Fight Aging! Newsletter, February 10th 2020

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  • Forcing Macrophages into Greater Clearance of Debris in Atherosclerotic Lesions
  • Chronic Inflammation is of Great Importance in the Progression of Aging
  • Macrophages Implicated in the Scarring of Heart Tissue Following Injury
  • More Data on the Tissue Specificity of Senescent Cell Accumulation with Age in Mice
  • Two Examples of UK Biotech Startups Focused on the Treatment of Aging
  • Evidence for the Epigenetic Clock to Underestimate Age in Later Life
  • A Discussion of Recent Work on Allotopic Expression of Mitochondrial Genes at the SENS Research Foundation
  • Mechanisms of Slowed Muscle Aging via Calorie Restriction in Rhesus Macaques
  • Modulating Macrophage Polarization as a Therapy for Atherosclerosis
  • An Example of Epigenetic Effects on Offspring Longevity
  • A Mechanism by which Chronic Inflammation Spurs Cancer Metastasis
  • Long Non-Coding RNAs and Macrophage Senescence in Age-Related Disease
  • Endothelial Cell Senescence can Impair Insulin Sensitivity
  • Reviewing Efforts to Develop NAD+ Therapies to Reverse Age-Related Loss of Mitochondrial Function
  • Exceptionally Long Lived Humans Exhibit Slower Epigenetic Aging, Measured by DNA Methlyation Clocks

Forcing Macrophages into Greater Clearance of Debris in Atherosclerotic Lesions

Atherosclerosis is the generation of fatty deposits in blood vessel walls, called plaques, atheromas, or lesions, that narrow and weaken important vessels. Sooner or later a vessel ruptures, or a plaque disintegrates and its fragments block the flow of blood, and this results in stroke or heart attack. In the public eye atherosclerosis is considered a disease of cholesterol, of blood lipids, and reducing cholesterol and other lipids in the blood remains the primary focus of treatment. This is despite the fact that this approach can only slow progression - it doesn't reverse existing lesions to a sizable degree.

Atherosclerosis is in fact a condition of macrophage dysfunction, not of cholesterol. Macrophages are the cells responsible for clearing out lipids from blood vessel walls. They ingest cholesterol, and then hand it off to HDL particles that can carry it back to the liver for excretion. This process works just fine in youth, but macrophages are unfortunately vulnerable to oxidized cholesterol. It makes them dysfunctional, inflammatory, and even kills them. As a result of other forms of age-related damage, such as mitochondrial dysfunction, levels of oxidized cholesterol increase significantly. A feedback loop forms in which macrophages are constantly drawn to a lesion, succumb to the oxidized cholesterol present there, and add their corpses to the growing deposit. Atherosclerotic lesions are macrophage graveyards.

Thus macrophages are, to my eyes, the right point of intervention for therapies to effectively treat atherosclerosis - to actually prevent and meaningfully reverse lesions. This might be achieved by making macrophages invulnerable to the oxidized cholesterol that challenge them, as Repair Biotechnologies is working towards, or by clearing out oxidized cholesterols, as Underdog Pharmaceuticals is working towards. The research noted here takes a different view of the opportunities presented by a tissue that is rich in macrophages, and reports on a way to force those macrophages to more aggressively clear debris and destroy harmful cells in a lesion, despite their impediments. The initial data seems promising.

Nanoparticle chomps away plaques that cause heart attacks

Researchers have demonstrated a nanoparticle that homes in on atherosclerotic plaque due to its high selectivity to a particular immune cell type - monocytes and macrophages. Once inside the macrophages in those plaques, it delivers a drug agent that stimulates the cell to engulf and eat cellular debris. Basically, it removes the diseased/dead cells in the plaque core. By reinvigorating the macrophages, plaque size is reduced and stabilized.

The research is focused on intercepting the signaling of the receptors in the macrophages and sending a message via small molecules using nano-immunotherapeutic platforms. Previous studies have acted on the surface of the cells, but this new approach works intracellularly and has been effective in stimulating macrophages. "We found we could stimulate the macrophages to selectively eat dead and dying cells - these inflammatory cells are precursor cells to atherosclerosis - that are part of the cause of heart attacks. We could deliver a small molecule inside the macrophages to tell them to begin eating again."

Pro-efferocytic nanoparticles are specifically taken up by lesional macrophages and prevent atherosclerosis

Atherosclerosis is the process that underlies heart attack and stroke. A characteristic feature of the atherosclerotic plaque is the accumulation of apoptotic cells in the necrotic core. Prophagocytic antibody-based therapies are currently being explored to stimulate the phagocytic clearance of apoptotic cells; however, these therapies can cause off-target clearance of healthy tissues, which leads to toxicities such as anaemia.

Here we developed a macrophage-specific nanotherapy based on single-walled carbon nanotubes loaded with a chemical inhibitor of the antiphagocytic CD47-SIRPα signalling axis. We demonstrate that these single-walled carbon nanotubes accumulate within the atherosclerotic plaque, reactivate lesional phagocytosis and reduce the plaque burden in atheroprone apolipoprotein-E-deficient mice without compromising safety, and thereby overcome a key translational barrier for this class of drugs.

Single-cell RNA sequencing analysis reveals that prophagocytic single-walled carbon nanotubes decrease the expression of inflammatory genes linked to cytokine and chemokine pathways in lesional macrophages, which demonstrates the potential of 'Trojan horse' nanoparticles to prevent atherosclerotic cardiovascular disease.

Chronic Inflammation is of Great Importance in the Progression of Aging

Aging is a process of damage and consequence. Damage to the molecular machinery of cells and the molecular structure of tissues accumulates as a normal consequence of the operation of healthy metabolism. This damage degrades function, producing a lengthy chain of downstream consequences that interact with one another, make one another steadily worse, and culminate in age-related disease. Some of these downstream consequences are more important than others. Among the most important are raised blood pressure, which converts low level molecular damage into actual structural pressure damage to tissues, and chronic inflammation, which converts low level molecular damage into sweeping failure and detrimental change of cell and tissue function.

These two downstream consequences of damage are so influential over the development and progression of age-related disease that some progress has been made in lowering age-related mortality by crudely forcing reductions in blood pressure and inflammation, with no effort to eliminate the causes of these issues. Most efforts to tackle inflammation involve sabotaging the cell signaling that drives it. This is a blunt tool, as transient inflammation is quite important to health. Only the inappropriate chronic inflammatory signaling should be suppressed, but the tools of the past are far from being discriminating enough in this matter.

A sizable degree of the chronic inflammation of aging is driven by the presence of senescent cells and the inflammatory signaling that they generate. Numerous mechanisms create senescent cells, and they are useful in the short term, a necessary part of wound healing, cancer suppression, and other mechanisms. The issue is that clearance of these cells fails with age, and thus their numbers grow inexorably. Fortunately, the advent of senolytic therapies to selectively destroy senescent cells offers considerable potential as a way to reduce only the undesirable chronic inflammation of aging, while preserving desirable transient inflammation. Given the importance of inflammation in aging, we might expect considerable benefits to emerge from the use of senolytics.

Scientists have identified the role of chronic inflammation as the cause of accelerated aging

"Today, chronic inflammatory diseases are at the top of the list of death causes. There is enough evidence that the effects of chronic inflammation can be observed throughout life and increase the risk of death. It's no surprise that scientists' efforts are focused on finding strategies for early diagnosis, prevention and treatment of chronic inflammation."

One of the serious results obtained to date has been the concept of immune aging, which enables researchers to characterize the immune function of an individual and to predict the causes of mortality much more accurately than by relying only on chronological age. In addition to well-known inflammation biomarkers, such as C-reactive protein, interleukin 1 and interleukin 6, tumor necrosis factor, scientists note the need to study additional biomarkers of the immune system, which differ very much from person to person, in particular, the subgroups of T-lymphocytes and B-lymphocytes, monocytes, etc.

Scientists have identified certain factors (social, environmental and lifestyle factors) that contribute to systemic chronic inflammation. Taken together, such factors are the main cause of disability and mortality worldwide. An integrative approach to the study of mechanisms of systemic chronic inflammation is being adopted by a growing number of scientists. Research is continuing, and scientists have a long way to go to fully understand the role of chronic inflammation in aging and mortality, and to be able to predict changes in a person's health throughout life.

Chronic inflammation in the etiology of disease across the life span

Although intermittent increases in inflammation are critical for survival during physical injury and infection, recent research has revealed that certain social, environmental and lifestyle factors can promote systemic chronic inflammation (SCI) that can, in turn, lead to several diseases that collectively represent the leading causes of disability and mortality worldwide, such as cardiovascular disease, cancer, diabetes mellitus, chronic kidney disease, non-alcoholic fatty liver disease, and autoimmune and neurodegenerative disorders. We describe the multi-level mechanisms underlying SCI and several risk factors that promote this health-damaging phenotype, including infections, physical inactivity, poor diet, environmental and industrial toxicants, and psychological stress. Furthermore, we suggest potential strategies for advancing the early diagnosis, prevention and treatment of SCI.

Macrophages Implicated in the Scarring of Heart Tissue Following Injury

A potentially important faction within the regenerative medicine community is engaged in trying to understand exactly how highly regenerative species such as salamanders and zebrafish can regenerate organs following injury, and do so repeatedly without scarring. There are also a few examples of adult mammals capable of regenerating a limited number of body parts without scarring, such as African spiny mice and the MRL mouse lineage. It seems plausible that mammalian species still carry much of the machinery of proficient regeneration, but that this machinery is suppressed in some way, possibly because that suppression acts to reduce cancer risk. Evidence in support of that thesis includes the ability of human tumor suppressor ARF to block zebrafish regeneration.

Thus understanding of the fine details of the ways in which highly regenerative species differ from near all mammals might lead to ways to induce limb and organ regrowth in humans. It is still a little early to say whether or not these differences will in fact include anything that could be the basis of a therapy. The differences might be too complex, or too fundamental to easily alter with today's tools. That said, there is compelling evidence for macrophage behavior to be fundamental to exceptional regeneration. All regeneration is an intricate dance between somatic cells, stem cells and progenitor cells of various types, transient senescent cells, and immune cells, particularly macrophages.

In this context, the research here is an interesting exploration of the activities of macrophages in heart injury in mice and zebrafish. Mice scar rather than regenerate from this type of injury, and the heart is one of the least regenerative organs in mammals. Zebrafish normally regenerate heart injuries without scarring, but this isn't the case if the injury is inflicted by freezing. These various circumstances give points of comparison to look into the behavior of macrophages in scarring and regeneration, and perhaps suggest lines of investigation that could lead to therapies to prevent scarring in human patients.

New target identified for repairing the heart after heart attack

Billions of cardiac muscle cells are lost during a heart attack. The human heart cannot replenish these lost cells, so the default mechanism of repair is to form a cardiac scar. While this scar works well initially to avoid ventricular rupture, the scar is permanent, so it will eventually lead to heart failure and the heart will not be able to pump as efficiently as before the damage caused by heart attack.

Zebrafish, a freshwater fish native to South Asia, is known to be able to fully regenerate its heart after damage due to the formation of a temporary scar as new cardiac muscle cells are formed. Researchers have been striving to understand and compare the composition of the cardiac scar in different animals as part of ongoing efforts to investigate whether it can be modulated to become a more transient scar like that of the zebrafish, and therefore potentially avoid heart failure in heart attack patients.

The team focused their efforts on studying the behaviour of macrophages, cells normally associated with inflammation and fighting infection in the body, when exposed to the three post-injury environments. They extracted macrophages from each model to examine their gene expression. In both mouse and fish macrophages, they found that they were showing signs of being directly involved in the creation of the molecules that form part of the cardiac scar, and particularly collagen, which is the main protein involved. "This information is important and quite striking because up to today, only cardiac myofibroblasts have been implicated in directly forming a scar in the heart. By showing that macrophages produce collagen, a key part of scar tissue, this research could lead to new ways to enhance repair after a heart attack."

Macrophages directly contribute collagen to scar formation during zebrafish heart regeneration and mouse heart repair

Canonical roles for macrophages in mediating the fibrotic response after a heart attack include extracellular matrix turnover and activation of cardiac fibroblasts to initiate collagen deposition. Here we reveal that macrophages directly contribute collagen to the forming post-injury scar. Unbiased transcriptomics shows an upregulation of collagens in both zebrafish and mouse macrophages following heart injury. Adoptive transfer of macrophages, from either collagen-tagged zebrafish or adult mouse collagen donors, enhances scar formation via cell autonomous production of collagen.

In zebrafish, the majority of tagged collagen localises proximal to the injury, within the overlying epicardial region, suggesting a possible distinction between macrophage-deposited collagen and that predominantly laid-down by myofibroblasts. Macrophage-specific targeting of col4a3bpa and cognate col4a1 in zebrafish significantly reduces scarring in cryoinjured hosts. Our findings contrast with the current model of scarring, whereby collagen deposition is exclusively attributed to myofibroblasts, and implicate macrophages as direct contributors to fibrosis during heart repair.

More Data on the Tissue Specificity of Senescent Cell Accumulation with Age in Mice

As work progresses on the clinical development of senolytic therapies to selectively destroy harmful senescent cells in old tissues, it is becoming ever more necessary to have a better understanding of just how many senescent cells are present in any given tissue with age. Not all tissues acquire lingering populations of these cells at the same pace. Further, most current senolytic therapies are quite tissue specific, either because of the biodistribution characteristics of the drug, or because effectiveness varies in destroying senescent cells of different cell types.

Prioritization of development efforts requires some idea as to which tissues are more burdened by senescent cells, and thus more subject to the senescence-associated secretory phenotype in producing dysfunction and age-related disease, at least in the small molecule portion of the field. It is possible that Oisin Biotechnologies at least could just power through this challenge by saturating all tissues in the body with their non-toxic, highly selective suicide gene therapy vector. Brute force is sometimes an option.

Non-invasive ways of quantitatively assessing the presence of senescent cells in different tissues are also much needed, because we'd all like some idea as to how effective a given therapy might be. The dasatinib and quercetin senolytic combination is readily available, and you'll bump into people who have used it at longevity industry conferences, but few of those have undergone the biopsies that are presently the only viable way to make before and after comparisons of senescent cell burden. Better methods are on the horizon, such as the circulating microRNA approach under development at TAmiRNA, but they are not on the market yet. These tools will be needed to enable a more rational design of the next generation of senolytics, and they would certainly help in the clinical development of the present generation of senolytics.

Tissue specificity of senescent cell accumulation during physiologic and accelerated aging of mice

In this study, we provide a comprehensive measure of senescence in aged wild type (WT) mice. Senescence was quantified in multiple tissues, using numerous methods and numerous molecular endpoints, and we compared measures with that of young adult WT mice. We used this as a benchmark to determine whether Ercc1-/∆ mice, that exhibit accelerated aging, accumulate senescent cells in physiologically relevant tissues.

As measured by qRT-PCR and p16LUC signal, levels of p16Ink4a were significantly increased in aged WT mice compared with younger adult mice, as expected, p16Ink4a and p21Cip1 expression are found in peripheral T cells and numerous tissues (10 of 14 total tested) with the exception of heart and skeletal muscles. The differences in senescent cell burden in tissues could be reflective of different levels of genotoxic stress and/or different responses to that stress (e.g., selection of cell fate decisions: senescence or apoptosis). Near complete concordance was found between the expression of senescence markers in aged WT (2.5 years) and progeroid Ercc1-/∆ (4-5 month) mice, in terms of tissue specificity and expression levels.

The systemic burden of senescent cells was equivalent at the halfway point of lifespan in each of Ercc1-/∆ and WT mice, although the strains have vastly different lifespans. This supports the notion that senescent cell burden correlates with organismal health and may prove to be useful in predicting health span, or the remaining fraction of life that is disease-free. The data also support the conclusion that Ercc1-/∆ mice spontaneously develop senescent cells in the same tissues and at similar levels as WT mice, albeit more rapidly, supporting the notion that these animals represent a model of accelerated aging.

Two Examples of UK Biotech Startups Focused on the Treatment of Aging

The biotech startups of the core longevity industry, founded by the entrepreneurs who are regulations in the English-language conference circuit, are largely US companies. This is the way of the world in the biotech industry in general. The non-US participants include startups of British, Russian, and other origins, though it is often the case that when a company achieves some success it moves to where its backers and allies are found. For reasons relating to the historical interests of local research communities, and current interests of local venture funds, companies tend to be clustered in a few countries rather than being more evenly spread. The United Kingdom includes a number of research communities working on portions of aging relevant to therapeutic development. It is also the case that Juvenescence is based in the UK, and London is the center of the sizable advocacy and funding network associated with its principals.

In these articles, takes a brief look at a couple of early stage UK-based biotech startups that are focused on the treatment of aging. They are not the only ones; I am aware of a few other groups at earlier stages in their progression from academic laboratory to launching a startup. It isn't unusual that both of the startups noted here have an interest in senescent cells: the role of cellular senescence in driving degenerative aging is a very active area of development. It is also the basis for a market that is potentially so large that dozens of companies could find success here in the years ahead. The animal data for elimination of senescent cells as a basis for rejuvenation therapies is very compelling, and the human data obtained to date is promising.

Five Alarm Bio targets Alzheimer's and skin aging

Five Alarm Bio is founded by Dr William Bains, a scientist and entrepreneur with a 30-year track record in research into the fundamentals of biology and commercialisation of those discoveries. Dr Bains attended an anti-aging conference in November 2014, which is where he had his eureka moment that led to the creation of Five Alarm Bio. "While thinking why the speaker in a talk had got it all wrong, I had a flash of insight how the chemistry and the aging fitted, and the basic idea was born. It took another year to flesh out why this was a business, and we incorporated in 2016."

"We are in the research stage, about to embark on a major program of chemical synthesis to optimise our initial probe molecule. All the data so far has been seed-funded research in vitro. We have got good data to show that the probe molecule we are using a) is non-toxic on prolonged use, b) slows the rate at which cells age in vitro, and c) may reduce the damaging effects that senescent cells can have on the cells around them."

Dr Bains explains that all the work is done on primary human cells, which he feels is the best model for human cellular aging (other than people), but it is still in the petri dish, not in an animal. Five Alarm Bio's next steps are to secure broad patent coverage of its mechanism of action and on the optimum molecules to take forward. In terms of specific targets for the company's technology, its initial proof of concept experiments were on cell senescence, and so is looking at other targets where cell senescence is important, but Dr Bains is also keen to explore its potential benefit in Alzheimer's.

Biosens raising £5m to support commercialisation

The market for the development of anti-senescence therapeutics is on fire and we've spoken to a number of companies working in this field in recent months. The latest of these to come to our attention is Biosens, a British-German biotech start-up focused on the discovery and production of performance and longevity enhancing products. Founded in 2013, Biosens originally focused on agricultural applications, but refocused on human therapeutics early 2017 with the goal of making longer and healthier lives an attainable and affordable reality.

While still relatively early stage, the company already has three therapeutic products in its pipeline, including therapies for cell rejuvenation, muscle regeneration and cognitive improvement. "Aging is characterized by accumulated damage in stem cells and somatic cells causing their senescence as well as pathogenic factors in blood causing chronic inflammation. Reactive oxygen species accumulation, DNA damage, epigenetic alterations, protein aggregation, and telomere shortening are major causes of cell senescence. Our lead candidate works by rejuvenating the stem cell pool while simultaneously removing damaging senescent cells."

Evidence for the Epigenetic Clock to Underestimate Age in Later Life

In recent years, the research community has put in ever more effort into the development of epigenetic clocks capable of assessing biological age. This focus has led to the discovery of various issues, as the nature of epigenetics of aging is further explored, challenges that will need to be understood and addressed in order to enable the practical use of epigenetic clocks. For example that degree of fitness doesn't appear to much affect epigenetic age, which is problematic to say the least, as we know that it affects the progression of aging. Here, researchers identify a systemic issue with assessment of epigenetic age in older individuals.

Subject age is a piece of data available in almost every study in which DNA methylation profiles are obtained. There is thus a huge amount of cross-sectional data in which it can be seen that the methylation level of many CpG sites varies with subject age, which, amongst other processes, could reflect developmental changes, cellular aging, cumulative environmental effects, and changes in cell-type composition.

Horvath used a large collection of publicly available DNA methylation data on multiple tissue types to train and test a model for age prediction from 353 CpG loci. This "epigenetic clock" continues to be widely used and is extremely valuable as a way of estimating ages of samples from unknown donors and possibly as an indicator of whether there are alterations in the aging rate of certain tissues or individuals. Although the epigenetic clock provides an estimate of age, the testing data used in generating this clock did not have a large representation of tissue from elderly individuals and as such it is unclear if the clock is accurate in older age groups, or those with age-related diseases.

The model systematically underestimates age in tissues from older people. This is seen in all examined tissues but most strongly in the cerebellum and is consistently observed in multiple datasets. Epigenetic age acceleration is thus age-dependent, and this can lead to spurious associations. The current literature includes examples of association tests with age acceleration calculated in a wide variety of ways. In conclusion, the concept of an epigenetic clock is compelling, but caution should be taken in interpreting associations with age acceleration. Association tests of age acceleration should include age as a covariate.

A Discussion of Recent Work on Allotopic Expression of Mitochondrial Genes at the SENS Research Foundation

A paper published last month outlines recent progress on allotopic expression of mitochondrial genes carried out by the SENS Research Foundation team. Allotopic expression is the name given to the process of putting copies of mitochondrial genes into the nuclear genome, suitably altered to allow proteins to be generated and shipped back to the mitochondria where they are needed. Mitochondria replicate like bacteria, and some forms of stochastic mitochondrial DNA damage can make mitochondria both dysfunctional and able to outcompete their undamaged peers. This is thought to be an important contribution to aging, resulting a small but damaging population of cells that are overtaken by broken mitochondria and which export harmful reactive molecules into the surrounding tissues.

Having a backup supply of mitochondrial proteins can in principle block these consequences of mitochondrial DNA damage, and thus remove this contribution to the aging process. Proof of concept has been demonstrated for a few of the thirteen proteins needed, and work proceeds on the rest. As noted here, one of the challenges in this project is that mitochondrial genetic machinery is of a different evolutionary origin to that of the cell nucleus, and thus the efficient production of equivalent proteins from nuclear genes is a much more challenging process than would otherwise be the case.

While the vast majority of mitochondrial proteins are encoded by the nuclear genome, translated in the cytosol, and imported into the mitochondrion, 13 core subunits of respiratory complexes are encoded by the reduced mitochondrial genome and synthesized within the mitochondrial matrix. Mutations in these 13 genes (or their associated non-protein-coding genes) tend to be especially severe, as all 13 proteins are core subunits of the oxidative phosphorylation chain, and any disruption to subunit structure, stability, or function may have grave biochemical and physiological consequences. Gene therapy to target affected mitochondrial subunits is a promising alternative strategy which circumvents some of the technical challenges faced by the above approaches. One issue that remains, however, relates to the prokaryotic origin of the organelle. Translation within the mitochondrion deviates from the universal genetic code, utilizing machinery and codon frequencies more similar to its α-proteobacterial ancestry than to the mammalian nuclear genome.

Subsequently, allotopic expression has been suggested as a therapeutic tool to genetically remedy deleterious mitochondrial DNA mutations through nuclear complementation of the affected genes. A critical, but often-overlooked consideration in these nuclear relocation studies is the influence of the primary coding sequence on protein production. The vast majority of these previous studies have utilized what may be considered "minimally-recoded" mitochondrial genes. While making these codon changes is essential to maintain amino acid sequence integrity during cytosolic translation, this minimal approach fails to account for other elements of primary sequence which can critically influence both gene and protein expression.

Many commercial algorithms have therefore been developed to determine the optimal sequence and conditions for expression of a gene from a particular host. Though there are concerns regarding the use of codon optimization to increase homologous expression of a nuclear gene, such as the generation of novel or immunogenic peptides or structural perturbations in the encoded protein, codon optimization continues to be widely utilized for the production of biotherapeutics. Applying this principle to allotopic expression, we hypothesize that, given the bacterial origin of the mitochondrial genome, the coding sequences of minimally-recoded mitochondrial genes are dissimilar from nuclear genes and are inefficiently translated by nuclear machinery, therefore resulting in poor allotopic expression.

Here we employed codon optimization as a tool to re-engineer the protein-coding genes of the human mitochondrial genome for robust, efficient expression from the nucleus. All 13 codon-optimized constructs exhibited substantially higher protein expression than minimally-recoded genes when expressed transiently, and steady-state mRNA levels for optimized gene constructs were 5-180 fold enriched over recoded versions in stably-selected wildtype cells. Eight of thirteen mitochondria-encoded oxidative phosphorylation proteins were observed to maintain protein expression following their stable selection, with mitochondrial localization of expression products. We also assessed the utility of this strategy in rescuing mitochondrial disease cell models and found the rescue capacity of allotopic expression constructs to be gene specific. Allotopic expression of codon optimized ATP8 in disease models could restore protein levels and respiratory function, however, rescue of the pathogenic phenotype for another gene, ND1, was only partially successful. These results imply that though codon-optimization alone is not sufficient for functional allotopic expression of most mitochondrial genes, it is an essential consideration in their design.

Mechanisms of Slowed Muscle Aging via Calorie Restriction in Rhesus Macaques

Many of the readers here will be familiar with the very long-running studies of calorie restriction in rhesus macaques. There was some discussion of the data a few years ago. The research has continued since then, and here researchers report on their investigation of the biochemistry of calorie restriction in connection to the slowed aging of muscle tissue observed in these animals. Calorie restriction produces sweeping changes in the operation of cellular metabolism, and aging is itself a very complex process, even though it stems from simpler root causes. Research into the tissue-specific details of how and why calorie restriction slows specific aspects of aging is thus a slow and complex undertaking.

Our studies of aging in rhesus monkey and calorie restriction (CR) include a comprehensive investigation of age-related change including physical parameters. Similar to humans, muscle mass loss begins in middle age in monkeys at ∼15 years of age, where age-related loss of quadricep bulk from ∼15 to +25 years of age is ∼23% for females and 27% for males. Vastus lateralis (VL) is one of the four constituent muscle groups within the quadriceps, and it is the one that is the most vulnerable to aging, with 40% lower tissue weight for old monkeys (∼30 years of age) at necropsy compared with young adults of full stature (∼8 years of age). VL comprises both oxidative and glycolytic fiber types and succumbs to fiber atrophy and increased fibrosis with age in humans and monkeys. Responses to aging are of a fiber-type-specific nature: slow twitch type I fibers are resistant to age-related atrophy, but fast twitch type II fibers exhibit a gradual decline in cross-sectional area beginning at middle age.

Defects in skeletal muscle energy metabolism with age have been documented in humans, rats, and mice. In humans, skeletal muscle mitochondrial activity declines with age. In rhesus monkeys, age-related changes in mitochondrial and redox metabolism occur in advance of the onset of muscle mass loss and before age-related declines in physical activity are detected, suggesting that metabolism could play a causal role in skeletal muscle aging. A separate study has demonstrated that energy metabolism pathways are uniformly but modestly induced with CR in mice, and pathway level analysis confirmed the same in rhesus monkey skeletal muscle.

To test this, we investigated the molecular and cellular phenotypes of delayed sarcopenia due to CR in rhesus monkeys and related these data to tissue, biometric, and functional outcomes. We show that CR induced profound changes in muscle composition and the cellular metabolic environment. Bioinformatic analysis linked these adaptations to proteostasis, RNA processing, and lipid synthetic pathways. At the tissue level, CR maintained contractile content and attenuated age-related metabolic shifts among individual fiber types with higher mitochondrial activity, altered redox metabolism, and smaller lipid droplet size. Biometric and metabolic rate data confirm preserved metabolic efficiency in CR animals that correlated with the attenuation of age-related muscle mass and physical activity. These data suggest that CR-induced reprogramming of metabolism plays a role in delayed aging of skeletal muscle in rhesus monkeys.

Modulating Macrophage Polarization as a Therapy for Atherosclerosis

Macrophages are the cells responsible for removing cholesterols from blood vessel walls, to prevent the formation of fatty lesions. Unfortunately they become dysfunctional and inflammatory with age, as a result of rising levels of oxidized cholesterol. This leads to atherosclerosis, an ultimately fatal condition in which lesions grow to the point of weakening and narrowing blood vessels. This condition is strongly affected by inflammation, as macrophages can adopt different behavioral types, known as polarizations, in response to circumstances. Greater inflammatory signaling will drive more macrophages to adopt the aggressive M1 phenotype, focused on destroying pathogens, rather than the regenerative M2 phenotype that is more useful in removing cholesterol from blood vessel walls.

A number of groups are working on ways to force macrophages to adopt a specific phenotype, overriding their usual reaction to surrounding circumstances. In the research here, an approach is demonstrated to be beneficial in a mouse model of atherosclerosis, presumably by putting more macrophages back to work in lesions, clearing out lipids rather than flailing and adding to the inflammatory environment.

Atherosclerosis-related cardiovascular disease is still the predominant cause of death worldwide. Araloside C (AsC), a natural saponin, exerts extensive anti-inflammatory properties. In this study, we explored the protective effects and mechanism of AsC on macrophage polarization in atherosclerosis in vivo and in vitro. Using a high-fat diet (HFD)-fed ApoE-/- mouse model and RAW 264.7 macrophages exposed to oxidized LDL, AsC was evaluated for its effects on polarization and autophagy.

AsC significantly reduced the plaque area in atherosclerotic mice and lipid accumulation in oxidized-LDL-treated macrophages, promoted M2 phenotype macrophage polarization, increased the number of autophagosomes and modulated the expression of autophagy-related proteins. Moreover, the autophagy inhibitor 3-methyladenine and BECN1 siRNA obviously abolished the antiatherosclerotic and M2 macrophage polarization effects of AsC. Mechanistically, AsC targeted Sirt1 and increased its expression, and this increase in expression was associated with increased autophagy and M2 phenotype polarization. Altogether, AsC attenuates foam cell formation and lessens atherosclerosis by modulating macrophage polarization via Sirt1-mediated autophagy.

An Example of Epigenetic Effects on Offspring Longevity

It was discovered only comparatively recently that epigenetic alterations, decorations attached to the genome rather than changes to the genome itself, can produce changes in offspring longevity. Not all epigenetic changes are erased during early embryonic development; some are retained and go on to influence development and metabolism throughout life. This is a mechanism by which species can improve their reproductive fitness via producing offspring better suited to the environment experienced by the parents. One of the best examples is that calorie restriction affects the metabolism and longevity of the offspring of animals, not just the calorie restricted parents. The research here is an example of ongoing investigations into this aspect of epigenetic regulation, focused on a single epigenetic mark that is shown to produce greater longevity in parents and offspring.

It is commonly accepted that genetic sequences coded within DNA are passed down through generations and can influence characteristics such as appearance, behavior, and health. However, emerging evidence suggests that some traits can also be inherited 'epigenetically' from information that is independent of the DNA sequence. One of the ways characteristics may be epigenetically passed down is through the temporary modification of histone proteins which help to package DNA into the cell. Histones are adorned with chemical marks that can regulate how and when a gene is expressed by changing how tightly the DNA is wrapped. These marks are typically removed before genetic information is passed on to the next generation, but some sites escape erasure.

It has previously been reported that genetic mutations in an enzyme complex called COMPASS increase the lifespan of tiny worms called Caenorhabditis elegans. This complex acts on histones and creates a chemical mark called H3K4me, which is typically associated with less compact DNA and higher gene expression. When these mutants mate with wild-type worms they generate descendants that no longer have COMPASS mutations. Although these wild-type offspring recover normal levels of H3K4me, they still inherit the long-lived phenotype which they sustain for several generations.

Previous work showed that one of the COMPASS complex mutants, known as wdr-5, has increased levels of another histone mark called H3K9me2. This epigenetic mark generally promotes DNA compaction and appears to antagonize the action of H3K4me. Researchers found that homozygous wdr-5 mutants, which had descended from ancestors carrying one copy of the mutated wdr-5 gene and one wild-type copy for multiple generations, did not live for longer than their non-mutant counterparts. This indicates that the mutation carried by wdr-5 worms did not immediately cause a lifespan change. However, future generations of worms that maintained the homozygous wdr-5 mutation had an increasingly longer lifespan, suggesting that the accumulation of an epigenetic signal across generations promotes longer living. These late generation wdr-5 mutants had higher levels of H3K9me2, and they were able to pass on this extended longevity to their progeny following mating with wild-type worms as previously reported.

A Mechanism by which Chronic Inflammation Spurs Cancer Metastasis

Chronic inflammation is a risk factor for cancer and cancer mortality. There are numerous reasons as to why this might be the case, some much more proven and settled than others, but the research here is focused on metastasis, the spread of cancerous cells throughout the body. Since cancer mortality is largely determined by whether or not a tumor progresses to the point of metastasis, we should not be surprised that researchers can identify mechanisms linking inflammation with metastasis.

Dysregulated inflammation is recognized as one of the hallmarks of cancer and is involved in tumor initiation, progression, and metastasis. Chronic inflammatory conditions, such as chronic obstructive pulmonary disease or ulcerative colitis, are strongly associated with elevated cancer incidence. Chronic use of aspirin or other non-steroidal anti-inflammatory drugs reduces mortality of esophageal, colorectal, and lung cancers.

Thus chronic inflammation facilitates tumor progression. We discovered that a subset of non-small cell lung cancer cells underwent a gradually progressing epithelial-to-mesenchymal (EMT) phenotype following a 21-day exposure to IL-1β, an abundant proinflammatory cytokine in individuals at-risk for lung cancer, and in the lung tumor microenvironments. Pathway analysis of the gene expression profile and in vitro functional studies revealed that the EMT and EMT-associated phenotypes, including enhanced cell invasion, PD-L1 upregulation, and chemoresistance, were sustained in the absence of continuous IL-1β exposure. We referred to this phenomenon as EMT memory.

Utilizing a doxycycline-controlled SLUG expression system, we found that high expression of the transcription factor SLUG was indispensable for the establishment of EMT memory. High SLUG expression in tumors of lung cancer patients was associated with poor survival. Chemical or genetic inhibition of SLUG upregulation prevented EMT following the acute IL-1β exposure but did not reverse EMT memory.

Although it is well known that EMT endows cells with metastatic capacity, analysis of tissue specimens from metastatic tumors often reveals cells with epithelial features. EMT plasticity therefore is proposed to temporally modify these properties by facilitating cellular responses to the microenvironmental stimuli that lead to mesenchymal phenotypes and metastatic behaviors. In the current study, fading of EMT memory, accompanied by a gradual elevation of E-cadherin expression, is consistent with a profound EMT plasticity. In a case of acquired EMT, increased migration and invasion of tumor cells enable them to travel away from primary tumor sites, which also distance them from EMT-promoting stimuli, such as inflammatory factors in the primary TME. We propose that because of the memorized EMT phenotypes, these migratory cells are able to seed the metastatic spread to distant organ sites.

Long Non-Coding RNAs and Macrophage Senescence in Age-Related Disease

Here, researchers review cellular senescence in macrophage cells and the biochemistry of long non-coding RNAs in macrophage senescence, a topic of great relevance to a number of age-related conditions, such as atherosclerosis. Cellular senescence takes place in most cell populations, in response to reaching the Hayflick limit on replication or in response to stress and damage. Senescent cells have important short-term roles to play, and near all destroy themselves or are destroyed by immune cells soon after entering the senescent state. These cells become harmful when they linger over long term, however, even in comparatively small numbers. They secrete a mix of signals, the senescence-associated secretory phenotype, that encourages other cells to become senescent, rouses the immune system to chronic inflammation, destructively remodels surrounding tissues, and more. The accumulation of senescent cells is one of the driving causes of degenerative aging.

Cellular senescence is a particularly stable state of permanent cell cycle arrest. Macrophages, although terminally differentiated cells, do not undergo this type of replicative senescence and may hence undergo stress-induced senescence. In healthy conditions, macrophages maintain homeostasis; however, in pathological states, different stresses including DNA damage, telomere shortening, oncogene activation, impairment of some key proteins, and infections activate the p53, AIM2, and NF-κB signal pathways, initiating macrophage senescence.

When these damage-associated molecule patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) are highly intensive or temporally irreversible, the balance between the production and clearance of proinflammatory factors is disrupted. At later stages of macrophage senescence, the net effect is senescence-associated secretory phenotype (SASP) expression. These factors not only aggravate macrophage senescence but are also extracellularly released, thus impairing the functions of surrounding cells. This process is called "paracrine senescence" and causes a wider range of inflammaging. With steady accumulation of senescent cells, senescence eventually occurs at the cellular level and then at the organ level, causing organ malfunction, consequently resulting in corresponding aging phenotypes.

Emerging data suggest that long non-coding RNA (lncRNA) plays a key role in regulating inflammatory responses. Alterations in various lncRNA expression levels are associated with a proinflammatory phenotype in various age-related diseases (ARDs). This leads to modification of cellular senescence through several diverse approaches, whether by mediating gene expression or protein function or functioning as competing endogenous RNA (ceRNA). Changes in lncRNAs in ARDs and the corresponding consequences have been widely studied, especially in cancer. However, the association between lncRNA and cellular senescence in ARDs remains an interesting and complex issue.

Atherosclerosis is a chronic inflammatory disease. Macrophages have been recently reported to display marked inflammatory plasticity, particularly polarization. They perpetuate chronic inflammation and growth of atherosclerotic plaques, thus being central to the initiation, growth, and ultimately the rupture of arterial plaques. Studies on atherosclerosis and macrophages have reported that lncRNAs majorly function as ceRNA in causing atherosclerosis. By sequestering microRNAs, MITA, GAS5, HOTAIR, and UCA1 promote M1 polarization, inducing proinflammatory cytokine, matrix metalloproteinase, and reactive oxygen species (ROS) levels. Atherosclerosis contributes to various lesions, especially cardiovascular disease. Current evidence suggests that the effect of lncRNAs on macrophages in coronary artery disease is the same as that on atherosclerosis, highlighting the consistency of its function and prompting its potential as a therapeutic target.

Endothelial Cell Senescence can Impair Insulin Sensitivity

The growing presence of senescent cells contributes to near all of the declines and tissue dysfunctions of aging, judging by the results produced in extensive research carried out in animal models of age-related disease. Senescent cells secrete a mix of inflammatory and other signals that, when present for the long term, cause considerable harm to tissue structure, function, and maintenance. The research here is focused on just one form of dysfunction, but is illustrative of many other studies in the field of senescence carried out in recent years.

Endothelial cells (ECs) line the inner surface of blood vessels, and plays an essential role in vascular biology, such as vasodilation, hormone trafficking, and neovessel formation. Moreover, EC produces many secreted angiocrine factors that are crucially involved in maintaining tissue homeostasis. Aging causes cellular senescence in various types of cells including EC, and cellular senescence plays an important role in age-related organ dysfunction.

Senescent cells produce senescence-messaging secretomes that have deleterious effects on the tissue microenvironment, referred as the senescence-associated secretory phenotype (SASP); therefore, cellular senescence is considered to be a primary cause for age-related diseases, such as diabetes, stroke, and heart attack. Because of the crucial roles of EC in tissue homeostasis, EC senescence is presumed to play significant roles in age-related organ dysfunction; however, whether and the mechanism by which EC senescence causes age-related diseases remained unknown.

Here we show that EC senescence induces metabolic disorders through the SASP. Senescence-messaging secretomes from senescent ECs induced a senescence-like state and reduced insulin receptor substrate-1 in adipocytes, which thereby impaired insulin signaling. We generated EC-specific progeroid mice. This EC-specific progeria impairment of systemic metabolic health in mice in association with adipose tissue dysfunction. Notably, shared circulation with EC-specific progeroid mice by parabiosis sufficiently transmitted the metabolic disorders into wild-type recipient mice. Our data provides direct evidence that EC senescence impairs systemic metabolic health, and thus establishes EC senescence as a bona fide risk for age-related metabolic disease.

Reviewing Efforts to Develop NAD+ Therapies to Reverse Age-Related Loss of Mitochondrial Function

Increasing levels of NAD+ in mitochondria, is a class of therapy that probably produces most of its benefits in animal models and human trials by restoring mitophagy. This may well be true of mitochondrially targeted antioxidants as well. Mitophagy removes damaged mitochondria, but is hampered by age-related changes in mitochondrial dynamics, among other reasons. Mitochondria are responsible for packaging chemical energy store molecules to power cellular operations. Mitochondrial function is critical to tissue function throughout the body, but is of particular note in the energy-hungry tissues of muscle and brain.

NAD+ declines with aging for causes that are not well understood, not well linked to the underlying molecular damage that causes aging. Methods of increasing NAD+ are operating on proximate causes at best. They can reverse some degree of the decline, as demonstrated in human trials focused on the function of smooth muscle in major blood vessels. Not all of these trials produced benefits, however, and in those that did, NAD+ upregulation so far doesn't achieve more than "some degree" of improvement. Thus assessment of the field of prospective NAD+ interventions is still very much an ongoing project.

Over the last decade, the importance of NAD+ in healthy ageing and longevity has been recognised, detailed molecular mechanisms unveiled, and many clinical trials explored. Studies from laboratory animals, such as in nematodes and mice, and in human primary cells and post-mortem tissues, as well as human brain imaging, indicate that there is an age-dependent reduction of NAD+ in cells and tissues. Mechanistically, it is suggested that ageing-induced NAD+ reduction may result from reduced production - as there is an age-dependent reduction of key enzymes involved in NAD+ metabolism - or increased consumption by NAD+-consuming enzymes, such as PARPs, CD38, and Sirtuins. All three classes of enzymes compete for NAD+ during ageing, ultimately leading to a bioavailability level insufficient to sustain all NAD+-requiring cellular activities.

Intriguingly, NAD+ repletion, by the supplementation of NAD+ precursors, such as nicotinamide riboside (NR), nicotinamide mononucleotide (NMN), nicotinamide (NAM), or even NAD+ itself, delay ageing phenotypes and promote healthy longevity in both normal and accelerated ageing models in Caenorhabditis elegans (roundworms), Drosophila melanogaster (fruit flies), and mice. Encouraged by animal studies, more than 20 clinical studies exploring whether NR may alleviate pathological ageing and age-predisposed diseases have been initiated.

At least 5 clinical trials have been completed showing that 1-2 g/day of NR for up to 1-3 months is safe. While there were encouraging results in some NR-based phase I clinical trials aiming to reduce blood pressure in healthy middle-aged and older adults and to slow disease progression in amyotrophic lateral sclerosis (ALS) (NR + pterostilbene), no effect was reported in trials of short-term (up to 2-3 months) NR supplementation in obese, insulin-resistant men and nondiabetic males with obesity, nor muscle-mitochondrial bioenergetics in aged men. Of note, all three reports were from the same study and reported different outcomes from the same set of obese men. Possible considerations include a much higher dose of NR (2 g/day) than other trials (mostly 1 g/day) and the sensitivity of the enzymatic, assay-based NAD+ detection method. Thus, these studies emphasise some of the challenges with clinical trials of NAD+-boosting compounds with regards to dose and assessment of NAD+ bioavailability.

Exceptionally Long Lived Humans Exhibit Slower Epigenetic Aging, Measured by DNA Methlyation Clocks

Epigenetic clocks are produced by examining age-related changes in DNA methylation, finding combinations of such changes that are consistent across populations, and predict chronological age. These clocks also predict mortality, in the sense that people with higher epigenetic than chronological age tend to have a higher mortality risk, or be more burdened by chronic age-related disease. The challenge here is that it remains very unclear as to what these epigenetic clocks are actually measuring, which of the underlying processes of aging they reflect, and to what degree. That makes it hard to use epigenetic clocks in any meaningful way - the results are not actionable.

There are other issues to be debugged as well. For example, that the first generation epigenetic clocks are unaffected by fitness differences, or that they appear to systemically underestimate age in older individuals. Given that second point, when looking at the results in the paper here, in which slower epigenetic aging is claimed for a cohort of exceptionally long lived individuals, we are left somewhat in the dark regarding the relevance of the data. These and other issues are not insurmountable problems, but they are standing in the way of broader application of epigenetic measures of biological aging.

Many studies are aimed at biomarker discovery and improvement for aging. The need for such characterization is of upmost importance in light of efforts to achieve longer health and lifespans across the world. Such biomarker detection would enable tracking and even reversal of aging processes and allow for drug targeting and development to benefit the already graying population. Molecular and genomic biomarkers for aging are still sparse and inaccurate with the exception of the very recent development of DNAmGrimAge. This DNA methylation biomarker outperforms all previously reported methylation age estimators and serves as a very accurate estimate of chronological age. Although this is expected due to the use of chronological as a surrogate for the age prediction, DNAmGrimAge also serves as an evaluation of health status, indicative of the rate of epigenetic aging. Use of such biomarkers as indication of rate of age acceleration could promote better understanding of the processes underlying progression of aging and replace use of chronological age in clinical assessments relating to those conditions.

We show here that, although accurate in offspring of exceptionally long lived individuals (ELLI) and unrelated controls, DNAmGrimAge underestimates the chronological age of our ELLI participants, predicting a younger epigenetic age. We believe that this represents a slower rate of aging processes occurring in ELLI, enabling them to reach such exceptional chronological age. This is in agreement with the methylation profile of semi-supercentenarians and their offspring, and replicates earlier results in our independent cohort.

Further, the DNA methylation based estimator of telomere length, DNAmTL, showed no correlation with qPCR measurement of telomere length, until adjusted by DNAmGrimAge. This masking effect of the physiological age (measured by DNAmGrimAge) adds support to the slower rate of aging. Telomere length has long been argued for and against use as an age indicator, but it is well-established to be decreased with age. Our qPCR measurements are consistent with previous observations of longer telomeres in ELLI. While telomere length of ELLI was expected to shorten in respect to offspring and controls because of their relatively advanced age, it remained unchanged, indicating a similar telomere length despite almost 30 years average age difference between group participants, demonstrating once again, a decreased aging rate. Taken together with the juvenile methylation rates in ELLI, we suggest that ELLI age slower than the general population through a beneficial methylation profile that may affect telomere length and other aspects of the hallmarks of aging.


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