The Fountain of Eternal Youth (2)
The Fountain of Youth: A tale of parabiosis, stem cells, and rejuvenation
Massimo Conese et al., Open Medicine, 2017.
For links, see the original article.
Translated by Evgenia Ryabtseva
A story about parabiosis, stem cells and rejuvenation
History of parabiosis (ending)
In general, the data presented at the beginning of the article indicate that in the conditions of a young organism, the cells of the osteoblastic niche that contribute to the homeostatic maintenance of the viability of stem cells undergo age-related changes and begin to contribute to the accumulation of non-functional hematopoietic stem cells. Apparently, these age-related changes in niche cells occur under the influence of uncoated circulating factors, one of the mechanisms of action of which is the effect on the signaling mechanism mediated by insulin-like growth factor-1 in the niche cells themselves (Figure 3). It is very likely that insulin-like growth factor-1 does not play an equally important role in the aging of different tissues, since, despite the aging-promoting effect on the osteoblastic niche, local expression of this factor in skeletal muscles, on the contrary, contributes to maintaining the regenerative ability of cells of aging animals.
Figure 3. The proposed model describing age-related changes in the osteoblastic cell niche and hematopoietic stem cells, as well as possible methods for restoring these changes using heterochronous parabiosis. Age-related changes in autocrine or paracrine effects of insulin-like growth factor-1 on osteoblastic niche cells occur under the influence of circulating soluble factors that change with age. The signaling mechanism mediated by the insulin-like growth factor-1 in the cells of the aging osteoblastic niche (a) directly contributes to age-related disorders of the functions of hematopoietic stem cells, including the accumulation of excess of these cells and a shift in the balance between cell differentiation into B-lymphoid (B cell) and myeloid (My) hematopoietic sprouts. With heterochronous parabiosis or after neutralization of the growth factor-1-mediated signaling mechanism in vivo (b), the activity of cells of the aging niche is restored to the level of a young organism, as a result of which they cease to induce excessive accumulation or shift in differentiation of hematopoietic stem cells.
In October 2010, three of the four authors, including Amy J. Wagers, withdrew this article. In particular, they refuted the role of osteoblastic niche cells in the rejuvenation of hematopoietic cells of old mice. It was found that the first author of the article manipulated images of bone nodules forming in the osteoblastic niche in young and old mice. Thus, this issue needs further study, especially considering that the parabiosis model was used to study the rejuvenation of other organs. Subsequently, two papers were published demonstrating that the effect of an aging systemic environment on a young mouse can inhibit myogenesis and neurogenesis.
In 2013, researchers led by Amy Wagers published another article in which they showed using a parabiotic model that age-related hypertrophy of the heart muscle can be reversed by exposure to a young circulatory environment in just 4 weeks in a state of parabiosis. The results of changes in blood pressure and levels of angiotensin II and aldosterone in the blood of animals of different groups clearly demonstrated that the reversal of the processes of hypertrophy of the heart muscle of old mice exposed to the blood of young animals cannot be explained only by a decrease in blood pressure or modulation of its effect on the body of an old animal. An interesting fact is that heterochronous parabiosis did not lead to changes in the values of the ratio of heart mass to the length of the tibia, the size of cardiomyocytes and blood pressure in young mice with old partners. These data support the existence of an antihypertrophic factor produced by the body of a young mouse (rather than breeding a prohypertrophic factor produced by the body of an old mouse) and triggering remodeling of the heart muscle under conditions of heterochronous parabiosis. In the model of "false parabiosis", that is, with the surgical connection of two animals without the formation of a common circulation, there was no decrease in the severity of age-related hypertrophy of the heart muscle in old mice, which served as an additional confirmation of the primary role of cross-circulation and the exchange of factors circulating in the bloodstream. As a result of the search for these factors using large-scale proteomic analysis based on optomer technology, 13 analytes were identified that reliably distinguish a young mouse from an old one. One of these candidates, growth and differentiation factor–11, belonging to the activin/transforming growth factor-beta superfamily, was confirmed in further experiments. Its reduced plasma content of old isochronous parabionts compared with young isochronous parabionts was restored to normal (young) values in old mice after their parabiosis with young animals. Daily administration of growth factor and differentiation factor-11 to old animals for 30 days resulted in a significant decrease in the ratio of heart mass to tibial length compared to control group animals that were injected with saline solution. These data indicate that at least one of the pathological components of age-related diastolic heart failure has a hormonal nature. However, the observed regression of cardiac muscle hypertrophy in old mice under the influence of young blood flow cannot be due to the restoration of the concentration of one factor, and other factors have yet to be identified.
In two subsequent studies, Wagers and her colleagues found that growth and differentiation factor-11 stimulates the growth of new blood vessels and neurons in the brain, and also improves the ability of stem cells to regenerate skeletal muscles in areas of damage. In one of these studies, a mouse model of heterochronous parabiosis demonstrated an increase in the volume of blood vessels in the brain and increased blood flow in response to the effects of systemic factors of a young organism with simultaneous stimulation of self-renewal and differentiation of a population of nerve stem cells in the subventricular zone, which provided an improvement in the ability to recognize odors (Figure 2). Moreover, the authors found that growth and differentiation factor-11 can improve the volume of blood vessels, as well as stimulate neurogenesis in old mice. Interestingly, the blood of a 15-month-old mouse did not cause a decrease in the population of nerve stem cells in the brain of a young animal, whereas the blood of an older individual (21 months) had a negative effect. This indicates in favor of the fact that the older the animal, the higher the concentration of harmful systemic factors and/or lower the levels of protective factors of the young organism. On the other hand, in an article written under the guidance of Rando and Tony Wyss-Corey, the chemokine CCL11/eotaxin was identified as an age-related blood factor associated with reduced neurogenesis and learning and memory disorders in mice. In the future, it remains to be seen whether CCL11 interacts directly with progenitor cells during aging, affecting their ability to differentiate, or whether it exerts its effect indirectly - through interactions with other types of neurogenic niche cells.
In another study, Wagers and her colleagues demonstrated that satellite cells isolated from the musculature of old heterochronous parabionts had an increased potential for myogenic differentiation, as well as a lower level of DNA damage compared to myosatellites of isochronous parabionts from the control group of the same age. As for reversing the processes of age-related myocardial hypertrophy, daily intraperitoneal administration of recombinant growth factor and differentiation-11 to old animals for 4 weeks increased the number of satellite cells with intact DNA compared to cells of elderly animals receiving placebo injections. Moreover, in the model of muscle injury, the administration of growth factor and differentiation-11 to old mice for 28 days before and 7 days after injury restored the diameter of regenerating fibrils to values characteristic of a younger age. Also, old mice treated with growth factor and differentiation factor-11 demonstrated endurance and grip strength exceeding the average values.
In this latest work, the authors also found that in vitro exposure to growth factor and differentiation-11 on aging myosatellite cells caused a dose-dependent increase in the activity of proliferation and differentiation of these cells, which did not occur when exposed to myostatin (another member of the superfamily of transforming growth factors-beta) or transforming growth factor-beta-1. This observation indicates that, unlike myostatin, growth and differentiation factor-11 can have a direct effect on myosatellite cells, changing their functions.
A story about a surprise and new drugs
The appearance of data on the role of growth factor and differentiation-11 in the rejuvenation of muscle tissue has not passed without consequences. The results of the revision of the earlier belief, according to which myostatin, a protein closely related to growth and differentiation factor–11, suppresses myogenesis, turned out to be a complete surprise. In a relatively recent study, this pattern was refuted. Egerman and colleagues conducted a thorough re-analysis of the hypothesis and found that the antibodies previously used to identify growth factor and differentiation-11 are non-specific, that is, they do not allow distinguishing myostatin from growth factor and differentiation-11. It also turned out that the overall levels of myostatin/growth factor and differentiation-11 increase with age, which contradicts earlier data. The use of a more specific reagent for the detection of growth factor and differentiation-11 revealed a tendency to increase the levels of this factor in the serum of aging rats and humans compared with young ones. Moreover, the concentrations of mRNA encoding this factor in the muscle tissue of rats increased as they aged. In vitro experiments have also shown that both growth and differentiation factor-11 and myostatin induce the same signaling pathways (activation of SMAD 2/3 and MAPK) to the same extent in primary and immortalized human skeletal muscle cells and that differentiation of primary human myoblasts into muscle tubules is inhibited by growth and differentiation factor-11 and myostatin. Using the same protocol developed by Sinha and colleagues, the authors found no differences in the regenerative capacity of skeletal muscles of old mice treated with growth factor and differentiation-11 or placebo 7 days after damage caused by cardiotoxin. Instead, elevated systemic levels of growth factor and differentiation-11 were associated with impaired regeneration in young animals, as evidenced by an increased number of very small muscle fibers in animal muscles after administration of this factor. Finally, they also found that the administration of growth and differentiation factor-11 dose-dependent inhibited the growth of adult and aging cultures of myosatellite cells. According to this study, which indicates an increase in the level of growth factor and differentiation-11 with age, the same group of scientists previously demonstrated that the activity of the myostatin/growth factor and differentiation-1 signaling pathway, which is characterized by phosphorylation of the SMAD3 gene, is also increased in aging rats. The essence of the revealed pattern is that if aging people have muscle tissue atrophy (sarcopenia) against the background of very high levels of growth factor and differentiation-11, they are candidates for selective blockade of growth factor and differentiation-11 or more general blockade, in addition to this factor extending to myostatin and their receptors.
Despite the fact that at first glance the data obtained by Egerman contradict the results of Amy Wagers, growth and differentiation factor-12 can be represented by different forms and only the level of one of them can decrease with age. This assumption was published in The Scientist magazine based on an email message received from Amy Wagers. Moreover, Novartis Group specialists inflicted more extensive damage to muscle tissue and subsequently applied higher doses of growth factor and differentiation-11 than the Wagers group, so the results they obtained are unsuitable for direct comparison (Novartis specialists used young animals and a three times higher dosage of the factor). In fact, Egerman's results may partly explain the mechanism of action of bimagrumab, an experimental Novartis drug designed to treat muscle weakness and muscle tissue depletion. Currently at the stage of clinical trials, the drug blocks myostatin and, possibly, growth and differentiation factor-1.
In short, there is no doubt that young blood rejuvenates old mice, but Novartis experts claim that the explanation proposed by Harvard scientists does not correspond to reality. Perhaps the truth is somewhere in the middle and maintaining the level of growth factor and differentiation-11 within the physiological norm is necessary to preserve the health of muscle tissue. It is also important not to forget the words of Amy Wagers: "We do not reverse the aging of animals. We are returning functionality to their tissues." In this context, other factors may also have an effect. In 2014, Irina and Michael Conboy identified one of the anti-aging factors circulating in the bloodstream. It turned out to be oxytocin, a nonapeptide synthesized in the hypothalamus, involved in the processes of labor and attachment formation. They found that in old mice (18-24 months) there is a decrease in the concentration of oxytocin, subcutaneous administration of which restores the regenerative ability of muscle cells of these animals in conditions of cardiotoxin damage.
In the search for specific anti-aging factors, many questions remain unanswered today. For example, it is largely unclear how CCL11 and growth and differentiation factor-11 improve the microenvironment of aging tissue-specific stem cells. Moreover, a number of observations are very poorly consistent with each other. For example, the blood of old animals with a reduced content of growth factor and differentiation-11 in serum has a negative effect on neurogenesis and cognitive function of young individuals. This contradicts the fact that old serum is diluted by young serum, calling into question the ability of microRNA or other protein factors to influence growth factor and differentiation-11 to reduce or increase its expression. In the context of this review, it should be noted that, at least, however, the company is trying to reproduce the results obtained by Wagers in a clinical setting, using plasma from healthy young people to treat patients with Alzheimer's disease. An interesting fact is that in 1972, researchers from the University of California studied the life expectancy of old and young rats connected in parabiotic pairs. The old partners lived 4-5 months longer than the control group animals, which for the first time indicated the ability of young blood to influence life expectancy. In September 2014, the California start-up Alkahestin began conducting an open clinical trial in one group, the purpose of which was to evaluate the safety and efficacy of a single dose of plasma from young donors (men aged 30 years or younger) as a treatment for mild or moderate Alzheimer's disease. Results concerning both primary (symptoms and adverse events) and secondary (magnetic resonance imaging and blood test results) are expected this year. However, despite the fact that this clinical trial is designed for 4 weeks, this practice may cause some concern. For example, there is a possibility that prolonged activation of stem cells may lead to an increased risk of cancer.
Pros and cons of using whole blood compared to specific factors
The advantage of introducing young blood to an old person is that it can contain various anti-aging factors that can have pleiotropic effects on many organs affected by the disease at the same time. In fact, whole blood is a mixture of cells, colloids and crystalloids. Each of these components can be separated from the others to obtain ready-made blood components, including concentrates of erythrocytes and platelets, freshly frozen plasma and cryoprecipitate, used in different situations. Transfusion of whole blood and its components is now a safe clinical practice and has a wide range of indications in regenerative medicine. However, some of its components, such as plasma proteins, leukocytes, erythrocyte antigens, plasma and pathogens, can trigger the development of undesirable side reactions ranging from moderate allergic manifestations to fatal complications. Moreover, we do not know, for example, whether the plasma of young donors contains factors useful for patients with muscular dystrophy or Alzheimer's disease. For this reason, a safer approach may be to identify specific factors that selectively contribute to the rejuvenation and restoration of the functions of certain organs or tissues. Some specialists would prefer to conduct clinical studies of certain blood factors or combinations of known factors synthesized in the laboratory. The disadvantage of this approach is that the mechanisms of action of the factors identified to date (CCL11/eotaxin, growth and differentiation factor-11 and oxytocin) still largely unclear. It may well be that these factors do not have a direct effect, and their activity depends on epigenetic mechanisms (for example, microRNAs), or that they exert their rejuvenating effect only in combination with other unknown blood components.
Conclusions
This article describes the long history of the human search for the "source of eternal youth". For many years, parabiosis has indicated that factors of young blood can contribute to the regeneration of diseased and aging tissues. Some would prefer to use whole blood or its derivatives, such as plasma. At the same time, others are supporters of the use of specific factors or their cocktails. The optimal scenario is to use the patient's own plasma isolated from platelets cytokines or growth factors for wound healing and tissue regeneration. A number of proofs of the appropriateness of the approach have been obtained in animal studies, but clinical results remain a challenge for the future.
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