Skip to main content

Rewinding the Clock

Treatment restores blood vessel growth, muscle vitality, boosts exercise endurance in aging animals  

By EKATERINA PESHEVA March 22, 2018 Research

We are as old as our arteries, the adage goes, so could reversing the aging of blood vessels hold the key to restoring youthful vitality?

The answer appears to be yes, at least in mice, according to a new study led by investigators at Harvard Medical School.

The research, published March 22 in Cell, identifies the key cellular mechanisms behind vascular aging and its effects on muscle health and has successfully reversed the process in animals.

The findings pinpoint a glitch in the normal crosstalk that occurs between muscles and blood vessels and keeps both tissues healthy.

Using the synthetic precursors of two molecules naturally present in the body, the scientists also managed to reverse blood vessel demise and muscle atrophy in aging mice, boosting their exercise endurance in the process.

The achievement, the team said, paves the way to identifying related therapies for humans.

“We’ve discovered a way to reverse vascular aging by boosting the presence of naturally occurring molecules in the body that augment the physiological response to exercise,” said study senior investigator David Sinclair, professor in the Department of Genetics at Harvard Medical School and co-director of the Paul F. Glenn Center for the Biology of Aging at Harvard Medical School.

“The approach stimulates blood vessel growth and boosts stamina and endurance in mice and sets the stage for therapies in humans to address the spectrum of diseases that arise from vascular aging,” added Sinclair, who is also a professor at the University of New South Wales School of Medical Sciences in Sydney, Australia.

The researchers caution that many promising treatments in mice don’t have the same effect in humans due to critical differences in biology. However, the results of the experiments were dramatic enough to prompt the research team to pursue experiments in humans. Clinical trials for safety are already under way, Sinclair said.

As old as our blood vessels

Sinclair and team set out to unravel the mechanisms behind one of biology’s inevitabilities: aging.

As we grow old, we become weak and frail. A constellation of physiological changes—some subtle, some dramatic—precipitate this inevitable decline. What exactly happens inside our cells to cause the biological shifts that lead to aging? It’s a question that has vexed Sinclair and team for years.

As we age, our tiniest blood vessels wither and die, causing reduced blood flow and compromised oxygenation of organs and tissues. Vascular aging is responsible for a constellation of disorders, such as cardiac and neurologic conditions, muscle loss, impaired wound healing and overall frailty, among others. Scientists have known that loss of blood flow to organs and tissues leads to the build-up of toxins and low oxygen levels. The so-called endothelial cells, which line blood vessels, are essential for the health and growth of blood vessels that supply oxygen-rich and nutrient-loaded blood to organs and tissues. But as these endothelial cells age, blood vessels atrophy, new blood vessels fail to form and blood flow to most parts of the body gradually diminishes. This dynamic is particularly striking in muscles, which are heavily vascularized and rely on robust blood supply to function.

Muscles begin to shrivel and grow weaker with age, a condition known as sarcopenia. The process can be slowed down with regular exercise, but gradually even exercise becomes less effective at holding off this weakening.

Sinclair and team wondered: What precisely curtails the blood flow and precipitates this unavoidable decline? Why does even exercise lose its protective power to sustain muscle vitality? Is this process reversible?

In a series of experiments, the team found that reduced blood flow develops as endothelial cells start to lose a critical protein known as sirtuin1, or SIRT1. Previous studies have shown that SIRT1 delays aging and extends life in yeast and mice.

SIRT1 loss is, in turn, precipitated by the loss of NAD+, a key regulator of protein interactions and DNA repair that was identified more than a century ago. Previous research by Sinclair and others has shown that NAD+, which also declines with age, boosts the activity of SIRT1.

A stimulating conversation

The study reveals that NAD+ and SIRT1 provide a critical interface that enables the conversation between endothelial cells in the walls of blood vessels and muscle cells.

Specifically, the experiments reveal that in young mouse muscle, SIRT1 signaling is activated and generates new capillaries, the tiniest blood vessels in the body that supply oxygen and nutrients to tissues and organs. However, as NAD+/SIRT1 activity diminishes over time, the study found, so does the blood flow, leaving muscle tissue nutrient-deprived and oxygen-starved.

Indeed, when researchers deleted SIRT1 in the endothelial cells of young mice, they observed markedly diminished capillary density and decreased number of capillaries, compared with mice that had intact SIRT1. Mice whose endothelial cells lacked SIRT1 had poor exercise tolerance, managing to run only half the distance covered by their SIRT1-intact peers. 

To determine SIRT1’s role in exercise-induced blood vessel growth, the researchers observed how SIRT1-deficient mice responded to exercise. After a month-long training regimen, the hind-leg muscles of SIRT1-deficient mice showed markedly diminished ability to form new blood vessels in response to exercise compared with same-age mice that had intact SIRT1 in their endothelial cells.

Exercise-induced blood vessel formation is known to occur in response to growth-stimulating proteins released by muscles under strain. SIRT1, however, appears to be the key messenger relaying growth-factor signaling from muscles to blood vessels, the study found. 

Experiments showed that endothelial cells lacking SIRT1 were desensitized to the growth-stimulating proteins released by exercised muscles.

“It’s as if these cells had grown deaf to the signals that muscles sent their way,” Sinclair said.

The observation, he added, explains why age-related loss of SIRT1 leads to muscle atrophy and blood vessel demise.

Since the experiments revealed the critical role of SIRT1 in exercise-induced blood vessel formation, the researchers wondered whether boosting SIRT1 levels would stimulate blood vessel growth and stave off muscle wasting.

Exercise in a pill?

The scientists set their sights on NAD+, a molecule conserved across many life forms, known to decline with age and previously shown to stimulate SIRT1 activity.

“We reasoned that declining NAD+ levels reduce SIRT1 activity and thus interfere with aging mice’s ability to grow new blood vessels,” said study first author Abhirup Das, who conducted the work as a post-doctoral fellow in Sinclair’s lab, currently a visiting scholar in genetics at Harvard Medical School and a post-doctoral research fellow at the University of South New Wales School of Medical Sciences.

To test this premise, scientists used a chemical compound called NMN, a NAD+ precursor, previously shown to play a role in repairing cellular DNA and maintaining cell vitality.

In lab dish experiments, endothelial cells from humans and mice treated with NMN showed enhanced growth capacity and reduced cell death.

Next, the team gave NMN over two months to a group of mice that were 20 months old—the rough equivalent of 70 in human years. NMN treatment restored the number of blood capillaries and capillary density to those seen in younger mice. Blood flow to the muscles also increased and was significantly higher than blood supply to the muscles seen in same-age mice that didn’t receive NMN.

The most striking effect, however, emerged in the aging mice’s ability to exercise. These animals showed between 56 and 80 percent greater exercise capacity, compared with untreated mice the study showed. The NMN-treated animals managed to run 430 meters, or about 1,400 feet, on average, compared with 240 meters, or 780 feet, on average, for their untreated peers.

To see whether the effects of NMN could be further augmented, the researchers added a second compound to the treatment regimen. The compound, sodium hydrosulfide (NaHS), is a precursor to hydrogen sulfide, which also boosts the activity of SIRT1.

A group of 32-month-old mice—the rough equivalent to 90 in human years—receiving the combo treatment for four weeks were able to run, on average, twice as long as untreated mice. In comparison, mice treated with NMN alone ran 1.6 times farther, on average, than untreated animals.

“These are really old mice so our finding that the combo treatment doubles their running capacity is nothing short of intriguing,” said study co-author James Mitchell, associate professor of genetics and complex diseases at the Harvard T. H. Chan School of Public Health.  Research led by Mitchell and published in the same issue of Cell also found sodium hydrosulfide to augment blood vessel formation in the muscles of mice.

Interestingly, the NMN treatment did not improve blood vessel density and exercise capacity in young sedentary mice. However, it did boost blood vessel formation and exercise capacity in young mice that had been exercising regularly for a month.

“This observation underscores the notion that age plays a critical role in the crosstalk between blood vessels and muscles and points to a loss of NAD+ and SIRT1 as the reason behind loss of exercise effectiveness after middle age,” Das said.

The researchers say their findings may pave the way to therapeutic advances that hold promise for the millions of older people for whom regular physical activity is not an option.

“Even if you’re an athlete, you eventually decline,” Sinclair said. “But there is another category of people—what about those who are in a wheelchair or those with otherwise reduced mobility?”

The team’s ultimate goal is to replicate the findings and, eventually, move toward developing small-molecule, NMN-based drugs that mimic the effects of exercise—enhanced blood flow and oxygenation of muscles and other tissues. Such therapies may even help with new vessel growth of organs that suffer tissue-damaging loss of blood supply and oxygen, a common scenario in heart attacks and ischemic strokes, the team said.

Neo-vascularization—the formation of new blood vessels—should be treated with caution, the researchers say, because increased blood supply could inadvertently fuel tumor growth.

“The last thing you want to do is provide extra blood and nourishment to a tumor if you already have one,” said study co-author Lindsay Wu, at the University of New South Wales School of Medical Sciences.

Sinclair and Wu point out that experiments done as part of the current study provide no evidence that treatment with NMN stimulated tumor development in animals treated with the compound.

Co-investigators included George Huang, Michael Bonkowski, Alban Longchamp, Catherine Li, Michael Schultz, Lynn-Jee Kim, Brenna Osborne, Sanket Joshi, Yuancheng Lu, Jose Humberto, Trevine-Villareal, Myung-Jin Kang, Tzong-tyng Hung, Brendan Lee, Eric Williams, Masaki Igarashi, James Mitchell, Nigel Turner, Zolt Arany and Leonard Guarente.

The work was supported by the Glenn Foundation for Medical Research (grants RO1 AG028730 and RO1 DK100263), and by the National Institutes of Health/National Heart, Lung and Blood Institute (grant RO1 HL094499).


Intravenous Glutathione Enhances Cycling Time Trial Performance

By William Misner Ph.D.


A healthy, fit 61-year old male endurance athlete received four (4 each) intravenous glutathione treatments consisting of 100 mg/cc, using 10cc (total 1000 milligrams) diluted total of 20cc normal saline solution slow IV-push delivery (15 minutes). The tests were recorded 36-days during a previously established aerobic cycling fitness on a common course. There were no changes in training or dietary protocols other than the administration of glutathione intravenously. The subject’s pre-treatment timed-trial was 57:30 (minutes:seconds). Performance improved progressively following each of 4 separate intravenous treatments to a peak performance of 52:21(minutes:seconds), representing a remarkable performance gain of 7.2%. The only variable to account for this performance gain was the intravenous glutathione treatments.


Whether antioxidant supplementation will enhance performance remains controversial. Exercise-induced changes in antioxidant scavengers and associated enzymes (e.g., glutathione, tocopherol, glutathione peroxidase) provide clues about demand imposed on the defense system. Exercise training is reported conclusively to result in an augmented antioxidant system including a reduction in lipid peroxidation. Supplementation with antioxidants appears to reduce lipid peroxidation but has not been shown to enhance exercise performance.

Endogenous glutathione, a tri-peptide referred to as “Reduced Glutathione”, is composed of the three amino acids: L-Cysteine, Glycine, and L-Glutamic Acid. Smaller peptides of 2 or 3 amino acids are absorbed without further reduction or delay, but there is some debate as to the effectiveness of taking peptides as opposed to free amino acids for enhancing endogenous glutathione stores. Most glutathione is found in the liver where it detoxifies harmful compounds later excreted in bile. Some glutathione is found in the red and white blood cells, the lungs, and the intestinal tract. The primary biological function of glutathione is to act as a nonenzymatic reducing agent to assist in cysteine thiol side chains in a reduced state on the surface of proteins.

Reduced glutathione is involved in the synthesis and repair of DNA, assists the recycling of vitamins C and E, blocks free radical damage, enhances the antioxidant activity of vitamin C, facilitates the transport of amino acids, and plays a critical role in detoxification. It is the base material for several other key antioxidant enzyme systems: glutathione-peroxidase, glutathione-reductase, and glutathione- transferase. Decline in glutathione concentrations in intracellular fluids correlate directly with indicators of longevity. Intravenous glutathione administration may provide the most direct and effective route for increasing intracellular glutathione levels. The glutathione (GSH) antioxidant system is foremost among the cellular protective mechanisms. Depletion of this small molecule is the common consequence of increased formation of reactive oxygen species during increased cellular activities. This phenomenon may occur in the lymphocytes during the development of an immune response or also in muscular cells during strenuous exercise. Time and transit of dietary oral GSH substrates may be a positive means of glutathione repletion. However, cysteine, found in milk proteins, is reported to be a crucial limiting amino acid for intracellular GSH4. Replenishing glutathione from dietary supplementation of Whey, N-Acetyl Cysteine, and Glutathione, are time- and dosedependant, but may further be inhibited by the influence of age, exercise stress, or environmental toxins. Bypassing gastric channels and hepatic intervention activity by intravenous loading protocol presents a model for resolving these deficits.

Exhaustive exercise depletes glutathione and simultaneously generates free radicals. This is evidenced by increases in lipid peroxidation, glutathione oxidation, and oxidative protein damage. It is well known that activity of cytosolic enzymes in blood plasma is increased as a result of exhaustive exercise.

Researchers accessed the antioxidant status and markers of oxidative damage in the members of the U.S. Men’s Alpine Ski Team during 10 days of intense training. Seven measures of antioxidant status were determined using Trolox equivalent antioxidant capacity, uric oxidase, alpha-tocopherol, total glutathione, cytosolic glutathione peroxidase, and superoxide dismutase. The results suggested that antioxidant status of elite alpine skiers declined during training. Further studies of blood values determined the importance of glutathione upon potentiating maximal oxygen carrying capacity. Higher glutathione levels influence red blood cell count, hematocrit, and hemoglobin. Conversely when red blood cells, hematocrit, and hemoglobin are reduced during anemia, low glutathione is implicated; the addition of GSH has been shown to resolve the aforementioned low blood markers.

Researchers reported the effects of reduced glutathione parenterally administered resolved the status in patients suffering from chronic renal failure and undergoing hemodialysis. Reduced glutathione and placebo were given for 120 days in a randomized double-blind fashion and the following measurements were performed: red blood cells reduced and oxidized glutathione, plasma reduced and oxidized glutathione, hematocrit, hemoglobin, reticulocytes, serum iron, transferrin, indirect bilirubin, urea, creatinine, calcium, phosphate, parathyroid hormone and alkaline phosphatase. In the treated group, during the supplementation period, there was an increase in the levels of red blood cells and plasma reduced glutathione, hematocrit and hemoglobin and a concomitant decrease in plasma oxidized glutathione and reticulocytes with a maximum effect on the 120th day of therapy. In the placebotreated group there were no significant variations of the parameters considered during the study period. When the therapy, on patients undergoing treatment, was terminated there was a drop in the analyzed parameters, which fell to pretreatment values at the subsequent controls. These findings indicate that reduced glutathione could represent a useful drug in the treatment and management of anemia in patients affected by chronic renal failure. Therefore, increased glutathione may enhance the blood oxygen carrying capacity and improve depleted glutathione stores, which hypothetically translates into enhanced performance outcome.


I hypothesized that performance gain would occur by increasing intravenous glutathione. The 61-year endurance athlete completed 1000 miles of aerobic cycling to establish base fitness, then completed a timed-trial over an 18.4 mile undulating hill course prior to receiving intravenous glutathione, 100 mg/cc, 10cc (total 1000 milligrams) dose diluted to a total volume of 20cc with normal saline by slow IVpush over a 15 minute time period. Subsequent treatments followed at 7-10 day intervals during 36 aerobic training days. Aerobic training levels were imposed as controls with the intent to maintain, not to increase performance outcome. Constant aerobic protocols do not contribute to enhanced time trial performance outcome. Constant aerobic cycling training did not exceed 75% maximum heart rate or maximum oxygen consumption, while diet and supplement protocols were also held constant throughout the test period. To evaluate the influence of IV-Glutathione on performance outcome, a cycling timed trial was pre-test recorded, then compared to a second time trial which was recorded between IV-Glutathione #1 and #2, then concluded after IV-Glutathione #3 & #4, with a comparison of a the final timedtrial recorded on the same 18.4 miles course, using the same bicycle, under similar conditions.

These results are shown in tables I, II, and III below:


Performance gain 7-10 days after treatment #1 was significant (+2.1%) with total performance gains from intravenous glutathione +7.2% over 36 days (57:30 to 52:21 minutes:seconds), averaging a remarkable +1.8% per treatment administered. The only variable to account for the remarkable improvement in performance was four glutathione injections. Measure of the efficiency of oxygen metabolization is closely tied to the mechanism of rapid glutathione repletion to resolve depleted levels. By increasing GSH serum levels, enhanced performance may result. Further research is needed with aging, fit, healthy subjects, measuring a variety of physiologic and metabolic endpoints to determine if the results of this case study prevail, and if so, the mechanism of any such benefits achieved.

Dr. Stanley B. Covert M.D., the Medical Director of High Road Clinic in Elk, Washington, (509) 292-2748 graciously provided expertise in glutathione pathway effects and medical administration of intravenous glutathione to this subject. Dr. Coverts work in resolving macular degeneration disorder using this slow push intravenous protocol is without equal.

The author was the subject of this open label case study. Dr. Covert was the supervising prescribing physician. The subject or the supervising physician reports no competing interests.


Emerging potential benefits of modulating NAD+ metabolism in cardiovascular disease


Nicotinamide adenine dinucleotide (NAD+) and related metabolites are central mediators of fuel oxidation and bioenergetics within cardiomyocytes. Additionally, NAD+ is required for the activity of multifunctional enzymes, including sirtuins and poly(ADP-ribose) polymerases that regulate posttranslational modifications, DNA damage responses, and Ca2+ signaling. Recent research has indicated that NAD+ participates in a multitude of processes dysregulated in cardiovascular diseases. Therefore, supplementation of NAD+ precursors, including nicotinamide riboside that boosts or repletes the NAD+ metabolome, may be cardioprotective. This review examines the molecular physiology and preclinical data with respect to NAD+ precursors in heart failure-related cardiac remodeling, ischemic-reperfusion injury, and arrhythmias. In addition, alternative NAD+-boosting strategies and potential systemic effects of NAD+ supplementation with implications on cardiovascular health and disease are surveyed.

Read the full article here:

NAD+ Metabolism as an Emerging Therapeutic Target for Cardiovascular Diseases Associated With Sudden Cardiac Death


In addition to its central role in mediating oxidation reduction in fuel metabolism and bioenergetics, nicotinamide adenine dinucleotide (NAD+) has emerged as a vital co-substrate for a number of proteins involved in diverse cellular processes, including sirtuins, poly(ADP-ribose) polymerases and cyclic ADP-ribose synthetases. The connection with aging and age-associated diseases has led to a new wave of research in the cardiovascular field. Here, we review the basics of NAD+ homeostasis, the molecular physiology and new advances in ischemic-reperfusion injury, heart failure, and arrhythmias, all of which are associated with increased risks for sudden cardiac death. Finally, we summarize the progress of NAD+-boosting therapy in human cardiovascular diseases and the challenges for future studies.

Read the full article here:

NAD+ and sirtuins in aging and disease


Nicotinamide adenine dinucleotide (NAD(+)) is a classical coenzyme mediating many redox reactions. NAD(+) also plays an important role in the regulation of NAD(+)-consuming enzymes, including sirtuins, poly-ADP-ribose polymerases (PARPs), and CD38/157 ectoenzymes. NAD(+) biosynthesis, particularly mediated by nicotinamide phosphoribosyltransferase (NAMPT), and SIRT1 function together to regulate metabolism and circadian rhythm. NAD(+) levels decline during the aging process and may be an Achilles’ heel, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies. Restoring NAD(+) by supplementing NAD(+) intermediates can dramatically ameliorate these age-associated functional defects, counteracting many diseases of aging, including neurodegenerative diseases. Thus, the combination of sirtuin activation and NAD(+) intermediate supplementation may be an effective antiaging intervention, providing hope to aging societies worldwide.

Read the full article here:

NAD + Depletion Triggers Macrophage Necroptosis, a Cell Death Pathway Exploited by Mycobacterium tuberculosis


Mycobacterium tuberculosis (Mtb) kills infected macrophages by inhibiting apoptosis and promoting necrosis. The tuberculosis necrotizing toxin (TNT) is a secreted nicotinamide adenine dinucleotide (NAD+) glycohydrolase that induces necrosis in infected macrophages. Here, we show that NAD+ depletion by TNT activates RIPK3 and MLKL, key mediators of necroptosis. Notably, Mtb bypasses the canonical necroptosis pathway since neither TNF-α nor RIPK1 are required for macrophage death. Macrophage necroptosis is associated with depolarized mitochondria and impaired ATP synthesis, known hallmarks of Mtb-induced cell death. These results identify TNT as the main trigger of necroptosis in Mtb-infected macrophages. Surprisingly, NAD+ depletion itself was sufficient to trigger necroptosis in a RIPK3- and MLKL-dependent manner by inhibiting the NAD+ salvage pathway in THP-1 cells or by TNT expression in Jurkat T cells. These findings suggest avenues for host-directed therapies to treat tuberculosis and other infectious and age-related diseases in which NAD+ deficiency is a pathological factor.

Link to full article: