Cross-Links Stiffen the Extracellular Matrix With Age
One of the root causes of degenerative aging is the accumulation of sugary metabolic wastes known as advanced glycation end-products that are in some cases very hard to for our evolved biochemistry to break down. Some types can form cross-links, gluing together important proteins such as those making up the supporting extracellular matrix scaffold. The properties of elastic tissues such as skin and blood vessel walls derive from the particular structure of the extracellular matrix, and cross-links degrade that structure, preventing it from functioning correctly. Their presence contributes to blood vessel stiffening with age and all the problems that result from that, for example, but there are plenty of other affected tissues.
The SENS approach to this contributing cause of aging is to build the necessary tools to work with the most common cross-link compound in human tissues, glucosepane. It is hoped that other research groups will pick up the work once they no longer have to start by building the very fundamental tools for the job. As things stand few research institutions are willing to start from scratch when there are so many other lines of research presently available that do not need a complete tool infrastructure built before anything can be accomplished.
Advanced age is associated with increases in muscle passive stiffness, but the contributors to the changes remain unclear. Our purpose was to determine the relative contributions of muscle fibers and extracellular matrix (ECM) to muscle passive stiffness in both adult and old animals. Passive mechanical properties were determined for isolated individual muscle fibers and bundles of muscle fibers that included their associated ECM, obtained from tibialis anterior muscles of adult (8-12 mo old) and old (28-30 mo old) mice. Maximum tangent moduli of individual muscle fibers from adult and old muscles were not different at any sarcomere length tested. In contrast, the moduli of bundles of fibers from old mice was more than twofold greater than that of fiber bundles from adult muscles at sarcomere lengths of more than 2.5 μm.
Because ECM mechanical behavior is determined by the composition and arrangement of its molecular constituents, we also examined the effect of aging on ECM collagen characteristics. With aging, muscle ECM hydroxyproline content increased twofold and advanced glycation end-product protein adducts increased threefold, whereas collagen fibril orientation and total ECM area were not different between muscles from adult and old mice. Taken together, these findings indicate that the ECM of tibialis anterior muscles from old mice has a higher modulus than the ECM of adult muscles, likely driven by an accumulation of densely packed extensively crosslinked collagen.
While looking at this research it is worth bearing in mind that short lived rodents have a different cross-link biochemistry in comparison to we long-lived humans. Early attempts to develop cross-link-breaking drugs floundered on this issue: promising results in rats didn't translate to human medicine at all. The overall picture of how this degeneration proceeds and why it happens is very similar, so there is much that can be learned, but the types of cross-link are different in ways that matter greatly for the development of treatments.
If there isn't yet a decent mouse model of collagen crosslinking, then I wonder how the FDA will ever approve a potential treatment to begin human trials? Do monkeys exhibit similar crosslinks to humans? Or would scientists have to created a Xenograft model with human skin, blood vessels, and muscle fibres being grafted into a mouse and then having their human collagen crosslinks removed?
Also it is rather curious that elderly mice suffer as badly as elderly humans do from collagen crosslinks, given that this is a purely chemical process, and as far as I know, mice do not have higher levels of glucose in their bodies than humans. You'd think that the rate of crosslinking per cubic millimeter of tissue would be the same in both species, and that mice would never live long enough to suffer the negative effects of ECM crosslinking, given that it takes so long to have negative effects in humans?
It's clear that rodents develop far more oxidatively-driven AGE crossinks than humans, and while many people think that the reason alagebrium worked in mice but not in humans was because they have significant levels of the α-diketone crosslinks that it targets, while we do not. But the fact that lab rodents have a different mixture of AGE crosslink species than humans doesn't mean they'll be useless for testing crosslink-breaking drugs: it's not a matter of what crosslinks they have in superabundance, but of whether they have enough of a crosslink targeted by the drug that is relevant to humans for that crosslink to impair their arterial (or other tissue) elasticity sufficiently to lead to pathology.
That said, we don't yet know that this will be the case for glucosepane in mice. Glucosepane is a very difficult structure to isolate from tissues; because of this, very little work has been done in quantitating it even in humans, and none in mice. So this should be determined before wasting resources in a study in mice that gives a misleading false negative.
Fortunately, we are now approaching the point where we can answer this key question. As you know, SENS Research Foundation has been funding research in David Spiegel's lab at Yale for several years now, developing enabling technologies for the development of glucosepane crosslink breakers. As I mentioned in reply to one of your previous questions, I can now report that they have recently successfully completed the first-ever synthesis of glucosepane, and are going on to work on its biologically-significant isomers (as well as pentosinane, another AGE crosslink that may have been mistakenly neglected).
The ability to synthesize glucosepane will in turn enable the development of synthetic glucosepane-crosslinked peptides that can be used to develop antibodies against glucosepane, which will make it orders of magnitude faster and cheaper to look for it in biological tissues, as well as to do high-throughput screening of potential crosslink-breakers and to test potential glucosepane-cleaving agents in vivo by administering the compounds to animals and evaluating the effects on glucosepane burden in the animals' tissues (something that was not ever possible for the α-diketone crosslinks that were thought to be the targets of alagebrium).
You can read a little bit more about this in the 2014 SENS Research Foundation Annual Report; Dr. Spiegel and his team are working on a formal scientific publication of their breakthrough as we "speak," and there will be extensive details then.
If it turns out that aging mice accumulate too little glucosepane in target tissues to have a meaningful effect on function, that isn't really a show-stopper: we'll just use our new antibodies to look for glucosepane in the tissues of a range of other model organisms (which researchers will want to do anyway for more classical, curiosity-driven correlative aging research), and pick one that accumulates a lot of it. Mice are convenient and widely-used, but there's no law that says you have to use them, specifically, for preclinical development.
To answer a couple of your incidental questions:
-A xenograft model wouldn't really be helpful: the grafted arterial and other tissues' proteins might more accurately reflect the intrinsic vulnerability to glucosepane crosslinking to the donor species, but remember that they'd be exposed to the glucose levels and other systemic factors of the recipient species, and also would gradually be remodeled by the recipient animal's enzymes, slowly converting them to a more host-like structure.
Jim: Also it is rather curious that elderly mice suffer as badly as elderly humans do from collagen crosslinks, given that this is a purely chemical process, and as far as I know, mice do not have higher levels of glucose in their bodies than humans. You'd think that the rate of crosslinking per cubic millimeter of tissue would be the same in both species, and that mice would never live long enough to suffer the negative effects of ECM crosslinking, given that it takes so long to have negative effects in humans?
Well, of course you could say the same thing about any (or very nearly any) of the various cellular and molecular lesions that accumulate in aging mice. Species vary in their rates of aging because of a mixture of factors, which include body temperature (higher temperatures speed up the rate of chemical reaction), intrinsic structural resistance (for example, longer-lived species' mitochondrial membranes contain lower proportions of highly-unsaturated fatty acids, notably DHA, which makes them less vulnerable to oxidative damage by mitochondrial free radicals), maintenance and repair machinery (DNA repair mechanisms, protein repair enzymes like methionine sulfoxide reductase, proteasomal and lysosomal activity and enzymatic complement, etc) — and circulating reactive intermediates, such as the blood sugar about which you specifically ask. Humans have longer lifespans than mice precisely because selective pressure has driven us to alter these metabolic and structural determinants of the rate of accumulation of aging damage in a way that slows the rate of accumulation of the various cellular and molecular damage of aging with age.
On glucose specifically: in fact, normal blood glucose levels in laboratory mice are significantly higher than humans', although it varies by strain and diet: among 72 strains of 8-week-old mice, fasting glucose averaged ≈165 mg/dL (normoglycemic humans Ending Aging (p. 175), this can have quite surprising effects on the particular crosslink species that develop in tissues.
Where are we now in 2017 with glucosepane crosslink breaking? I wasn't aware that it had been synthesized finally. Is anyone working on it now? Is it a funding issue? What would it cost annually to fund a concerted effort?
It was synthesized in 2015 and the procedure simplified and made cheaper in 2016. And this year the Spiegel lab received a 5-year grant of ~$350,000 per year from the American Diabetes Association for developing glucosepane breakers, so I think they don't need more funding for some time now.
I get old and get young again, I don't know exactly what you are doing on animals experimentation. You can experiment in yourself and you'll find out that not all things were done scientifically, sometimes a simple thing missed out in science or overcomplicated things that can be done in an ordinary way.
Aging is caused by the accumulation of waste inside the body and it can flush out in many ways without even costing you a cents.
Goodluck! Do the experiment and you will stay young and healthy.
Do we know for sure that alagebrium (Alteon-711) failed or was it simply a matter of resources and patenting? It became an orphan drug. This is a highly important thread. It is the basis of aging in diabetics with high glycation levels and activity. HgA1c levels is a very simple biomarker. And pulse wave or reflected wave blood pressure monitoring is another simple biomarker.
I eat a diet low in AGEs, I fast 5 days a month, and I take supplements to prevent and/or protect against the damage caused by AGEs. Benfotiamine, R Lipoic Acid, pyridoxal-5-phosphate, and. Carnosine.