Assessing the Prevalence of Sarcopenia

Sarcopenia is the name given to the characteristic loss of muscle mass and strength that accompanies aging, though formal definitions under development tend towards including only those with the greatest degree of loss. This is something of a political problem in the research and medical community; the tendency to describe some level of aging as normal and therefore not treatable, while classifying greater degrees of exactly the same process and symptoms as a disease. Along with the failure of the immune system and loss of bone strength, sarcopenia is one of the most evident forms of age-related frailty. A good many research groups are involved in the attempt to find ways to slow or reverse this decline, most of which are focused on mechanisms of stem cell activity and tissue regeneration rather than fundamental damage after the SENS model of aging. Of the present options outside the SENS portfolio, gene therapies or antibody therapies that target the muscle growth regulators of myostatin and follistatin appear most promising in the short term, given the rapid progress taking place in the broader field of genetic editing.

Sarcopenia, an age-related decline in muscle mass and function, is one of the most important health problems in elderly with a high rate of adverse outcomes. However, several studies have investigated the prevalence of sarcopenia in the world, the results have been inconsistent. The current systematic review and meta- analysis study was conducted to estimate the overall prevalence of sarcopenia in both genders in different regions of the world.

Electronic databases were searched between January 2009 and December 2016. The population- based studies that reported the prevalence of sarcopenia in healthy adults aged ≥ 60 years using the European Working Group on Sarcopenia in Older People (EWGSOP), the International Working Group on Sarcopenia (IWGS) and Asian Working Group for Sarcopenia (AWGS) definitions, were selected. According to these consensual definitions, sarcopenia was defined by presence of low muscle mass (adjusted appendicular muscle mass for height) and muscle strength (handgrip strength) or physical performance (the usual gait speed). The random effect model was used for estimation the prevalence of sarcopenia.

Thirty-five articles met our inclusion criteria, with a total of 58,404 individuals. The overall estimates of prevalence was 10% in men and 10% in women, respectively. The prevalence was higher among non-Asian than Asian individuals in both genders especially, when the Bio-electrical Impedance Analysis (BIA) was used to measure muscle mass (19% vs 10% in men; 20% vs 11% in women). Despite the differences encountered between the studies, regarding diagnostic tools used to measure of muscle mass and different regions of the world for estimating parameters of sarcopenia, present systematic review revealed that a substantial proportion of the old people has sarcopenia, even in healthy populations. However, despite sarcopenia being a consequence of the aging progress, early diagnosis can prevent some adverse outcomes.

Link: https://dx.doi.org/10.1186/s40200-017-0302-x

A View of How Senescent Cells Disrupt Tissue Regeneration

Normal tissue regeneration is disrupted in various ways in later life, such as the tendency for increased fibrosis, scar tissue formation rather than normal regrowth. Researchers here theorize on the role of growing numbers of lingering senescent cells in this age-related loss of function, a complex situation because the transient creation of senescent cells, soon destroyed, is an important part of the normal wound healing process. Despite their positive function in that scenario, the accumulation of long-lasting senescent cells is nonetheless one of the root causes of aging. These cells produce a harmful effect on surrounding tissue through the potent mix of signals they generate, known as the senescence-associated secretory phenotype (SASP), which drives chronic inflammation, among other items.

The inability of adult tissues to transitorily generate cells with functional stem cell-like properties is a major obstacle to tissue self-repair. Nuclear reprogramming-like phenomena that induce a transient acquisition of epigenetic plasticity and phenotype malleability may constitute a reparative route through which human tissues respond to injury, stress, and disease. However, tissue rejuvenation should involve not only the transient epigenetic reprogramming of differentiated cells, but also the committed re-acquisition of the original or alternative committed cell fate. Chronic or unrestrained epigenetic plasticity would drive aging phenotypes by impairing the repair or the replacement of damaged cells; such uncontrolled phenomena of in vivo reprogramming might also generate cancer-like cellular states. We herein propose that the ability of senescence-associated inflammatory signaling to regulate in vivo reprogramming cycles of tissue repair outlines a threshold model of aging and cancer.

The degree of senescence/inflammation-associated deviation from the homeostatic state may delineate a type of thresholding algorithm distinguishing beneficial from deleterious effects of in vivo reprogramming. First, transient activation of NF-κB-related innate immunity and senescence-associated inflammatory components (e.g., IL-6) might facilitate reparative cellular reprogramming in response to acute inflammatory events. Second, para-inflammation switches might promote long-lasting but reversible refractoriness to reparative cellular reprogramming. Third, chronic senescence-associated inflammatory signaling might lock cells in highly plastic epigenetic states disabled for reparative differentiation. The consideration of a cellular reprogramming-centered view of epigenetic plasticity as a fundamental element of a tissue's capacity to undergo successful repair, aging degeneration or malignant transformation should provide challenging stochastic insights into the current deterministic genetic paradigm for most chronic diseases, thereby increasing the spectrum of therapeutic approaches for physiological aging and cancer.

If the loss of differentiation features following reprogramming is not accompanied by re-acquisition of the original or alternative differentiated cell fate, the resulting tissue plasticity might impair the repair or replacement of damaged cells. The ability of SASP-associated pro-inflammatory cytokines to regulate stemness and nuclear reprogramming raises the notion that a SASP-impaired local environment could interfere with tissue rejuvenation by imposing the so-called "stem-lock" state. Chronic inflammatory conditions via exposure to IL-1, which normally functions as a key "emergency" signal and a master regulator of SASP by inducing downstream effectors such as IL-6, has been shown to impair tissue homeostasis and to induce an aged appearance of the hematopoietic system by restricting stem cell differentiation.

While counterintuitive, given the ability of SASP factors including IL-6 to transiently create a permissive environment for in vivo reprogramming capable of inducing cellular plasticity and tissue regeneration, a prolonged promotion of such progenerative response might reduce tissue rejuvenation and promote aging by self-enhancing futile cycles of SASP/IL-6-driven reparative cellular reprogramming. Compared with young tissues containing few senescent cells where transient creation of senescent cells might cause temporary reprogramming and differentiation/proliferation to replenish cells, the prolonged accumulation of senescent cells in tissues that are old or under high levels of stress (e.g., following medical procedures such as chemotherapy) might be accompanied by a defective clearance of damaged, senescent cells, which can promote further SASP accumulation. A situation of chronic SASP secretion might not only counter the continued regenerative stimuli by promoting cell-intrinsic senescence arrest in single damaged cells but also paradoxically impose a permanent, locked gain of stem cell-like cellular states with blocked differentiation capabilities in surrounding cells.

Link: https://doi.org/10.3389/fcell.2017.00049

Are there Commonalities Between Neurodegenerative Conditions that can be Targeted to Produce General Therapies?

Cancer research will only progress meaningfully towards control of all cancer when the research community puts significant time and effort into finding common mechanisms shared by many or all cancers - or better still, attacking the one known mechanism shared by all cancers, which is abuse of telomere lengthening. The reason that the cancer community struggles with progress is that there are hundreds of forms of cancer, and researchers largely continue to try to address them one by one. There is a lot of cancer, but only so much funding and only so many scientists. A better way forward is needed. The question for today, however, is whether or not this principle of action extends to another broad class of widely varied conditions, the neurodegenerative diseases that corrode the aging brain. Are there faster paths forward here as well, built on common mechanisms? I'm on the fence on this topic. I think it easy to argue that any two different forms of neurodegeneration are far more distinct from one another than any two types of cancer; they involve completely different ways to disrupt cellular activity in the brain or kill brain cells. There is no one mechanism with a clear analogy to the central role of abuse of telomere lengthening in cancer when it comes to neurodegenerative disease.

Still, it is tempting to speculate on mechanisms that might be shared between many different types of neurodegenerative disease, because if they do exist, that offers the same prospect of faster progress, if only the research community better directed its efforts. Obviously, at root, many layers of cause and consequence removed from the disease state, we can look to the forms of tissue and cell damage outlined in the SENS rejuvenation research proposals - the root causes of aging. Most neurodegeration is age-related because it is caused by aging, and thus the first resort should probably be attempts at first principles rejuvenation, therapies based on repair of root cause damage. Sadly, few in the research community agree with that statement; persuading them to see the light is an ongoing project. Further along the chain of damage and dysfunction can be found other examples. We might, for example, consider the failure of cerebral spinal fluid drainage channels as a possible common factor in all conditions involving the build-up of aggregates and other unwanted molecular waste in the brain. Equally, there may be other, more esoteric points at which intervention is possible, though in general the later in the disease process the intervention occurs, the less likely it is to produce more than marginal benefits, if the past century of medicine is any guide to what the future holds. You might look at this work as an example of the type:

Alzheimer's, Parkinson's, and Huntington's diseases share common crucial feature

Abnormal proteins found in Alzheimer's disease, Parkinson's disease, and Huntington's disease all share a similar ability to cause damage when they invade brain cells. The finding potentially could explain the mechanism by which Alzheimer's, Parkinson's, Huntington's, and other neurodegenerative diseases spread within the brain and disrupt normal brain functions. The finding also suggests that an effective treatment for one neurodegenerative disease might work for other neurodegenerative diseases as well. "A possible therapy would involve boosting a brain cell's ability to degrade a clump of proteins and damaged vesicles. If we could do this in one disease, it's a good bet the therapy would be effective in the other two diseases."

Previous research has suggested that in all three diseases, proteins that are folded abnormally form clumps inside brain cells. These clumps spread from cell to cell, eventually leading to cell deaths. Different proteins are implicated in each disease: tau in Alzheimer's, alpha-synuclein in Parkinson's and huntingtin in Huntington's disease. The new study focused on how these misfolded protein clumps invade a healthy brain cell. The authors observed that once proteins get inside the cell, they enter vesicles (small compartments that are encased in membranes). The proteins damage or rupture the vesicle membranes, allowing the proteins to then invade the cytoplasm and cause additional dysfunction. When protein clumps invade vesicles the cell gathers the ruptured vesicles and protein clumps together so the vesicles and proteins can be destroyed. However, the proteins are resistant to degradation. "The cell's attempt to degrade the proteins is somewhat like a stomach trying to digest a clump of nails."

Endocytic vesicle rupture is a conserved mechanism of cellular invasion by amyloid proteins

Numerous pathological amyloid proteins spread from cell to cell during neurodegenerative disease, facilitating the propagation of cellular pathology and disease progression. Understanding the mechanism by which disease-associated amyloid protein assemblies enter target cells and induce cellular dysfunction is, therefore, key to understanding the progressive nature of such neurodegenerative diseases. In this study, we utilized an imaging-based assay to monitor the ability of disease-associated amyloid assemblies to rupture intracellular vesicles following endocytosis. We observe that the ability to induce vesicle rupture is a common feature of α-synuclein (α-syn) assemblies, as assemblies derived from wild type (WT) or familial disease-associated mutant α-syn all exhibited the ability to induce vesicle rupture. Similarly, different conformational strains of WT α-syn assemblies, but not monomeric or oligomeric forms, efficiently induced vesicle rupture following endocytosis.

The ability to induce vesicle rupture was not specific to α-syn, as amyloid assemblies of tau and huntingtin Exon1 with pathologic polyglutamine repeats also exhibited the ability to induce vesicle rupture. We also observe that vesicles ruptured by α-syn are positive for the autophagic marker LC3 and can accumulate and fuse into large, intracellular structures resembling Lewy bodies in vitro. Finally, we show that the same markers of vesicle rupture surround Lewy bodies in brain sections from PD patients. These data underscore the importance of this conserved endocytic vesicle rupture event as a damaging mechanism of cellular invasion by amyloid assemblies of multiple neurodegenerative disease-associated proteins, and suggest that proteinaceous inclusions such as Lewy bodies form as a consequence of continued fusion of autophagic vesicles in cells unable to degrade ruptured vesicles and their amyloid contents.

Complicating the Bigger Picture of Protein Aggregation in Aging

A number of different proteins can misfold or otherwise be altered in ways that cause them to precipitate into solid deposits. The best known of these are best known because they are are a contributing cause of age-related disease, through their disruption of normal tissue function or via a surrounding halo of biochemistry that is in some way toxic to cells. There are twenty or so forms of amyloid deposits, for example, and of these the most attention is given to the amyloid-β involved in Alzheimer's disease - though transthyretin amyloid is catching up, given the growing evidence for its role in heart failure. In the paper here, the authors suggest that the way in which amyloids and other similar deposits get started involves interactions between the aggregation of potentially many other proteins: in other words that proteins A and B might aggregate without any great evidence for a link to resulting harm, but their aggregation acts to seed the aggregation of protein C that is very definitely harmful to health over the years.

A variety of neurodegenerative diseases are associated with the misfolding and aggregation of specific proteins. In Alzheimer's disease (AD), amyloid-β (Aβ) peptides and tau proteins aggregate and ultimately form the characteristic pathological hallmarks: amyloid plaques and neurofibrillary tangles (NTFs) respectively. In recent years, understanding the initiation and spread of these hallmark protein aggregates has become a central area of investigation. The current model stipulates that aggregation in disease is initiated by a protein seed that forms a template for further protein aggregation. Support for this model comes from research showing that the exogenous addition of minute amounts of Aβ or tau seeds greatly accelerates the onset of aggregation both in vitro and in vivo. An important and currently understudied question is how aging influences protein aggregation in neurodegeneration. Recently, physiological protein insolubility in the context of aging has become a hot topic of research. Indeed, numerous publications demonstrate that protein aggregation is not restricted to disease but a normal consequence and possibly cause of aging.

Until now, it remains unclear whether and how age-dependent protein aggregation and disease-associated protein aggregation influence each other. One possibility is that age-dependent aggregates indirectly accelerate disease-associated protein aggregation by stressing the cell and/or titrating away anti-aggregation factors. Another possibility is a direct interaction whereby disease-associated proteins and age-dependent aggregation-prone proteins co-aggregate. In support of this latter hypothesis, proteins prone to aggregate during normal aging are significantly overrepresented as minor protein components in amyloid plaques and NFTs. Recent research reveals that the sequestration of these age-dependent aggregation-prone proteins in the disease aggregates is a source of toxicity. However, whether misfolded proteins aggregating with age can form heterologous seeds that initiate Aβ aggregation has not been investigated.

Although current research focuses on homologous seeding, there are a few examples of cross-seeding (or heterologous seeding) mostly between different disease-aggregating proteins. For instance, Aβ is a potent seed for the aggregation of human islet amyloid polypeptide (hIAPP) involved in type II diabetes; Aβ and prion protein PrPSc cross-seed each other and accelerate neuropathology; and both α-synuclein and Aβ co-aggregate with tau and enhance tau pathology in vivo. Finally, we recently showed that cross-seeding between different age-dependent aggregating proteins is possible in the absence of disease. Here, we demonstrate that cross-seeding during aging is likely to be an important mechanism underlying protein aggregation in AD.

We show for the first time that highly insoluble proteins from aged Caenorhabditis elegans or aged mouse brains, but not from young individuals, can initiate amyloid-β aggregation in vitro. We tested the seeding potential at four different ages across the adult lifespan of C. elegans. Significantly, protein aggregates formed during the early stages of aging did not act as seeds for amyloid-β aggregation. Instead, we found that changes in protein aggregation occurring during middle-age initiated amyloid-β aggregation. Mass spectrometry analysis revealed several late-aggregating proteins that were previously identified as minor components of amyloid-β plaques and neurofibrillary tangles such as 14-3-3, Ubiquitin-like modifier-activating enzyme 1 and Lamin A/C, highlighting these as strong candidates for cross-seeding. Overall, we demonstrate that widespread protein misfolding and aggregation with age could be critical for the initiation of pathogenesis, and thus should be targeted by therapeutic strategies to alleviate neurodegenerative diseases.

Link: https://doi.org/10.3389/fnagi.2017.00138

Why is the Postfertile Longevity Exhibited by Humans so Unusual?

Humans are an unusually long-lived species when compared to other mammals of a similar size, and even in comparison to our near relative primates. Further, we exhibit an extended period of life following loss of fertility, a rare form of life history that is only observed in a few other species. The grandmother hypothesis is one of the possible explanations for the evolution of extended longevity without fertility; it is a selection effect based on the ability of older individuals to assist in the survival of their descendants. Given the existence of such a mechanism, however, why is it not more widespread?

Data on historical agricultural populations and modern hunter-gatherers show that these groups enjoy significant postfertile periods. Taking an evolutionary approach, the Grandmother Hypothesis proposes that this reproductive inactivity is in fact adaptive. With the sacrifice of continued reproduction, an individual may increase their inclusive fitness by decreasing the interbirth intervals of their offspring. The care that would otherwise be put into one's own children can now be put into weaned (and increasingly independent) grandchildren, allowing their own offspring to reproduce again sooner. Otherwise put, the cost of a reduced relatedness coefficient may be outweighed by an increase in total number of grandchildren resulting from the diverted care.

A valid objection to the Grandmother Hypothesis, however, is if grandmothering can result in a higher fitness, why are significant postfertile life stages so rare? Among vertebrates in the wild, only humans, Globicephala macrorhynchus (pilot whales) and Orcinus orca (resident killer whales), have a significant proportion of individuals with such a life history. In this study, we present a model to investigate this objection. Our model assumes only that individuals transition through various life stages and that there is an average time to conception and gestation. In one of those stages, individuals have the option to provide care for a certain number of their grandchildren thereby allowing their own offspring to reproduce again sooner.

By comparing inclusive fitnesses of individuals that provide intergenerational care with those that instead continue to reproduce into old age, we arrive at a necessary condition for grandmothering to be an evolutionarily stable strategy (ESS). This condition, or stability threshold, relates the number of grandchildren that care must be given to with the ratio of the length of the first two life stages. It tells us nothing about when or how grandmothering may arise initially in a population, but places restrictions on when it will persist. We then make the observation that if a grandmother is to provide care for even one set of grandchildren, their expected postfertile stage must be sufficiently long. More precisely, for grandmothering to be adaptive, it must be the case that postfertile life exceeds the time taken to raise a weaned child to independence. If this were not the case, grandmothers would not be able to shorten their offspring's time between births by caring for some infants themselves. In this way, we derive an eligibility threshold that tells us when grandmothering is a strategy with any possible evolutionary advantage. These eligibility and stability criteria must both be satisfied for grandmothering to evolve and then, most importantly for our purposes, to persist.

Our analyses show that there is conflict between the stability and eligibility thresholds. As it becomes increasingly easier to meet one of them, it becomes increasingly harder to fulfill the other and vice versa. This conflict is, at its core, a grandparent-grandoffspring conflict analogous to parent-offspring conflicts. The result of this is that there is a narrow range over which we should expect grandmothering to evolve and then to persist. In other words, we should in fact expect grandmothering to be rare.

Link: https://dx.doi.org/10.1002/ece3.2958

Planning a Single Person Trial of Senolytic Drug Candidates

This post should be considered as part of an ongoing and yet to be concluded process of thinking out loud on the topic of self-experimentation with senolytic drug candidates. These are compounds that to some degree selectively destroy senescent cells in animal studies. Some have been shown to have positive effects in animal studies of various sorts in the years prior to the present wave of interest in senescent cell clearance, and some of those effects might be plausibly linked to removal of senescent cells. Some were tested as cancer therapeutics, or analgesics, or for other uses. Some have serious and harmful side effects, as is the case for most prospective chemotherapeutics. They are intended to destroy cells, and they are nowhere near as discriminating as we'd all like them to be. Nonetheless, all of these drug candidates are to varying degrees available for purchase, and thus available for self-experimentation.

Now, self-experimentation has a long and storied history in the scientific community. Many noted researchers at some point obtained the first human data from their own bodies, and that seems to me the most ethical of approaches: the researcher assumes the risks. Setting aside for a moment the question of risk, the point to take away from this history is that there is absolutely no point in doing this unless you measure and publish what you did and what happened. Guessing at outcomes or using drug candidates merely in the hope that effects will carry over from studies in mice helps no-one. The same goes for picking easily measured outcomes just because they are easy to measure. The objective here is to learn something and transmit that learning, which is possible even in an environment of single person tests without controls, provided we are seeking effects that are both large and reliable, and provided we go about this is a sensible manner. In this context, self-experimentation can help to point the way for those with the resources to run more rigorous experiments capable of better quantifying effect size, optimal dosage, and the like.

Obtain a Cooperative Physician

The first step is to ensure that you have a physician who understands what you are intending to do and achieve, and is willing to order up the required tests. You will need an interface and guide to the local medical establishment, especially for the more expensive scanning and testing. This usually isn't all that hard to obtain, since you'll be paying.

Obtain a Cooperative Laboratory Company

You will need a company to act as an interface with suppliers, as many of them will not accept orders for senolytics from individuals. In this age of drug prohibition, it also smooths the way for biochemical deliveries across national borders for them to be between laboratory companies. You will also need a company with laboratory resources, or that can act as an interface to laboratory services for some of the work you might want carried out. The ideal situation here is to work with someone within the community, via your connections, as it would otherwise require some legwork to find a company willing to work with you.

Determine the Health Metrics to be Assessed

The ideal set of data desired at the end of a short test of senolytics includes (a) the degree to which senescent cells were removed, and (b) the degree to which relevant measures of aging were reversed. The reality is that both require some compromises given the current state of medical testing. After some reading around and thinking on what would likely be affected by cellular senescence, given what is presently known, I settled on the following tests for consideration. One important item is that the normal values obtained from healthy individuals for a given test must vary to a large enough degree across the age range of 30 to 60 to make it useful to run the test if you are something other than very old. This is definitely not true for as many of the available tests as you might think would be the case.

Firstly, there is standard bloodwork and urinalysis. This is actually not all that likely show anything interesting if comparing before and after measures, especially in people who are not in their 60s or later, but it is cheap and a useful demonstration to show that nothing terrible took place. Further, some of the measures in bloodwork are needed for other parts of the testing. In particular, it is possible to see indications of tumor lysis syndrome resulting from senescent cell destruction. If there is a characteristic change in such measures immediately following use of a senolytic drug, it is an indication that something is happening, which is useful evidence.

When looking at liver function, none of the values obtained from normal bloodwork are particularly helpful. The numbers for normal function don't vary enough with age, and do vary a fair amount with circumstances and lifestyle choices. However, hepatobiliary scintigraphy results do change characteristically with age. This is a nuclear medicine procedure involving use of a radioactive tracer, so expect to pay accordingly.

For kidney function, the desired measure is glomular filtration rate. Now there are numerous ways of obtaining this result. There is the direct and expensive nuclear medicine approach with tracers, but also estimated approaches using data obtained from standard bloodwork. There are a number of resources that explain the differences in some detail, such as a PDF from the National Kidney Foundation. The estimated approaches suffer from various degrees of inaccuracy for the levels one would expect to find in a healthy individual, sad to say. The MDRD Study equation method should not be used, but the alternative CKD-EPI equation seems worth trying.

Given the evidence for a relationship between cellular senescence and calcification of blood vessels, calcium scans and scoring at first seem interesting. This is especially the case since it is apparently very hard to reduce a calcium score; it is something achieved only gradually over years, and with great attention to lifestyle changes. Calcium scans are just a standard CT scan followed by semi-automated analysis, producing an Agatston score or lesion-specific calcium score. Unfortunately, even later in life a large percentage of people score zero - as many as half or more in the late 40s and early 50s, for example. There is an online calculator from one of the research groups involved in this work if you are interested in exploring the numbers. All of this makes calcium scoring nowhere near as helpful as it might be, given the cost of a CT scan. It is probably only worth trying for people in their 70s and later, or who already have a score to hand and know it is non-zero.

Tests in lung tissue suggest that removal of senescent cells can somewhat reverse loss of tissue elasticity. So it seems worth looking at measures of skin elasticity. These can be obtained using cutometer or ballistometer commercial devices, with a number of papers commenting on reliability of the results. You might have to find a plastic surgeon or one of those dubious anti-aging clinics, however, rather than a standard dermatology practice. Possibly more useful is the indirect measure of blood vessel elasticity via pulse wave velocity, which is an easy test to arrange, and which does have a significant degree of change over the middle years of life. The question there, as with all matters cardiovascular, is the degree to which normal readings change because of primary (including the effects of senescent cells) versus secondary (weight gain and lack of exercise) causes of aging. The testing that is being accomplished here is as much of the relevance of the tests as it is of the effects of senolytic therapies. For that and other reasons, you can't just pick one test.

Another cardiovascular measure with a useful profile of changes over time is heart rate variabilility. Measurement here is also easily arranged. Of note, the Palo Alto Prize founders chose heart rate variability as their measure of aging for the interventions produced by competing teams.

Biomarkers of aging based on DNA methylation are well on the way towards becoming a practical possibility these days, though there is as yet no one consensus approach that everyone agrees upon. Nonetheless, Osiris Green is offering a DNA methylation biomarker of aging implementation at an affordable price. This is cheap enough to put into contention, even though there is as much validation of the test needed as validation of senolytics.

If you can stretch to custom lab work, then it is worth looking into the existing cellular senescence tests, or the skin sample test noted this morning, both of which require a biopsy. In the former case there are kits and the tests are well established, at least in the research community, with a question mark on how the biopsy process will interact with the role of cellular senescence in wound healing to make the results unhelpful. In the latter case, the paper provides enough details for someone to repeat the protocol, but it is anyone's guess as to how useful it will be in practice. This is another case where calibrating the test is as much the goal as calibrating the effects of a senolytic.

Pick the Senolytic Drug Candidates

Right at the start, let us throw out dasanitib, navitoclax, and similar items targeting the Bcl-2 family. They are comparatively indiscriminate chemotherapeutics, and almost everything else that the research community has identified as a potential senolytic drug is better, judging from the animal data: either more discriminating, less harmful, or both. Of the remaining compounds, it makes sense to try a combination, as some studies have suggested synergies exist between drug candidates, or that different senolytics work on different types of senescent cell. Also, the academic and corporate studies will not at the outset tend to run trials for drug combinations. It is better to raise the odds of finding interesting new data.

The compounds that seem worth looking into fall into two categories. The first are easily obtained supplement-like items that are comparatively cheap, taken orally, and well characterized for safety. In this category are fisetin and quercetin, though there is some debate over whether or not the latter is in fact senolytic. The second are more recently identified senolytics that are less easily obtained and used, in some cases with little to no human data on safety and usage, but that seem promising given recent research. Here, I'd include piperlongumine and FOXO4-DRI. In each case, you would want to read around on what is known of the pharmacology, the studies that used the compound, current thinking on how it works, and make a call on whether or not you are willing to take the risk of trying it. This will certainly involve digging through research papers, and will certainly be an individual choice. Don't blindly follow anyone's recommendations: choose for yourself.

Establish Dosage and Schedule

Figuring the likely dose for a human study involves reading the existing literature on animal studies to find the most relevant dose used there, usually expressed in mg/kg, and then multiply accordingly. You will quickly find that for most senolytics there is no easy way to come to a recommended dose, and you'll be forced to use your best judgement. For example, piperlongumine has so far only been studied in cell cultures for its effects on senescent cells. Looking at the literature, it was tested as an analgesic at levels of 1-250 mg/kg, for cancer suppression at 2.5-5 mg/kg, for sensitizing cancer to other treatments at 1 mg/kg, and for more direct cancer ablation at 2.5 mg/kg. In some cases these were single doses followed by an assessment, in others they continued for weeks.

Similarly for fisetin, there are no published animal studies for effects on senescent cells. For other purposes in past years, however, you'll find data on the pharmacokinetics for doses of 10-250 mg/kg, another study providing 10-45 mg/kg, twice a day for weeks, and yet another for cancer suppression at 5 mg/kg twice over a period of a few weeks.

For quercetin, one can look at the original study identifying it as senolytic to see that the researchers used a single dose of 50 mg/kg. For FOXO4-DRI, there is a very little data beyond the one recent study announcing its effects and another equally recent focused on cancer. Both are paywalled and unfortunately the details of the dosage are not in the main body of the original paper, but rather in the supplemental materials that I've yet to obtain. Still, it is there for consideration when I get to it.

Bear in mind that you are certainly going to want to try a very tiny dose at the outset, and then work your way up to the final dose. This precaution is only sensible and is done for a variety of reasons. In some cases these senolytic compounds are poorly or not at all tested in humans. Secondly, how certain are you that the suppliers did everything absolutely correctly, and that the testing of their compounds worked as desired? Further, if trying combinations yet to be tested in any published paper, there is always the possibility of unforeseen interactions. Lastly, if things actually work well and you started out with a high load of senescent cells, you do have to worry about the possibility of tumor lysis syndrome due to too many cells dying at once. All of these are very good reasons to ease into the desired dosage over time.

There is very definitely a spectrum of safety in the compounds I've mentioned here, from quercetin (sold in stores, manufactured by many supplement companies, in existence for years) through to FOXO4-DRI (comparatively new, barely manufactured at all, must be custom ordered, with no published human data, and only a couple of papers for animal studies). When you pick your poison, do so in full knowledge of the level of risk you undertake.

Figure out the Logistics

Quercetin and fisetin are things you put in bottles on a shelf and can leave there for months. You take the pills orally. That is all pretty easy. Piperlongumine requires freezer storage, and possibly powering or compounding to be taken orally. FOXO4-DRI is a short lifespan protein, must be keep in freezer storage, then reconstituted and given via injection: intraperitoneal injection in mice, but most likely intravenous injection would be the most desirable option in humans. If you are familiar at all with how diabetics manage their insulin supplies, the situation is very similar.

Management of injection logistics is something that you want a lab company and probably a physician to help with, rather than embark upon it alone. In this context it is very much worth noting that, given the drug war nonsense that has gripped the world these past few decades, you want to be careful as to how you go about obtaining needles for any compounds that must be injected. This is another good reason to arrange everything in conjunction with a friendly lab company and physician.

Determine Suppliers and Order Products

Finding suppliers for the chosen senolytics varies considerably in difficulty. For quercetin, you walk across the street to pick up a few bottles from the nearest supplement store, and by going with a trusted brand can probably feel good about skipping the step of validating that the contents are what they say they are. Or you may be able to find an existing review of the supplier's products online. Fisetin can still be ordered in bottles, but here the number and quality of suppliers is more of an unknown, so the need to test the product comes into play.

For piperlongumine, you will be ordering from a chemical supplier and paying a considerable amount - hundreds of dollars for a single dose, going by the levels used in animal studies. For FOXO4-DRI, it is likely that the best course, given the very small number of suppliers, is to have it synthesized as a custom batch by a company that specializes in protein synthesis. This is expensive, and is where you will need the lab company. In both cases, suppliers will be reluctant to supply anyone they think is going to use it for human testing outside the formal trial system or a research institution.

Test the Products

You will also need the friendly lab company for the task of determining how to validate the quality of products when they arrive from the suppliers. Validation of quality is not a completely straightforward process, and may require digging up specialist services, which is better done through a company already in that ecosystem than to try it yourself. It is a matter of great importance to establish that you are getting what you pay for, both to avoid wasting the time and resources spent on the exercise of self-experimentation, as well as for reasons of personal safety. Even with the best of intentions, compounds that are not mass manufactured can have bad batches.

Run the Experiment

The first step is to run all the desired tests to obtain a set of initial baseline values. For many of these, such as standard bloodwork, it makes sense to run them twice, perhaps a few weeks apart, since numbers tend to vary with circumstances. Then follow the dosage schedule. Then run two more sets of tests, one a few days after the end of dosage, and one a month later. Precisely because many of the measures can vary with lifestyle, it is important to be consistent in your diet, exercise, and so forth across this period of time.

Then, once done, wrap it all up by publishing the data online for the community to look over.

Considering the Easy versus the Not So Easy Options

It should be apparent from reading the above notes and the linked materials that the choice of candidate senolytics and assays makes a big difference to the amount of work required to run a useful exercise in self-experimentation. It also makes a big difference to the level of personal risk undertaken. I picked the senolytics discussed in this post in part to make this point. To cut down to the easiest and safest level of self-experimentation, it would be possible to try only fisetin and quercetin and largely avoid the need for laboratory services, just relying upon a friendly physician to order bloodwork, cardiovascular, and other established tests. One could also be fairly confident that the risk of adverse effects in that scenario is lower than it is in the others. Sadly these are also the more dubious senolytic candidates; there is no such thing as a free lunch, it seems.

SIWA Therapeutics Obtains Funding to Continue with an Immunotherapy Approach to Clearance of Senescent Cells

SIWA Therapeutics is one of the older companies in the field of cellular senescence, among the small number of ventures that made an attempt to target senescent cells for destruction a decade ago and didn't really get all that far before funding ran out. Times have changed, however, and these groups have now been invigorated by progress in the science of cellular senescence and demonstrations of turning back aging and age-related disease in animal studies. One of these older ventures transformed into Unity Biotechnology, and Unity's success in raising a very large amount of venture funding has made it that much easier for everyone else with a credible approach to find backers. Between the established groups and newer ventures like Oisin Biotechnologies a wide range of potential approaches to senescent cell destruction are covered. It remains to be seen how well they all do on the later stages of the path to the clinic.

SIWA Therapeutics announced that it has successfully humanized its SIWA 318 monoclonal antibody, a significant milestone in the race to treat cancer and numerous other diseases by removing senescent cells, which become increasingly problematic as humans age. Senescent cells lose their ability to divide or replicate for a variety of reasons and also secrete chemicals which interfere with the normal functions of other cells as well as contribute to inflammation. When too many senescent cells accumulate, they can cause or exacerbate a variety of age-related and degenerative diseases.

In previous research in mice, SIWA 318 has targeted and successfully removed senescent cells, and it also increased muscle mass. Other testing showed that mice treated with SIWA 318 had fewer metastatic lung cancer occurrences as well as possible suppression of tumor growth. No adverse effects were observed from the antibody treatment in either study. The humanized form of SIWA 318 has demonstrated strong and significant binding to senescent cells in preclinical studies, critical to accurately targeting and removing them. SIWA Therapeutics just completed a new round of funding and is planning to submit an IND to the FDA, with the ultimate goal of conducting the first human clinical trials for senescent cell removal. Based on initial results, the primary focus likely will be pancreatic cancer metastasis.

"With SIWA 318 now available in humanized form, we have moved closer to determining if removing senescent cells could become a common therapeutic approach in the fight against metastatic cancers. Based on data that we and others in the scientific community have generated over the last few years, evidence is clearly mounting that many diseases, including cancer metastasis, will be treatable through senescent cell removal."

Link: http://www.businesswire.com/news/home/20170518005384/en/SIWA-Therapeutics-Takes-Key-Step-Efforts-Treat

The Basis for a Skin Sample Test of Level of Cellular Senescence

Researchers here set forth the basis for a novel approach to assessing the level of cellular senescence present in a patient, using a skin sample as a starting point. The current situation for assays of cellular senescence is very biased towards laboratory research needs, with little innovation over the past twenty years. The present standard assays are unfortunately not a suitable basis for the efficient, discriminating, and above all easy and low-cost clinical tests that will be needed in the years ahread. Senolytic therapies capable of clearing senescent cells as a form of rejuvenation treatment will become available in the next few years, and adventurous souls can already self-experiment with some of the drug candidates. Tests capable of clearly establishing the results of such experimentation are much needed.

Fibroblasts form one the most important cellular components of the skin derma. During aging, skin fibroblasts undergo substantial changes in their functional activity, morphology and proliferative potential. The number of dermal fibroblasts decreases with aging, along with their ability to synthesize active soluble factors and to maintain proteostasis of components of the intercellular dermal matrix. The skin thinning, the loss of skin flexibility and elasticity, and wrinkle formation are natural consequences of such a decline. Therefore, we suggested that evaluating the proliferative potential of dermal fibroblasts is of great significance.

Measuring the ability to form colonies in vitro represents one of the "gold standard" methods for the assessment of the clonogenic survival of cells. The method was initially developed to evaluate the loss of reproductive capacity (reproductive death) of cells after exposure to damaging agents, particularly ionizing radiation. Later it was shown that cells isolated from biopsy material from different patients had varying ability for colony formation. This allows for comparative assessment of different patient's cell capacity to proliferate and may represent a promising avenue for personalized medicine.

Beside a colony-forming efficiency of fibroblasts, defined as percentage of plated cells that are able to form colonies, the evaluation of colony size/type distribution provides additional important information especially for heterogenic cell populations such as primary fibroblasts. In this case, the size of the colony depends directly on the proliferative capacity of cell-precursors: cells can form morphologically distinct colonies that can be broken down into the following three types: dense (or compact), diffuse and mixed colonies. If the fractions of each of these colony phenotypes are known, one can evaluate the proliferative potential of the entire fibroblasts culture. Cellular aging, traditionally assessed by the fraction of senescence associated β-galactosidase (SA-βgal) positive cells, along with the degree of differentiation are closely associated with the proliferative capacity of cells. With aging, intracellular β-galactosidase accumulates in lysosomes and a sharp increase in the β-galactosidase activity in older cells is traditionally considered to be a classic marker of cellular aging. Therefore, it could be anticipated that the fraction of aging cells in colonies of the diffuse phenotype would be larger than that in the colonies of the dense phenotype.

The aim of this work was to verify the assumptions regarding the relationship of cellular aging with the formation of fibroblast colonies of different phenotypes, and to examine whether such enriched analysis of colony formation may be used for evaluating the degree of cellular senescence. To this end, we measured the fraction of SA-βgal positive cells (SA-βgal+) in the three types of colonies (dense, mixed and diffuse) of human skin fibroblasts from donors of various ages. Although the donors were chosen to be within the same age group (33-54 years), the colony forming efficiency of their fibroblasts (ECO-f) and the percentage of dense, mixed and diffuse colonies varied greatly among the donors. We showed, for the first time, that the SA-βgal positive fraction was the largest in diffuse colonies, confirming that they originated from cells with the least proliferative capacity. The percentage of diffuse colonies was also found to correlate with the SA-βgal positive cells in mass culture. Moreover, a significant inverse correlation between the percentage of diffuse colonies and ECO-f was found. Our data indicate that quantification of a fraction of diffuse colonies may provide a simple and useful method to evaluate the extent of cellular senescence in human skin fibroblasts.

Link: http://dx.doi.org/10.18632/aging.101240

A Broadening of Efforts to Clear Senescent Cells

The accumulation of senescent cells over time is one of the causes of aging. It is one of the limited number of root cause mechanisms that collectively distinguish old tissue from young tissue. Cells become senescent constantly, most because they have reached the Hayflick limit on replication, but senescence also occurs in response to cell damage, tissue injury, or a harmful tissue environment. Near all of these cells are destroyed shortly after becoming senescent, either through the programmed cell death process of apoptosis, or by the immune system. A tiny fraction linger, however. These cells generate a mix of signals and other proteins that promote inflammation, destructively remodel the nearby extracellular matrix, and change the behavior of normal cells for the worse, among other things. This all makes sense in the context of their presence in embryonic development, wound healing, and cancer suppression - and when there are comparatively few such senescent cells. When there are many senescent cells, however, and when they are not destroyed as they should be, this behavior adds up to cause significant harm. Destructive processes such as fibrosis, arterial calcification, development of atherosclerotic plaques in blood vessels, loss of tissue elasticity, chronic inflammation in joints, and many more can all be directly tied to the presence of senescent cells, and can be improved by removing those cells.

Targeted removal of senescent cells to at least some degree is in fact now fairly easy to accomplish in a laboratory setting through the methodology of targeting known suppressors of apoptosis. As a consequence a whole range of drug candidates of varying quality are emerging. The senescent cells that linger in old tissue are remain primed for the fate of apoptosis, but are held back by a few mechanisms that are increasingly well characterized. Near any established medical research group with experience in cellular biochemistry can jump in and try their hand. Clearly a growing number of researchers are doing just this, managing to raise funding and join the field. There is plenty of room for them. Clearance of senescent cells - as a rejuvenation therapy capable of turning back some of the consequences of aging - has a target market of every human much over the age of 40, for treatments undertaken once every few years. This is such an enormous potential industry that no one company or methodology will win it all. In the next few years, we'll probably see sizable and successful companies emerge in many different countries, all of which have different regulatory regimes, and thus there will be comparatively little direct competition between these ventures.

The publicity materials below are really just banging the drum for work published last year, in which researchers used ABT-737 to inhibit BCL-W and BCL-XL. These two members of the Bcl-2 family suppress the process of apoptosis. Targeting them thus selectively destroys senescent cells by removing one of the blocks to undergoing apoptosis - a manipulation that should have comparatively little effect in normal cells. Many of the apoptosis inducing drug candidates at this time have significant side-effects, however, and so it is likely that success in the market will only be achieved by those lacking that problem. At this point, the researchers here are somewhere in the early stages of commercializing their approach, and hence the emergence of extra publicity from their supporting institution. There will be a lot more of this sort of thing going on in the next few years.

Understanding why cells refuse to die may lead to treatments for age-related disease

One of the things that happens to our bodies as we age is that certain cells start to accumulate. So-called senescent cells - cells that "retire" and stop dividing but refuse to undergo cellular death - are always present, and they even serve some important functions, in wound repair, for example. But in aging organs, these cells don't get cleared away as they should, and they can clutter up the place. Researchers are revealing just how these cells are tied to disorders of aging and why they refuse to go away. The work is not only opening new windows onto the aging process, but is pointing to new directions in treatments for many of these disorders and diseases.

Research into cellular senescence has taken off in recent years, due to findings that clearing these cells from various parts of the body can reverse certain aspects of aging and disease processes. Pharmaceutical industries have taken note, as well, of research that could lead to the development of drugs that might target senescent cells in specific organs or tissues. In basic research conducted on human cell culture and on mice, researchers have asked exactly what ties senescent cells to aging. Are they, for example, a primary cause of age-related disease, or a side effect? And why don't these cells die, despite being damaged, so that the "clean-up crews" of the immune system have to clear them away?

The researchers hypothesized that the answer to the second question might lie in a family of cellular proteins that regulate a type of cell suicide known as apoptosis. They identified two proteins in this family that prevent apoptosis and which were overproduced in the senescent cells, BCL-W and BCL-XL. When they injected mice that had an extra supply of senescent cells with ABT-737 molecules that inhibit these two proteins, the cells underwent apoptosis and were then eliminated, and there were signs of improvement in the tissue. "In small amounts, these cells can prevent tumors from growing, help wounds clot and start the healing process. But as they amass, they trigger inflammation and even cancer."

Certain common age-related diseases have been shown to be associated with this build-up of senescent cells, for example, chronic obstructive pulmonary disease (COPD), and researchers hope to apply these findings to research into treatments for such diseases. The trick will be to target the offensive cells without causing undue side effects. Researchers have been developing mouse models of COPD and asking whether clearing senescent cells just from the lungs can prevent or ease the disease. They are now working to patent and license these discoveries.

Alzheimer's Disease as Laminopathy

The lack of tangible progress over the last fifteen years towards working therapies for Alzheimer's disease that are based on clearing amyloid has led to a great diversity of alternative thinking on the causes and pathology of the condition, as well as on other approaches to treatment. It is easier to theorize than it is to push therapies through trials, so this sort of thing is to be expected whenever the road ahead turns out to be much harder than expected. Some of the recent theorizing on Alzheimer's disease is quite promising, and some of it is quite dubious. From a first reading, this one falls somewhere in the middle. It should probably be read in the context of what has been discovered of the role of lamins in progeria versus in normal aging, the latter a work of investigation still very much in progress.

The cell nucleus is typically depicted as a sphere encircled by a smooth surface of nuclear envelope. For most cell types, this depiction is accurate. In other cell types and in some pathological conditions, however, the smooth nuclear exterior is interrupted by tubular invaginations of the nuclear envelope, often referred to as a "nucleoplasmic reticulum," into the deep nuclear interior. We have recently reported a significant expansion of the nucleoplasmic reticulum in postmortem human Alzheimer's disease brain tissue. We found that dysfunction of the nucleoskeleton, a lamin-rich meshwork that coats the inner nuclear membrane and associated invaginations, is causal for Alzheimer's disease-related neurodegeneration in vivo.

Neurons of tau transgenic Drosophila and of postmortem human Alzheimer's disease brains harbor significant invaginations of the nuclear envelope and have reduced levels of B-type lamin protein compared to controls. Dysfunction of B-type lamins has functional consequences in adult neurons in regard to heterochromatin formation, cell cycle activation, and neuronal survival. Taken together, our results suggest that pathological tau-induced stabilization of filamentous actin disrupts the LINC complex, which reduces lamin protein levels and causes the nuclear envelope to invaginate. Lamin reduction or dysfunction, in turn, causes constitutive heterochromatin to relax, allowing expression of genes that are normally silenced by heterochromatin and activating the cell cycle in postmitotic neurons, which causes their death.

Our findings suggest that Alzheimer's disease and associated tauopathies are, in fact, acquired neurodegenerative laminopathies. We demonstrate that loss of lamin function can lead directly to age-related neurodegeneration, indicating that basic mechanisms of aging are conserved between neurons and other somatic tissues. The lamin nucleoskeleton is thus a plausible molecular link between aging, the single most important risk factor for developing common neurodegenerative diseases, including Alzheimer's disease, and basic mechanisms of cellular senescence. Functional consequences of nucleoplasmic reticulum expansion in physiological aging and pathological conditions including cancer and Alzheimer's disease remain to be determined, however.

Link: http://dx.doi.org/10.1080/19491034.2016.1183859

Reviewing the Aging of Heart Tissue

This open access paper takes a brief tour of the dominant themes in the aging of heart tissue, viewed structurally and biochemically. These are some of the changes that have yet to be assembled into a coherent and generally agreed upon chain of events, starting with fundamental cellular damage, and proceeding through successive layers of cause and consequence in reaction to that damage. Most of the research community begins a line of inquiry with an investigation of one facet of the aged, diseased state. Researchers then attempt to work backwards to identify and address proximate causes of the observed problems, one by one, producing marginal improvements. The alternative approach of starting with fundamental damage and attempting to fix it in order to observe a resulting sweeping improvement all the way down the chain of consequences has far too little support. Note the links to the list of fundamental damage from the SENS rejuvenation research portfolio in the items below: mitochondrial damage and amyloid are mentioned directly; senescent cells and cross-linking drive harmful extracellular matrix changes; cross-linking also stiffens arteries, which produces hypertension, which in turn drives remodeling of heart structure.

The average lifespan of the human population is increasing worldwide, mostly because of declining fertility and increasing longevity. It has been predicted that, in 2035, nearly one in four individuals will be 65 years or older. With age being the dominant risk factor for the development of cardiovascular diseases, their prevalence increases dramatically with increasing age. At the end of the twentieth century, researchers announced the emergence of two new epidemics of cardiovascular disease: heart failure and atrial fibrillation. The prevalence of heart failure in the adult population in developed countries is 1-2%, which rises to more than 10% among persons 70 years or older. The same trend is seen for atrial fibrillation, with a prevalence rising from 0.12 - 0.16% in persons younger than 49 years, to 3.7-4.2% in persons aged 60-70 years, to 10-17% in persons aged 80 years or older. Since there is a clear association between aging of the population and increasing prevalence of cardiovascular disease, cardiovascular aging most likely affects pathophysiological pathways also implicated in the development of cardiovascular disease. Therefore, a better insight into cardiac aging may unravel factors implicated in cardiac pathophysiology and help towards improved prevention of human cardiovascular disease.

On a structural level, the most striking phenomenon seen with age is an increase in the thickness of the left ventricle (LV) wall as a result of increased cardiomyocyte size. This hypertrophy affects the LV in an asymmetrical way, leading to a redistribution of cardiac muscle. In the elderly, atrial contraction plays a much greater role in LV filling during diastole than in the young population. This change in function is associated with the development of atrial hypertrophy and dilation. Left atrial size has been associated with the presence of atrial fibrillation, indicating that atrial remodeling favors the development of this arrhythmia.

Remodeling at the cellular level includes a loss of cardiomyocytes and sinoatrial node pacemaker cells with age, and may contribute to the compensatory development of hypertrophy. This compensatory remodeling process may also involve changes in the composition of the extracellular matrix. The function of the extracellular matrix is to maintain the myocardial structure throughout the cardiac cycle. Hereby it plays an important role in the elastic and viscous properties of the LV. Changes in both the quantity of fibrosis and in the type of collagen fibers have been associated with old age in human hearts. It is easy to imagine that changes in the elastic properties of the LV caused by fibrosis may eventually lead to diastolic dysfunction. Indeed, in hypertensive heart disease patients, more severe diastolic dysfunction has been associated with a more active fibrotic process.

Another histopathological change found in cardiac tissue of old people is amyloid deposition. An autopsy study on a Finnish population aged 85 or over showed the presence of amyloid deposits in 25%, with a strong correlation between the presence of amyloid and the age at time of death. Amyloid found in heart of the elderly is derived from the transthyretin molecule. With age, this molecule may become structurally unstable and result in the development of misfolded intermediates that aggregate and precipitate as amyloid, mainly in the heart. In some cases, amyloid deposition in the heart occurs at a level that will lead to the progressive development of heart failure. This infiltrative cardiomyopathy is defined as systemic senile amyloidosis (SSA).

Cardiac function requires an enormous amount of energy and mitochondria are critical for the required ATP production in the myocardium. They also play a fundamental role in the survival and function of cardiomyocytes. Cardiac senescence is accompanied by a general decline in mitochondrial function, clonal expansion of dysfunctional mitochondria, increased production of reactive oxygen species (ROS), suppressed mitophagy and dysregulation of mitochondrial quality processes such as fusion and fission. Of these processes, the development of oxidative stress as a consequence of excessive ROS generation is the most frequently described phenomenon. The mitochondrial free radical theory of aging is debated, but in the context of cardiac disease, ample evidence exists for the existence of a pathogenic link between enhanced ROS production, mitochondrial dysfunction and the development of heart failure.

Link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418492/

Replacement Heart Valve Structures that Mimic Natural Extracellular Matrix

Over the past few years, there have been a number of important advances in the infrastructure technologies needed for tissue engineering and related fields such as the construction of scaffolds to support and guide cell growth. Along these lines, researchers have recently demonstrated a rapid jet spinning approach to the construction of scaffold materials that mimic the properties of natural extracellular matrix. This allows for the construction of - to pick one example - heart valve implants, structures that will be populated by cells to form living tissue, capable of regeneration and growth, after implantation in a patient. This has been tested in animal models, and represents an improvement in cost and time over the prior standard approaches to constructing scaffolds.

Implanting scaffolds that carry chemical cues similar to those of the extracellular matrix, but lack any cells, is one of many different approaches to tissue engineering that chiefly differ from one another in where the tissue growth is expected to occur. There is a lot to be said for pushing the tissue growth stage into the body, as this works around many of the challenges inherent in trying to grow tissues outside the body: establishing all of the correct signals and environmental factors; growing blood vessel networks needed to support larger tissue sections; designing and maintaining a suitable custom bioreactor for the time it takes tissue to assemble itself; that intrusive rather than minimal surgery is required to transplant new tissue; and so on. Ultimately, I think it likely that the end goal for the tissue engineering field is to attain sufficient control over cells and cell signaling to direct the desired behavior inside the body without the need for scaffolds, bioreactors, transplantation, and other related technologies. That lies some way in the future, however. At the present time, all viable approaches that enable creation of tissue without the need for donors represent a great leap forward, a dramatic improvement over current limitations.

Engineering heart valves for the many

The human heart beats approximately 35 million times every year, effectively pumping blood into the circulation via four different heart valves. Unfortunately, in over four million people each year, these delicate tissues malfunction due to birth defects, age-related deteriorations, and infections, causing cardiac valve disease. Today, clinicians use either artificial prostheses or fixed animal and cadaver-sourced tissues to replace defective valves. While these prostheses can restore the function of the heart for a while, they are associated with adverse comorbidity and wear down and need to be replaced during invasive and expensive surgeries.

A team lead recently developed a nanofiber fabrication technique to rapidly manufacture heart valves with regenerative and growth potential. The researchers fabricated a valve-shaped nanofiber network that mimics the mechanical and chemical properties of the native valve extracellular matrix (ECM). To achieve this, the team used a rotary jet spinning technology in which a rotating nozzle extrudes an ECM solution into nanofibers that wrap themselves around heart-valve-shaped mandrels. "Our setup is like a very fast cotton candy machine that can spin a range of synthetic and natural occurring materials. In this study, we used a combination of synthetic polymers and ECM proteins to fabricate biocompatible JetValves that are hemodynamically competent upon implantation and support cell migration and re-population in vitro. Importantly, we can make human-sized JetValves in minutes - much faster than possible for other regenerative prostheses."

Another group of researchers have previously developed regenerative, tissue-engineered heart valves to replace mechanical and fixed-tissue heart valves. In their approach, human cells directly deposit a regenerative layer of complex ECM on biodegradable scaffolds shaped as heart valves and vessels. The living cells are then eliminated from the scaffolds resulting in an "off-the-shelf" human matrix-based prostheses ready for implantation. In collaboration the two teams successfully implanted JetValves in sheep using a minimally invasive technique and demonstrated that the valves functioned properly in the circulation and regenerated new tissue. "In our previous studies, the cell-derived ECM-coated scaffolds could recruit cells from the receiving animal's heart and support cell proliferation, matrix remodeling, tissue regeneration, and even animal growth. While these valves are safe and effective, their manufacturing remains complex and expensive as human cells must be cultured for a long time under heavily regulated conditions. The JetValve's much faster manufacturing process can be a game-changer in this respect."

In support of these translational efforts, a larger initiative will commence to generate a functional heart valve replacement with the capacity for repair, regeneration, and growth. The team is also working towards a GMP-grade version of their customizable, scalable, and cost-effective manufacturing process that would enable deployment to a large patient population. In addition, the new heart valve would be compatible with minimally invasive procedures to serve both pediatric and adult patients.

JetValve: Rapid manufacturing of biohybrid scaffolds for biomimetic heart valve replacement

Tissue engineered scaffolds have emerged as a promising solution for heart valve replacement because of their potential for regeneration. However, traditional heart valve tissue engineering has relied on resource-intensive, cell-based manufacturing, which increases cost and hinders clinical translation. To overcome these limitations, in situ tissue engineering approaches aim to develop scaffold materials and manufacturing processes that elicit endogenous tissue remodeling and repair. Yet despite recent advances in synthetic materials manufacturing, there remains a lack of cell-free, automated approaches for rapidly producing biomimetic heart valve scaffolds.

Here, we designed a jet spinning process for the rapid and automated fabrication of fibrous heart valve scaffolds. The composition, multiscale architecture, and mechanical properties of the scaffolds were tailored to mimic that of the native leaflet fibrosa and assembled into three dimensional, semilunar valve structures. We demonstrated controlled modulation of these scaffold parameters and show initial biocompatibility and functionality in vitro. Valves were minimally-invasively deployed via transapical access to the pulmonary valve position in an ovine model and shown to be functional for 15 h.

Suggesting Mitochondrial Dysfunction Contributes to Age-Related Hair Loss

Researchers here investigate declining mitochondrial function in the context of hair growth, suggesting that age-related mitochondrial dysfunction is one of the causes of loss of hair in later life. Lower levels of - and less efficient - mitochondrial activity is implicated in a number of age-related diseases, especially those of the brain, where correct function requires large amounts of the energy store molecules produced by mitochondria. There appear to be several processes at work, ranging from mitochondrial DNA damage thought important in the SENS view of aging to a general and broader mitochondrial malaise that might result from dysfunctional regulation of cellular metabolism, a reaction to other forms of cell and tissue damage.

Emerging research revealed the essential role of mitochondria in regulating stem/progenitor cell differentiation of neural progenitor cells and other stem cells through reactive oxygen species (ROS), Notch or other signaling pathway. Inhibition of mitochondrial protein synthesis results in hair loss upon injury. However, alteration of mitochondrial morphology and metabolic function during hair follicle stem cells (HFSCs) differentiation and how they affect hair regeneration has not been elaborated upon.

Hair follicle (HF) is a cystic tissue surrounding the hair root, controlling hair growth. It consists of two parts: an epithelial part (hair matrix and outer root sheath) and a dermal part (dermal papilla and connective tissue sheath). The hair follicle goes through cycles of anagen phase (growth), catagen phase (degeneration) and telogen phase (rest). In the late telogen phase, hair follicle bulge stem cells differentiate into matrix cells upon stimulation, to re-enter the anagen phase. While in the catagen phase, proliferation and differentiation of hair follicle cells gradually attenuates, leaving with HFSCs and a dormant hair germ, re-entering the telogen phase.

As an essential organelle for anaerobic respiration, mitochondria attracted more research attention to its morphology and function during stem cell differentiation. Mitochondria show less mass in embryonic stem cells (ESCs) than that in differentiated cells, with a reduced oxygen consumption rate and less ROS produced. Effective control of mitochondrial morphology and function is critical for the maintenance of energy production and the prevention of oxidative stress-induced damage resulting from ROS. Besides, mitochondria play an essential role in determining hair cell differentiation and proliferation upon injury though regulating energy metabolism. In addition, ROS inhibit stem cell differentiation and proliferation through redox signaling pathway. Therefore, to counteract the adverse effect of ROS, the level of enzymes such as SOD2 is subsequently up-regulated.

We compared the difference in mitochondrial morphology and activity between telogen bulge cells and anagen matrix cells. Expression levels of mitochondrial ROS and superoxide dismutase 2 (SOD2) were measured to evaluate redox balance. In addition, the level of pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase (PDH) were estimated to present the change in energetic metabolism during differentiation. To explore the effect of the mitochondrial metabolism on regulating hair regeneration, hair growth was observed after application of a mitochondrial respiratory inhibitor upon hair plucking. The results revealed that disrupting mitochondrial respiration delays hair regrowth. It is possible that hair regeneration might be retarded due to insufficient energy supply. Another possibility is that mitochondrial dysfunction affects HFSCs differentiation through regulating redox balance or other signaling pathways, leading to the delay of hair growth.

Link: https://doi.org/10.7717/peerj.1821

Considering the Future of Academic Aging Research

Noted researcher Gordon Lithgow is here interviewed on the future of the aging research field. The focus is on academic funding, career, and whether or not current mainstream efforts to slow aging via alteration of the operation of metabolism in order to slow damage are the right way to go. It can be argued that the major problem in aging research is that there simply is next to no funding in comparison to other fields of medical research. The research is thus stuck moving slowly, at a point of great potential but with limited progress towards a coherent community of researchers all heading in what is definitively agreed to be the right direction for therapies to control aging. This is not because the field is currently divided and that there is much left to determine about cellular metabolism in aging, but because the funding isn't large enough to plow through these problems in a reasonable amount of time and thus quickly determine and prove which of the available options for development are actually the basis for viable human therapies.

It was odd that I ended up studying aging. I got into it not really knowing that, just seeing a profoundly mysterious process that there was no papers on, as far as I could tell. In the last 25 years, we've got textbooks on worm aging, we have signaling pathways and hormones and so, so much, it's great. But I still struggle to tell people what aging is. I tell them narratives about protein and protein insolubility during aging and how that could be driving dysfunction, but it's still hard to really say to someone, "This is what aging is". And now more than ever, beyond curiosity it's this idea that while it's been a great privilege to just be able to mess around and do science and find stuff out, actually what we've found out could be useful for people. It motivates the research somewhat, but also how I talk about the research, and my willingness to go off and do public stuff to try and turn people's heads to thinking about this. And it drives me crazy that we're training a group of scientists who are very comfortable with the biology of aging and the idea that it causes multiple diseases, who are very comfortable moving from discipline to discipline as you have to do in aging research, and unfortunately there's no jobs for these people.

Funding has been flat for 15 years in aging research. We're still here, the institute's growing. It wasn't for a while, but we're gonna be hiring again and creating some new jobs, so it's not like nothing's happening, but compared to what should be happening, and what the science is telling us we should be doing, it can be a little frustrating. We've seen our own people go to Calico and Unity Biotechnology, which is a spinout biotech from the Buck Institute that's doing very well. There have been many false dawns of aging companies and aging biotechnology going back 15 or more years, but with Calico and Unity it feels different. It feels like they're serious about finding cures to diseases based on aging technologies. And I hope they're going to be big employers.

The biggest obstacles right now is funding at every level. Translation. We've got a lot of information and compounds that we need to move forward. Obviously those two things are tightly related. Funding also is at the heart of the inability to grow the field with these new scientists. It's just so sad, people with fantastic skillsets leaving science or going into industry, and not in an aging context at all. I don't think that there's a problem with the science. In past years we could have said that there's a big problem because people don't understand the evolutionary origins of aging, or problems in the past where people were very dogmatic about it being down to one mechanism or another. And there was literally a time when many people in the field thought cellular senescence was an artefact of the culture dish and couldn't really be important in aging, because it didn't happen frequently enough in animals. And now we're at a point where we're thinking no, chances are it's really important. So a lot of the factions are melting away and you're seeing much more unity in this paradigm of what aging is.

One possibility is that most of the modifications that we've made or interventions that we've made are really just optimizing interventions. That they're not really affecting the underlying biology of aging. It's hard to draw a hard distinction between optimization and changing the underlying biology, but essentially all the models that we use, flies, yeast and worms, they all come from the same ecological niche. They all have laboratory drift and we use lab strains that aren't the same as wild strains, and during that process we may have been creating problems and shortening lifespan for years, and now all we're doing is fixing some of those laboratory-based problems. That's one view of a lot of what we've done. If that were true, it would be a bit of a crisis. It's certainly the case that we seem to be hitting some sort of upper limit with things. We don't see lifespan being extended in mice by two- or threefold, like we've seen in worms. Even in flies we haven't seen twofold life extension. It's possible that we're hitting limits in our ability to extend lifespan. I don't know.

Yet there is no biological upper limit on lifespan. We have clams living over 500 years, bristlecone pines that are living hundreds of years and things. In theory, we could all live to 122, because one human has done that. So in theory we can at least do that well, which is amazing in itself. In theory, there are mammals that live even longer than that, so we should be able to live longer than the oldest human. Clams have a circulatory system, there's a beating heart, so if there are hearts on earth that have been beating for 500 years, why not our hearts. I don't believe in biological limits, because even in human life expectancy, every time someone says there's an upper limit, someone breaks it. I don't believe in limits of that sort, but how much you have to change the human condition to attain greatly extended longevity, I don't think we know. The empirical observation so far is that it's harder to produce strong effects in more complex animals. It could be because it's just that the experiments in more complex animals are more expensive, so a tiny fraction of the experiments we've done in worms have been done in mice. It may be that we just haven't hit on it yet.

Link: http://geroscience.com/dr-gordon-lithgow-biotech-geroscience/

Excess Weight Increases Disease Risk and Shortens Life

No-one wants to hear that they are responsible for their own ill health, or that they are destroying the prospects for their own future. Thus, human nature being what it is in this era of cheap calories, there exists a thriving cottage industry based upon telling people that their excess weight is just fine and can be managed in such a way as to cause no harm. Unfortunately, that just isn't the case. Carrying excess visceral fat tissue does cause considerable personal harm: it reduces life expectancy, significantly increases risk of disease, and for all intents and purposes essentially accelerates the downward spiral of degenerative aging. You won't just be less healthy, you'll also spend more on medical services despite living a shorter life. The amount and quality of evidence that exists to support these conclusions is very hard to argue with. Nonetheless, people try, Canute against the tide.

The visceral fat tissue packed around internal organs is metabolically active, and by this point I think most people are at least passingly familiar with the idea that too much fat tissue distorts the operation of metabolism in ways that lead to metabolic syndrome and type 2 diabetes. These conditions are harmful enough over the long term that scientists have long used diabetes as a stand-in for aging in laboratory animals, a way to induce most of the consequences and conditions of aging more rapidly and thus more cheaply. In our species, type 2 diabetes is a self-inflicted condition for the vast majority of those who suffer it, caused by being overweight. It can even be turned back simply through the exercise of will power, through losing weight via a low calorie diet. It is amazing that this isn't the first thing done by every patient, rather than suffering through years of disability and medications with significant side-effects.

An excessive amount of fat tissue causes many other issues, however. It spurs chronic inflammation through its interactions with the immune system, and inflammation drives all of the common age-related diseases, especially those related to the decline in function and structure of the cardiovascular system. Excess weight also contributes to the development of hypertension, increased blood pressure, which puts further stress on blood vessels and heart tissue. Raised blood pressure is an important determinant of age-related mortality. Fat tissue also clearly drives the corrosion of the mind, as conditions such as Alzheimer's disease are strongly correlated with weight. Some of these links are mediated through the increased levels of cellular senescence produced by the presence of visceral fat tissue - recall that senescent cells are one of the root causes of aging, and more of them is a bad thing. Along the same lines, fat tissue and its activities can be linked to dysfunction of the immune system. It is just a really bad idea to get fat or stay fat: you are damaging yourself in so many ways.

'Fat but fit is a big fat myth'

The idea that people can be fat but medically fit is a myth. Early work, as yet unpublished, involved looking at the GP records of 3.5 million people in the UK. The researchers say people who were obese but who had no initial signs of heart disease, diabetes or high cholesterol were not protected from ill health in later life, contradicting previous research. A summary of their study was discussed at the European Congress on Obesity.

The term "fat but fit" refers to the alluring theory that if people are obese but all their other metabolic factors such as blood pressure and blood sugar are within recommended limits then the extra weight will not be harmful. In this study, researchers analysed data of millions of British patients between 1995 and 2015 to see if this claim held true. They tracked people who were obese at the start of the study, defined as people with a body mass index (BMI) of 30 or more, who had no evidence of heart disease, high blood pressure, high cholesterol or diabetes at this point. They found these people who were obese but "metabolically healthy" were at higher risk of developing heart disease, strokes and heart failure than people of normal weight.

No such thing as 'fat but fit', major study finds

Several studies in the past have suggested that the idea of "metabolically healthy" obese individuals is an illusion, but they have been smaller than this one. The new study involved 3.5 million people, approximately 61,000 of whom developed coronary heart disease. The scientists examined electronic health records from 1995 to 2015 in the Health Improvement Network - a large UK general practice database. They found records for 3.5 million people who were free of coronary heart disease at the starting point of the study and divided them into groups according to their BMI and whether they had diabetes, high blood pressure (hypertension), and abnormal blood fats (hyperlipidemia), which are all classed as metabolic abnormalities. Anyone who had none of those was classed as "metabolically healthy obese".

The study found that those obese individuals who appeared healthy in fact had a 50% higher risk of coronary heart disease than people who were of normal weight. They had a 7% increased risk of cerebrovascular disease - problems affecting the blood supply to the brain - which can cause a stroke, and double the risk of heart failure. While BMI results for particular individuals could be misleading, the study showed that on a population level, the idea that large numbers of people can be obese and yet metabolically healthy and at no risk of heart disease was wrong. "So-called metabolically healthy obesity is not a harmless condition and perhaps it is better not to use this term to describe an obese person, regardless of how many metabolic complications they have."

Bioprinted Artificial Ovaries Demonstrated to be Fully Functional in Mice

Researchers cannot yet produce large amounts of tissue using tissue engineering approaches such as bioprinting, as there is still no good solution for the creation of a suitable blood vessel network to support sizable tissue sections. However, that hasn't stopped the research community from forging ahead to develop the necessary recipes to produce functional tissue of various types, just in very small amounts. In many cases this artificial tissue isn't exactly the same in structure as the tissue it replaces, but it is nonetheless still capable of carrying out the desired functions. Some organs or crucial parts of organs are small enough to be produced in entirety, however, and hence researchers are now able to carry out demonstrations such the one here, in which artificial mouse ovaries are created, transplanted, and shown to be fully functional. The engineered ovaries produce the desired hormones and are capable of supporting the full process of mammalian reproduction. It is a good example of the quality of tissue being produced these days; once the blood vessel hurdle is overcome, the generation of entire organs will follow shortly thereafter.

Patients undergoing treatment regimens that eradicate their disease, such as cancer, may be left with diminished ovary function. Therefore, the oncofertility field is tasked to develop a whole organ replacement that restores long-term hormone function and fertility for all patients. In past work, we and others have sought to create an engineered ovary with biomaterials and isolated follicles. Ovarian follicles are spherical, multicellular aggregates that include a centralized oocyte (female gamete) and surrounding support cells, granulosa and theca, that produce hormones in response to stimulation from the pituitary. The spheroid shape of a follicle is critical to its survival in that the support cells must maintain contact with the oocyte until it has matured and is ready for ovulation. Consequently, a three-dimensional (3D) material environment is critical to maintaining these cell-cell interactions and follicle shape.

Thus far, there have been several reports of live births from biomaterial implants in mice, and all have used isolated follicles or whole ovarian tissue encapsulated in a plasma clot or similar fibrin hydrogel bead containing growth factor components or purified vascular endothelial growth factor. These results are very encouraging and have validated both the model procedure and the need for graft vascularization for complete restorative organ function of isolated follicles in a biomaterial. However, hydrogel encapsulation of follicles poses several challenges, especially with respect to the size of anticipated transplants. Specifically, when translating this work to a large animal or human, the implant must house a significantly larger population of follicles and therefore must be considerably larger than those used in mice. At these scales, diffusion limits may become a concern.

Future strategies must permit channels within the hydrogels (to facilitate host vasculature infiltration) or including pre-embedded vasculature to sustain follicle viability and circulate follicular hormones. Moreover, the ovary is a heterogeneous organ that compartmentalizes different follicle pools (quiescent and growing) into the cortex and medulla regions that have varying stiffness. It is believed that this compartmentalization will be critical to providing long-term (multiple decades) function with an implant. Therefore, a biomaterial strategy that can produce a mimetic construct of spatially varying material properties may be required for optimal implant function and longevity.

3D printing can be used to address all of these future implant requirements for creating a human bioprosthetic ovary, a bioengineered functional tissue replacement. As the first steps towards this goal, here, we investigated porous hydrogel scaffolds with murine follicles seeded throughout the full depth of the scaffold layers to create a murine bioprosthetic ovary. Microporous architectures were achieved through 3D printing partially crosslinked, thermally regulated gelatin. We found that specific scaffold architectures created a 3D feel by providing appropriate depth and multiple contact sites for the ovarian follicle, which resulted in optimal murine follicle survival and differentiation in vitro. The open micropores within the hydrogel scaffold provided sufficient space and nutrient diffusion for follicle survival and maturation in vitro and in vivo, as well as space for vasculature to infiltrate when implanted in vivo without the need for significant scaffold degradation as is required when using hydrogel encapsulation.

Follicle-seeded scaffolds become highly vascularized and ovarian function is fully restored when implanted in surgically sterilized mice. Moreover, pups are born through natural mating and thrive through maternal lactation. These findings present an in vivo functional ovarian implant designed with 3D printing, and indicate that scaffold pore architecture is a critical variable in additively manufactured scaffold design for functional tissue engineering.

Link: https://dx.doi.org/10.1038/ncomms15261

Comparing Regeneration of Fingertips Between Species

As a sidebar to yesterday's post on regeneration in mammals, here is a review paper that just considers fingertip regeneration in various species. This can occur in mammals, and even on rare occasions in adult humans, though it isn't well understood as to why it happens at all given the inability to regenerate most other lost appendages. It is possible that this is a useful point of investigation in order to better understand why mammals do not regenerate like salamanders, and how that state of affairs might be changed for the better.

Mammalian fingertips and toes can partially regrow under certain conditions; however, regeneration is greatly limited compared to urodele amphibians such as newts and salamanders that can completely regrow an amputated limb. The question is why there is such a difference between the regenerative potentials of mammals and amphibians. Embryonic, neonatal, and adult mice can regenerate digit tips if the amputation is midway through the third phalanx; however, if the amputation occurs proximal to the midway point of the third phalanx in mice, regeneration of the digit tip does not typically occur. Similarly, young patients have also been documented to regrow the tips of amputated fingers if treated conservatively. Although adults and even elderly individuals have potentially regenerated amputated digit tips, the regenerative process may not be as efficient as it is in younger patients and usually results in fibrous scars in adults. The regeneration process of the digit following injury may be related to the age of the host, with decreased restoration in adults compared to fetal or neonatal mammals. Injured adult mammalian tissues are usually replaced with fibrotic scar tissue, whereas scarless healing typically occurs in fetal wound healing which results in complete tissue recovery. Stem cell activation and scarless wound healing are considered to be essential requisites for quality tissue regeneration; however, for some regenerative processes a dedifferentiation process, but not stem cell activation, is required.

Many theories have been proposed to explain why successful regeneration occurs in urodele amphibians but not in mammals. First, the immune system has been shown to play a major role in the regeneration process of amputated limbs in newts. In mammals, fetal wounds can regenerate because they have an immature immune system; however, in adults, clearing pathogens appears to be evolutionarily favored compared to retaining the ability to regenerate a limb or digit. Second, amphibians have retained limb regeneration-specific genes not found in mammals, which allow their cells to dedifferentiate. A related theory is that mammals have evolved tumor suppression genes that inhibit regeneration. The Ink4a locus is present in mammals but not amphibians; this region encodes the tumor suppression genes p16ink4a and Alternative Reading Frame (ARF). Inactivation of both tumor suppressors retinoblastoma (Rb) and ARF allows terminally differentiated mammalian muscle cells to dedifferentiate. An extension of this theory is that differentiated mammalian tissues can regenerate if the cells are induced to reenter the cell cycle, which occurs in the Murphy Roths Large (MRL) mouse and the p21-deficient mouse. Third, bioelectric signaling (e.g., membrane voltage polarity, ionic channels) may also play a role in the tissues' regeneration potential. Nonregenerating wounds display a positive polarity throughout the healing process, whereas in regenerating animals the polarity is initially positive but then quickly changes to negative polarity with the peak voltage occurring at the time of maximum cellular proliferation.

Link: https://doi.org/10.1155/2017/5312951

Macrophages, and Possibly Senescent Cells, are the Keys to the Exceptional Regeneration of African Spiny Mice

In recent years, researchers have assembled a number of what appear to be important pieces of the puzzle when it comes to understanding regeneration and scarring. Why do mammals scar rather than regenerate like salamanders, and how do the exceptions to that rule function? Mutant MRL mice can heal small injuries without scarring, African spiny mice can regrow large sections of their skin without scarring, the liver can regrow sections of itself, and people can sometimes regenerate lost fingertips. It is of great interest to the medical community to come to a deeper understanding of the mechanisms of regeneration in our species and other mammals, as in principle anything that an MRL mouse can achieve in the healing of injury can be induced through suitable changes in the regulation of human regeneration. In principle, if fingertips can regenerate without scarring in some rare occasions, why can't the root causes be identified and applied to larger injuries? A fair number of research groups have for years tackled various approaches to these questions, investigating the biochemistry of regeneration in a variety of mammalian lineages and other species capable of proficient regeneration.

A picture is beginning to emerge in which the activities of senescent cells and the immune cells called macrophages are the most important players. The final assembly and details of a theory that explains all of the observed variation in regeneration remains to be accomplished, but there is a good deal of evidence to speculate upon. For example, senescent cells are known to play a temporary role in wound healing; some of their signaling is important in this respect. One of the side-effects of the recent focus on removal of lingering senescent cells as a treatment for aging is that researchers have found wound healing to be impaired when these cells are constantly cleared. Senescent cells are created in wounded tissue and serve some transient purpose before destroying themselves; if they are removed before the healing process can get underway, this slows it down. Separately, researchers have found that salamanders, known for their ability to regenerate, have a much more efficient and energetic ability to create and then entirely clear out senescent cells during regeneration.

In salamanders, the clearance of senescent cells is accomplished by macrophages, and without their presence the process of efficient regeneration runs awry. This has been shown to be the case in zebrafish as well, another species capable of healing without scarring and regeneration of body parts. Macrophages respond to injuries in mammals, and play their part in regenerative processes. There is evidence to suggest that their activities can be improved upon - researchers have altered macrophage behavior to enhance nerve regeneration, for example. Similarly, and as is the case in the research noted below, there is good evidence for macrophages to be both beneficial and detrimental to healing depending on their characteristics; some spur regeneration, others spur scarring. Given that the evidence below makes proficient regeneration in African spiny mice look very much like proficient regeneration in salamanders and zebrafish, it now seems plausible that there is a lever in here somewhere that could be used to tilt mammalian regeneration in the direction of greater capacity and lesser degrees of scarring.

Researchers Identify Macrophages as Key Factor for Regeneration in Mammals

Researchers have discovered that macrophages, a type of immune cell that clears debris at injury sites during normal wound healing and helps produce scar tissue, are required for complex tissue regeneration in mammals. Their findings shed light on how immune cells might be harnessed to someday help stimulate tissue regeneration in humans. "With few examples to study, we know very little about how regeneration works in mammals; most of what we know about organ regeneration comes from studying invertebrates or from research in amphibians and fish. If we want to apply what we learn from basic regenerative biology to humans, it would be helpful to understand what cell types and molecules regulate regeneration in a mammal where it occurs naturally."

Scientists have been trying to learn for years why some animals, like salamanders and zebrafish, are able to regrow body parts following injury, while others - like humans - can only produce scar tissue in response. Researchers learned nearly eight years ago that African spiny mice are one of the few mammalian models capable of complex tissue regeneration, making them particularly fascinating subjects. But what remained unclear was exactly how an identical injury in spiny mice and non-regenerating lab mice could produce dramatically different healing responses. The researchers decided to investigate how the inflammatory environment might differ between the regenerative response observed in spiny mice compared to the typical scarring response observed in lab mice. Although white blood cell profiles were the same in uninjured animals from both species, injury elicited different local responses. "Comparing spiny mice to common house mice, we discovered that subtypes of macrophages active during regeneration are different than those active during scarring."

When the team looked at different types of macrophages in healing tissue they found that a pro-inflammatory type of macrophage was highly abundant during scarring, but very rare during regeneration. "Our findings imply that macrophage activation in our model favors regeneration. The next step is to identify the components of macrophage activation that are necessary for regeneration. Since we are actively developing clinically feasible therapies that selectively activate macrophages, identifying targetable components of macrophage activation opens new areas of discovery with real potential for improving tissue regeneration in humans."

Macrophages are necessary for epimorphic regeneration in African spiny mice

When an animal is injured, immune cells such as macrophages rush to the wounded site to clear debris and help repair the damage. Macrophages come in different forms and subtypes, and express different protein markers on their surface, depending on where in the body they reside. Few mammals can completely renew or regrow a damaged tissue - a process known as tissue regeneration. Instead, humans and most other mammals repair injuries by producing scar tissue, which has different properties compared to the original tissue it replaces. One exception is the African spiny mouse (Acomys cahirinus), which, unlike other rodents studied, can regrow skin and fur, nerves, muscles, and even cartilage. It has been shown that in highly regenerative animals such as salamanders and zebrafish, macrophages are necessary to initiate tissue regeneration. Documented cases of tissue regeneration in mammals are rare and therefore less understood. Until now, it was not clear why two species as closely related as spiny mice and house mice would heal identical injuries in different ways.

Here, we report how the two main orchestrators of inflammation, neutrophils and macrophages, respond to injury during regeneration in Acomys cahirinus compared to scarring in the house mouse (Mus musculus). Acomys and Mus exhibit the same circulating leukocyte profiles, and we demonstrate a robust acute inflammatory response in both species. We demonstrate higher neutrophil activity in the scarring system compared to higher reactive oxygen species (ROS) activity in the regenerative system. We show that macrophages between the two species display similar in vitro properties providing a comparable baseline prior to and following injury. We also observed distinct differences in the spatiotemporal distribution of macrophage subtypes during regeneration and scarring. Finally, depletion of macrophages, prior to and during injury, inhibited blastema formation and regeneration, thus demonstrating a necessity for these cells.

A popular hypothesis to explain why most mammals heal injuries with scar tissue is that they evolved a strong inflammatory and adaptive immune response that induces intense fibrosis in lieu of regeneration. Yet, the fact that some mammals exhibit epimorphic regeneration (e.g. rodent and primate digit tips, rabbit and spiny mice ear punches and skin) suggests that regeneration can occur despite a complex adaptive immune system. It is possible that macrophages provide an initiating signal for regeneration or remove subpopulations of local cells secreting inhibitory signals (e.g. senescent cells). In support of the first idea, ROS production has been suggested as an essential early signal for regeneration based on studies in zebrafish tail models of regeneration. Macrophages are a major source of ROS after injury, and we observed significantly stronger and prolonged ROS production during regeneration compared to scarring. In support of the idea that macrophages may limit inhibitory signals through selective removal of senescent cells, recent work in salamanders suggested that clearance of senescent cells is important for limb regeneration and persistence of senescent cells during liver regeneration leads to excessive fibrosis. Furthermore, the accumulation of senescent cells with age has been suggested to shorten lifespan, degrade tissue function, and increase the expression of pro-inflammatory cytokines in mammals. These and other studies suggest that proper clearance of senescent cells from damaged tissues may promote regenerative outcomes.

Researchers Generate Improved Lung Tissue Organoids

In tissue engineering this is the age of organoids: while the challenge of generating a blood vessel network sufficient to grow large tissue sections is not yet solved, researchers are nonetheless establishing the diverse set of methodologies needed to grow functional organ tissue from a cell sample. The recipe is different for every tissue type, and there are many forms of tissue in the body. The resulting small tissue sections are known as organoids. At this time organoids are largely used to speed up further research, but for some tissue types there is the potential to produce therapies based on transplantation of multiple organoids to patch or augment failing organs. Sadly, that is probably not an option for lung disease due to the highly structured nature of lung tissue, and here the focus is on using organoids to improve the state of research. A number of groups have demonstrated functional lung organoids of increasing sophistication in the past few years, and here is the latest example in this line of research:

New lung "organoids" have been created from human pluripotent stem cells. Researchers used the organoids to generate models of human lung diseases in a lab dish, which could be used to advance our understanding of a variety of respiratory diseases. Organoids are 3-D structures containing multiple cell types that look and function like a full-sized organ. By reproducing an organ in a dish, researchers hope to develop better models of human diseases and find new ways of testing drugs and regenerating damaged tissue. "Researchers have taken up the challenge of creating organoids to help us understand and treat a variety of diseases. But we have been tested by our limited ability to create organoids that can replicate key features of human disease."

The lung organoids created in this study are the first to include branching airway and alveolar structures, similar to human lungs. To demonstrate the functionality of the organoids, the researchers showed that the organoids reacted in much the same way as a real lung does when infected with respiratory syncytial virus (RSV). Additional experiments revealed that the organoids also responded as a human lung would when carrying a gene mutation linked to pulmonary fibrosis. RSV is a major cause of lower respiratory tract infection in infants and has no vaccine or effective antiviral therapy. Idiopathic pulmonary fibrosis, a condition that causes scarring in the lungs, causes 30,000 to 40,000 deaths in the United States each year. A lung transplant is the only cure for this condition. "Organoids, created with human pluripotent or genome-edited embryonic stem cells, may be the best, and perhaps only, way to gain insight into the pathogenesis of these diseases."

Link: http://newsroom.cumc.columbia.edu/blog/2017/05/11/a-new-3d-model-for-lung-disease-made-from-stem-cells/

Dysfunction of the GABAergic System and the Aging of the Brain

Perhaps the most fearsome aspect of aging is that it degrades and ultimately destroys the function of the mind. With the exception of those who suffer neurodegenerative conditions - such as Alzheimer's disease - that in their late stages cause widespread cell death in the brain, most of the infrastructure of the mind remains largely intact even in very late life, however. This is despite the widespread small-scale damage due to broken blood vessels. The operation of that infrastructure is disrupted, however, and that disruption manifests as a progression of the various forms of cognitive decline. Analogously to the situation observed in aging stem cell populations, in which the cells are still present but not functioning as they did in youth, this suggests that some degree of restoration of lost cognitive function could be achieved rapidly if the right underlying damage could be repaired, the right signaling changed.

Cognitive aging is a consequence of molecular and biochemical aging. Alterations in gene expression, influencing the levels of proteins in many biological pathways, can be regarded as a hallmark of molecular aging. Changes in the biochemical composition of neural cells, which affect the efficiency of their synapses and whole circuits, impair the plasticity of the brain, that is the ability to reorganize, learn and remember. In this way, the disturbances of synaptic machinery profoundly contribute to the cognitive impairments as well as to the age-related brain disorders.

The majority of studies concerning the plasticity of neural circuits have focused on excitatory synapses. However, the role of inhibitory interactions in neuroplastic changes has recently been widely recognized. The most basic role of inhibitory neurons is to control the excitability of the principal cells, ensuring a proper homeostatic balance and preventing runaway excitation. Strong network inhibition suppresses the excitatory population response, providing the circuit with an intrinsic mechanism enabling precise contrast-gain control. Therefore, even though excitatory neurons are a large majority of cortical neurons, local inhibitory interneurons shape their firing and timing. There is increasing support for the hypothesis that disruption of inhibitory circuits is responsible for some of the clinical features of many neurodegenerative disorders. Many of them have been proposed to be synaptopathies - diseases related to the dysfunction of synapses. Brain aging is, in this context, considered a phenomenon promoting biological alterations associated with the above-mentioned disorders, resulting in so-called late-onset diseases.

The difficulty in understanding the mechanisms of interneurons aging, along with its relationship to plasticity impairments, cognitive decline and brain disorders, lies in the tremendous diversity of inhibitory neurons. Inhibition can be performed by perisomatically, dendritically or axonally targeting interneurons, which can be devoted to different inhibitory tasks. Furthermore, over 20 subtypes of potentially inhibitory neurons using GABA as a neurotransmitter have been recognized. Nevertheless, this diversity makes interneurons a potent and complex regulatory machinery controlling the physiology of neural circuits, and their molecular and biochemical aging can significantly contribute to the cognitive deficits observed in the aged brain. The role of neuroplasticity is to compensate for those age-related changes and to maintain the proper function of inhibitory circuits, supporting the balance between excitation and inhibition and the correct cognitive performance.

Age-related loss of synaptic contacts, decreased neurotransmitter release and reduced postsynaptic responsiveness to neurotransmitters result in a decline in synaptic strength, contributing to age-related cognitive decline. Molecular aging, defined as age-related transcriptome changes, and biochemical protein-related alterations within synapses weaken the plastic potential of neurons. Inhibitory neurons, despite being in the minority, are powerful regulators of neuronal excitability and, being particularly susceptible to aging-related alterations, are involved in many aging-induced cognitive impairments and brain disorders.

In the aged mouse somatosensory cortex, we have shown that although potential for learning-related plasticity is preserved there, the corresponding mechanisms are weakened and need longer stimulation to trigger plastic changes. We have postulated that the decreased effectiveness of the GABAergic system in the aged mouse somatosensory cortex contributes to the deficits in learning-induced plasticity. We posit that aging-induced impairments of the GABAergic system lead to an inhibitory/excitatory imbalance, thereby decreasing neuron's ability to respond with plastic changes to environmental and cellular challenges, leaving the brain more vulnerable to cognitive decline and damage by synaptopathic diseases. This is an intermediate stage of the transition from healthy aging to age-related cognitive decline and then to disease. Pharmacological and/or environmental reinforcement of the GABAergic system thus seems to be a promising therapeutic target for aging-related brain disorders.

Link: https://dx.doi.org/10.1111/acel.12605

Moving Forward with the Maximally Modifiable Mouse

One of the past projects undertaken by the SENS Research Foundation was the groundwork for a better methodology of carrying out investigative gene therapies in mice. This is called the Maximally Modifiable Mouse, and it might be thought of as a sort of mirror image of CRISPR gene editing technology: instead of bacterial genetic mechanisms normally used to defend against viruses being adapted to insert DNA into cells, is is the case for CRISPR, in the Maximally Modifiable Mouse viral genetic mechanisms normally used to attack bacteria are adapted and placed into the mouse genome to act as a docking station for the later insertion of arbitrary genetic material.

The point of the exercise is that the Maximally Modifiable Mouse technology makes it possible, or at the very least easier and less costly, to make precise genetic alterations in mice at any point in life, young or old. Most research into cellular mechanisms involves genetic engineering at some point, even if the end result for human medicine is usually some other form of intervention. It is the most effective way, and sometimes the only way, to make progress in understanding the inner workings of specific cellular processes. This engineering is still largely accomplished through the creation of altered lineages of mice rather than the application of gene therapies to normal adult mice, however. Building those lineages takes time and money, and it might be possible to cut this cost from the picture via the Maximally Modifiable Mouse. Cheaper research is faster research, and that is one of the goals of this tool.

The other important goal here is to build a system that can be used to cost-effectively test therapeutic genetic alterations aimed at rejuvenation. The obvious candidate is allotopic expression of mitochondrial genes, which requires genetic material to be delivered to the cell nucleus in order to bypass the consequences of damage to mitochondrial DNA. This is one of the root causes of aging, and allotopic expression has the potential to eliminate it. There will likely be other gene therapies to help with other forms of damage as this age of genetics moves on; perhaps the insertion of artificial enzymes capable of safely breaking down forms of metabolic waste that presently accumulate, for example. Almost any therapy that involves adding novel proteins or changing levels of existing proteins might in the future be accomplished with gene therapies at least as efficiently as via small molecule drugs - or at least once the research and development community has moved beyond its current reluctance regarding elective genetic alteration.

Creation of a "Maximally Modifiable Mouse"

We hope this project will demonstrate the feasibility of bona fide rejuvenation biotechnologies - therapies that remove, replace, repair or render harmless the pre-existing burden of cellular and molecular damage of aging in persons who have already suffered substantially from the degenerative aging process. It requires that new therapies be tested in animal models that have already undergone significant biological aging. Many of these therapies will be best demonstrated using gene therapy in animal models, and may ultimately require gene therapy for maximal efficacy in humans. Conventional transgenic animals bear their novel genes in the germ line, and although convenient methods for inducing the expression of therapeutic transgenes late in life exist, doing so still requires the custom generation of a line of transgenic animal for each new tested gene, and then allowing it to age, typically for two or more years, before the induced transgene's effects can be tested. This greatly slows down the development cycle of testing, refining, and iteratively re-testing therapeutic genes.

A promising alternative is the use of integrases from bacteriophages (or "phages,"), a class of virus whose hosts in nature are bacteria. Phage integrases are enzymes that catalyze precisely-targeted, unidirectional recombination between paired DNA recognition sequences: one (attB) a specific site in the bacterial host where the viral DNA is inserted, and another (attP) in the phage genome, from which the viral DNA is copied. Moreover, phage integrases can be used to insert arbitrary amounts of DNA into the host genome. To exploit phage integrases for gene therapy in mammals, one plasmid is generated containing the gene(s) to be inserted linked to an attB site, and another is generated containing the phage integrase; the plasmid DNA is translated in the host cell, generating the integrase, which then inserts the attB-bearing gene of interest into the host genome, with essentially no risk of gene disruption; the attP and attB sites are both destroyed in the process. The serine integrase from the mycobacteriophage Bxb1, in particular, is extremely precise: it will only mediate integration at specific attB sites. The Bxb1 integrase has already been demonstrated as a highly effective tool for somatic gene therapy in Drosophila, and has been shown to allow repeated, high-titer delivery of novel genes.

Unfortunately, mammals lack attP sites in their genomes, and thus the Bxb1 integrase cannot be used to insert new genes into mammalian model organisms such as the mouse. This limitation could be overcome with a one-time germline insertion of the Bxb1 insertion sequence into a transcriptionally-active but safe genomic location in the mouse genome: in such mice, the Bxb1 integrase system could be used at any time during the lifespan to insert therapeutic genes of any size, and with repeated rounds of gene dosing with multiple delivery methods to hit all the relevant tissues in the animals' body, with only a very low risk of mutagenesis. The effects of such genes on age-related disease could then be rapidly evaluated, and if improvements need to be made, a new transgene constructed and tested immediately in mice who are already the same age, without having to wait for a new generation of transgenic animal to be generated, born, mature, and age with every round of testing.

I'm pleased to see that the SENS Research Foundation, with funding from the Forever Healthy Foundation and other donors in our community, has started a collaboration with the Buck Institute for Research on Aging to move ahead with field testing of the Maximally Modifiable Mouse. Infrastructure projects with the potential to greatly reduce cost and time in research are one of the most important activities in any field of research. Few people pay enough attention to such work, and it rarely results in the headlines it deserves, but this sort of thing is what drives the pace of progress over the longer term.

SRF and Buck Institute to Collaborate on Gene Therapy

SENS Research Foundation (SRF) has launched a new research program focused on somatic gene therapy in collaboration with the Buck Institute for Research on Aging. Brian Kennedy, PhD, a leading expert on the biology of aging, will be running the project in his lab at the Buck. Many potential treatments of age related diseases require the addition of new genes to the genome of cells in the body, a technology known as somatic gene therapy. The technology has been hampered, up until now, by the inability to control where the gene is inserted. That lack of control resulted in a significant risk of insertion in a location that encourages the cell to become malignant.

SRF has devised a new method for inserting genes into a pre-defined location. In this program, this will be done as a two-step process, in which first CRISPR is used to create a "landing pad" for the gene, and then the gene is inserted using an enzyme that only recognizes the landing pad. SRF has created "maximally modifiable mice" that already have the landing pad, and this project will evaluate how well the insertion step works in different tissues. "Somatic gene therapy has been a goal of medicine for decades. Being able to add new healthy genes will enable us to address treatments of such age-related diseases as atherosclerosis and macular degeneration. Our collaboration with SRF will substantially move us toward finding effective treatments to genetically based age related diseases."