stem cells we have, uh, impaired cell cell communication, cellular senescence, and low grade inflammation. So this is uh where purines come from and uh it all starts with uh the mitochondria and ATP. ATP is uh generated at high concentrations in the cell. It's probably the, the molecule that's most abundant in the cell after potassium, and um it's some of it is transported out of the cell into the extracellular space. And in the extracellular space on most cell types, um, it's converted ultimately to adenosine by these cell surface enzymes, CD39 and CD 73. And adenosine um can bind to its receptors on its own cell or on a on that cell or uh to adenosine receptors on adjacent cells. Adenosine itself has a very short half-life, uh, so it's measured in seconds so that uh the adenosine that's generated doesn't last very long. It's converted ultimately to uric acid, um. But most of it is actually taken, taken up and recycled by uh uh by the cell and reconverted to ATP. Uh, I've spent most of my career looking at uh both uh the metabolism and the receptors for adenosine. Uh, there are 44 receptors. They're all members of the G protein coupled receptor family of, uh, of, uh, receptors, and there are two high affinity receptors. These are all names historically, A1 and A2A. And then um low affinity receptors A2B and A3 uh uh are also uh functional at and and more functional in some cells than others. Uh, we first described the anti-inflammatory effects of uh of adenosine. Uh, um, over 40 years ago, and, um, it's most of that is mediated by the adenosine H2A receptor, although some effects are mediated by A2B and A3 receptors. Uh, these receptors are highly conserved, uh, through evolution so that, uh, there's anywhere from 85 to 98% uh homology between human and uh uh and other uh species. Um, adenosine receptors. So let me tell you a, a couple of uh stories about uh the role of adenosine metabolism and adenosine receptors in uh uh in aging and tissues that we commonly deal with the musculoskeletal system. Um, and, um, CD 73 has been shown to be, uh, diminished in a, in, uh, its expression whether that's because it's, uh, more rapidly degraded or, uh, because it less is made as we age and um. In some tissues, uh, this is, uh, uh, this is particularly relevant, and this is work, uh, from Shay Vargasse's lab, uh, at, uh, Duke in which she showed that, um, if you, uh, overectomize animals you find that there's a reduction in both CD 73 and CD 39 expression, uh, as compared to uh to the sham of ectroized animals. And um this uh this is important. It turns out that estrogen controls the uh or regulates the expression of CD 73 and CD39 and so there's a reduction in adenosine that that's associated with ovarectomy in mice and presumably uh in uh uh in women as they age uh with lower estrogen levels uh at the time of menopause. And she, she went on to show that, uh, estrogen is important if you knock down the, uh, estrogen receptors in uh um in uh cells uh whether it's well both brands of estrogen receptor, uh, you find that, uh, there's uh less CD 39 and CD 73 expression. And the single knockdowns are uh as good as the dual knockdowns in terms of uh diminishing the expression and diminishing the amount of adenosine that's present in the uh extracellular space. And so, um, she asked whether that this was, uh, important, uh, we'd previously shown that H2A receptors were important in bone metabolism uh, others had shown that H2B receptors were important in promoting bone growth and so, uh, what, what she looked at was, uh, whether or not you could treat these animals with an A2B receptor agonist and reverse the, um. Uh, reverse the, uh, osteopenia and this is a control mouse, uh, bone. Uh, there's less bone in the, uh, overectomized animal, but if you treat the overectomized animal with, um, uh, with, uh, the compound, it's a bear compound, it's an A2B receptor agonist, you see that there's a partial um uh restoration of the normal bone. And that's shown here, uh, that there are fewer trap cells uh in after treatment with A2B receptors and down here, um, we were discussing uh tetracyclines before, uh, the way you measure bone growth is by administering a dose of tetracycline or or one of its analogs a week later and measuring the difference um between. The two and this is the amount of osteoid that's made in a mouse. Um, this is what happens if you overectomize them they're making far less bone and if you treat with this compound, uh, with the A2B receptor agonist, you find that, uh, you can restore bone, bone formation. And we, um, we've been working on this, uh, trying to, uh, uh, reverse some aging, uh, alendronate is a very, uh, useful carrier for, uh, for, for compounds that you want to get into bone and in collaboration with, um, with Ken Jacobson at the NIH, uh, we tested, uh, an alendronate A2A receptor agonist, uh, conjugate. Uh, on, um, on, uh, bone metabolism and, uh, we, we've looked at, uh, uh, overectomized mice as well, as well as, uh, inflammatory bone destruction, and we see that, uh, uh, if we give the, uh, uh, the alendronate uh A2A agonist that there's more bone than in, uh, just the alendronate treated or the saline treated. Um, um, mice and, um. Here you can see it, uh, this is the number of osteoclasts uh is uh reduced by landrenate we knew that and it's reduced by the, uh, uh, by this compound, but the number of osteoblasts is also increased by, uh by this compound as well and um. The uh uh bone deposition is, uh, is restored to uh to control levels and overectomized mice uh by uh this H2A receptor agonist alendronate conjudant. So we think that uh Alendronate or that uh uh adenosine production which is diminished with age uh contributes to postmenopausal bone loss so there is, you know, because of uh mitochondrial damage, uh, uh, because of, uh, there's less internal ATP less of it is externalized or transported out and. Uh, there's obviously less, uh, less available to become, uh, converted to adenosine and less uh activation of the adenosine receptor. So let, let me, uh, so another, uh, burden that we have of, uh, aging, um, is osteoarthritis. Uh, this occurs in 7% of the US population and as you can see it's enriched in the, uh, population over 65, and I think you all know this from your, from your practice. There are a variety of risk factors that have been. Um, that have been described, including genetic factors, bowel alignment of the joints, prior injury, and obesity, and the care of patients with OA is, uh, is obviously expensive. We do about 3 million knee and hip replacements in the United States in 2022. It's expected to increase to 5 million. By 2030. As a result of the aging of the population, and, and so, um. This, uh, uh, this is, uh, a, a real problem for all of us. So we looked at uh chondrocytes and uh in in vitro model if you will of osteoarthritis uh we treated them with interleukin one and um you'll notice that uh there's ATP and uh chondrocytes as expected, it's reduced by about 40%, uh, um, in the cells by IL1 treatment. And uh this leads to a uh coordinate uh reduction in extracellular ATP uh by about 40%. This also this clearly leads to a reduction in in adenosine uh you can see this is about a 40% reduction in adenosine uh by these cells, uh, so, um. So treatment with a uh osteoarthritis uh uh initiator leads to a leads to a reduction in adenine release. And so, uh, we, we asked whether mice, uh, who lacked, uh, CD 73 and were unable to convert, uh, um, extracellular ATP to adenosine, uh, developed osteoarthritis, uh, prematurely and in fact they do and here you can see the, uh, cartilage which is kind of torn up looking here you can see an osteophyte and a CD 73 knockout mouse. But more importantly this happens to CD 73 knock out humans. Um, these are, there are about 15 patients who have been described who lack CD73 activity. Uh, they develop, uh, uh, vascular calcification which is, uh, quite remarkable leads to, uh, vascular insufficiency and leads to amputation, so at least you don't get in the way of the uh joints that have been amputated. But it also leads to premature, um osteoarthritis and uh osteoarthritis um in the hands and feet and, um, in, uh, uh, people under the age of 20. Which is quite striking. And we were struck a number of years ago after Hurricane Sandy drowned all of our mice, um, we, uh, we found, uh, we had a couple of cages of very old A2A knockout mice, uh, uh, who remained on the very top shelf and survived the deluge and, uh, so we had a chance to look at their joints and this is a normal knee and a mouse, um, and this is a mouse, uh. Uh, an 82A receptor knockout mouse, it's about a year old, which is kind of middle aged for for a mouse, and what you can see is that there's subchondral sclerosis, there's loss of uh uh joint space or joint space narrowing, uh, and there's even an osteophyte present uh in this mouse. So they develop spontaneous osteoarthritis. Uh, it occurs increasingly over time, starts at about the age of 3 months. And um Was responsible, uh, you know, most of us, uh, many of us who work at at wet benches, uh, find that we're essentially mouse farmers, and one of the problems that we had was that the, uh, male mice would stop breeding when they were 3 months old of the Bay to a knockouts and, um, I didn't really care why frankly, but, uh, uh, but it turns out, uh. That when their knees hurt they they uh are no longer I guess in the mood um and so they stopped breeding. This was a real problem for us because we obviously wanted more mice to uh uh for work. But you can see here again this is the loss of proteoglycan uh in the A2 receptor knockout mouse, um, as compared to a wild type mouse. And here uh we've we've, uh, subsequently had the opportunity uh to treat these mice with, uh, um, liposomal adenosine, um, and we find that we were able to, uh, almost completely reverse the osteoarthritis in mice and rats, and here we see that uh uh that uh the um. Liposomal adenosine also seems to, uh, also seems to help uh uh dogs as well with uh with traumatic, uh, post traumatic OA. So, uh, when we look at the function, um, it improves over time, um, in the, uh, dogs that have been given the, uh, uh, treatment, these are just two different formulations, the, uh, range of motion, um. This actually does show an improvement in the range of motion, um, in these dogs. Uh, there's a marked reduction in pain. It was this was really surprising and then that's actually maintained, uh, for actually, uh, I think we only gave them two injections, so, uh, for, uh, about 6 months, but that's when we ended the experiments and these were done all by blinded observers, uh, actually at a, at another institution. Um, so, uh, so this seems to work in, in larger animals as well, where there's a biomechanical, uh, uh, effect. And we see slowing of uh radiographic changes. These are the radiographic changes in the placebo treated um mice, and you can see that uh that they're less. I mean there there's still some degeneration, but there's less, um, OA change in the uh in the uh dogs, uh, who received uh treatment with liposomal adenosine. And the same is true for MRI scoring as well. We, we've, uh, investigated, uh, these chondrocytes fairly extensively from 8 to a knockouts and, uh, uh, and wild type animals, and you can see when we do a heat map there are quite a few changes in uh in the chondrocytes that we collect from neonatal mice and these are mice that never actually walks so, um, so there's no biomechanical contribution here, um. And you can see that there are a number of uh uh of responses uh or pathways that are involved, um, if you are uh lacking an A2A receptor or if your mouse is um they uh have a marked pro pro-inflammatory response nitric oxide biosynthesis, which is again part of the inflammation that we see in osteoarthritis, aging. Metalloproteases, uh, reactive oxygen, uh, uh, metabolites, etc. And we, we've looked at, uh, most of the other factors that have been associated with aging. We haven't looked at genomic damage, um, and the effect of uh adenosine H2A receptors on genomic damage, but, uh, we clearly are able to reverse epigenetic alterations, this regulation of uh protein uh homeostasis and nutrient sensing, uh, mitochondrial function, we find that, uh, there. Uh, mitochondrial, uh, mitochondria are lost in OAchondrocytes, and, um, there's evidence that they are, are regenerated, um, and we, we have evidence that, uh, they're regenerated in, uh, uh, in the chondrocytes from, um. Uh, they've been treated with, uh, adenosinate receptor agonists. Uh, adenosinate2A receptors are important for promoting stem cell, uh, stem cell, uh, uh, proliferation, and in fact, uh, one of the markers for, uh, stem cells is CD 73. Uh, we've seen effects, uh, on, uh, TGF beta signaling in cells. Uh, we also see, uh, uh, loss of, uh, cellular senescence and, um, in inhibition of low grade inflammation as well. So I, and I won't show you the data. This is all published and I'm happy to send you the reprints if, uh, if you're interested. I guess, uh, a couple of years ago, um, Bill Robinson was here and, and some of the people from his group, uh, did an observational study in which they looked at, uh, uh, they looked at whether or not this might be relevant and what they did was very clever. They looked at patients who were starting on tiagolor versus patients who were starting on clopidogrel. These uh agents both uh target uh uh platelet ADP receptors and um inhibit uh platelet aggregation, but uh icagalor has the additional property of, uh, blocking adenosine uptake and um, thereby increasing uh extracellular adenosine levels. And what they showed in their observational study was that patients started on icagular had significantly less uh or were significantly less likely to develop uh osteoarthritis than those started on uh clopidogrel and um I think this uh provides some supporting evidence for this, uh, for this claim. So what about the the other part of the musculoskeletal system we've uh described skeletal and uh joint uh issues but adenosine uh acting and A2B receptors also, uh, reverses the effect of aging on skeletal muscle and uh this is work from a a group in uh Bonn and Germany and um. What they showed was that uh A2B receptor signaling ameliorates the effects of aging and counteracts obesity by it counteracts obesity by increasing uh uh brown fat uh accumulation or browning of fat. uh, we showed this with H2A receptors as well and um. They also show that uh uh they prevent sarcopenia by treating with uh adenosine A2B receptor agonists. And the highlights of their, they've had a couple of papers on this, the highlights include the um the fact that A2B receptors regulate two major energy dissipating tissues, muscle and brown fat. And um activation of A2B receptors counteracts sarcopenia as well as obesity in mice and A2B forms uh receptors from uh heterodimers which are cru crucial for physiologic uh adenosineyline. And the um the expression of these uh receptors correlates with uh uh energy expenditure in human muscle and brown fat. And um Rancho Medro who was in my lab, uh, is now back in Madrid, uh, showed reported recently and then a couple of uh papers that uh dipyriamol or proin which also blocks adenosine uptake, um, uh, increases uh extracellular adenosine and diminishes, uh, inflammation-related sarcopenia. And uh she looked at models of, of arthritis and looked at the sarcopenia that results in um in mice that uh that are induced to uh undergo uh arthritis and they um. They uh. Uh, seemed to be stronger, they had, uh, uh, more stamina, and they were able to, uh, they lost, uh, less uh muscle mass. So To summarize to this point in the musculosketal system, extracellular adenosine is, uh, uh, is a homogeneous uh or homeostatic factor, and it's reduction during aging, uh, contributes to the effects of aging on the musculoskeletal system. And so, uh, the metabolism which starts with the mitochondrial uh synthesis of ATP, um, and, uh, it's, uh, uh, it's transport to the extracellular space, uh, with subsequent metabolism to adenosine, um, is homeostatic and with aging, um, what we see in many tissues is there's reduction of mitochondria. Some of which is reversible by uh adenosine uh adenosine receptor stimulation that leads to uh less ATPs less ATP in the cell, uh less ATP transported out of the cell, um, and therefore less, uh, uh, AMP to be converted to adenosine by CD 73 and so all of this, uh, we think, uh, contributes to uh agent. So if you think I'm the only one who's thought of this, I'm not, uh, uh, Adenosine has been looked at in skin. This is, it's kind of peculiar looking at these websites. uh, this is, uh, uh, Keh's product, but there are a number of other brands, um. And I, I've never received any money from L'Oreal who owns, uh, Kiel, uh, although, uh, I have been to their, uh, headquarters here in New York, uh, for the Arthritis Foundation board, uh, meetings, so, so I have, uh, I guess I've taken a. You know, I, uh, some pate from them or something, uh, but there are two blinded and controlled studies that demonstrate the topical application of adenosine produces wrinkles. This actually all came from work that we did on wound healing and adenosine receptors. Adenosine promotes, uh, matrix production and wounds. It also, uh, stimulates angiogenesis and, um. So we think it's the uh stimulation of fibroblast matrix production that uh uh that leads to reduced wrinkles. And I love the stuff, you know, the names that they have for men's cosmetics are really wonderful. The age defender power serum is, uh, just, just what we need. There, there are obviously some exceptions to this, um, in the immune system adenosine is an immunosuppressive, um, and, um, it right now actually, uh, there are a number of companies that are developing adenosine A2A receptor antagonists to, uh, as, uh, checkpoint inhibitors, uh, to treat, uh, cancer, um. But, uh, recent studies from, uh, I think, uh, Connie Wyan and Yoni, uh, suggest that adenosine release, uh, accounts for the diminished vaccine responses, uh, associated with aging. In the liver adenosine is actually probably not so good for you. Uh, we've shown that, uh, both A1 and A2B receptor stimulation promotes lipid accumulation by very different mechanisms and A2A receptors, uh, uh, stimulate uh hepatic fibrosis, um, and, uh, this is probably. You know, there's, uh, there have been a number of studies over the years uh on caffeine intake. Caffeine is an adenosine receptor antagonist, uh, and, um, the development of liver disease, and they all seem to uniformly show that the more caffeine you take, uh, the less likely you are to develop liver disease, whether it's uh fibrosis or uh fatty liver, um, and, uh, this may account for. Uh, the effects of caffeine on, uh, on liver metabolism. And in the CNS, uh, uh, adenosine appears to mediate, uh, inflammatory damage in the CNS, um, and also, uh, because adenosine receptors are sort of, uh, linked at the hip to dopamine receptors, uh, and they the adenosine H2A receptors turn them off, uh, turn off those receptors for dopamine. Uh, treatments for Parkinson's, uh, uh, have included, uh, adenosine H2 receptor antagonists that are available in China and Japan. I think became available in the US recently. And whether they promote the aging, uh, it's hard to tell, you know, most people with Parkinson's tend to be older, so. So what's in the fountain of youth and uh this is my take on it, which is adenosine. Um And uh these are the people who did the work Carmen Corsi did most of the work on uh uh on osteoarthritis. Christina did work on uh on mitochondria and Ben Friedman, uh, uh, did most of the work on, um, uh, senescence of, uh, of, uh, cells and dennosine receptors and a Ron I mentioned her work earlier. And thank you for your attention. Published March 14, 2024 Created by Related Presenters Bruce Cronstein, MD View full profile
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