Utilizing Developmentally Essential Secreted Peptides Such as Thymosin Beta-4 to Remind the Adult Organs of Their Embryonic State—New Directions in Anti-Aging Regenerative Therapies

Our dream of defeating the processes of aging has occupied the curious and has challenged scientists globally for hundreds of years. The history is long, and sadly, the solution is still elusive. Our endeavors to reverse the magnitude of damaging cellular and molecular alterations resulted in only a few, yet significant advancements. Furthermore, as our lifespan increases, physicians are facing more mind-bending questions in their routine practice than ever before. Although the ultimate goal is to successfully treat the body as a whole, steps towards regenerating individual organs are even considered significant. As our initial approach to enhance the endogenous restorative capacity by delivering exogenous progenitor cells appears limited, we propose, utilizing small molecules critical during embryonic development may prove to be a powerful tool to increase regeneration and to reverse the processes associated with aging. In this review, we introduce Thymosin beta-4, a 43aa secreted peptide fulfilling our hopes and capable of numerous regenerative achievements via systemic administration in the heart. Observing the broad capacity of this small, secreted peptide, we believe it is not the only molecule which nature conceals to our benefit. Hence, the discovery and postnatal administration of developmentally relevant agents along with other approaches may result in reversing the aging process.

In accordance with our current scientific knowledge, numerous cellular attributes of aging were identified and summarized in various review articles throughout the literature [ 1 , 7 , 8 , 9 , 10 ], all of which are eventually contributing to the decay of the cells and the organism. In addition to identifying the microscopic alterations, there is also an urgent drive to understand the molecular interchanges and to define the molecular markers to depict the pathology behind senescence and recovery. As stated by Niedernhofer and colleagues, while several molecules which were potentially considered for testing and implementation have been successfully identified, the main challenge is to find a unifying mechanism of age-related tissue damage and to prioritize which molecular endpoints to pursue and validate [ 10 ]. In following the chronology, investigations first began with initial observations, in which late reproducing organisms live longer than early reproducers. These findings supported that genes may have a significant role in lifespan determination [ 11 ]. Today, the actual list of potential influencer genes numbers over 800 [ 12 ], jump-starting the identification of numerous well conserved age-related molecular pathways such as insulin-like signaling, mTOR pathway, the discovery of Sirtuins and the importance of NAD + [ 12 ]. In the 1950s, research turned towards identifying the role of free radicals and the relevance of the mitochondria in which all results suggested these organelles may be intimately linked to a wide range of processes associated with aging [ 13 ]. Animal studies suggested however, that while the levels of mitochondrial mutations increase with age, it remains unclear whether this alteration does truly play a fundamental and significant role in the process [ 14 , 15 ]. In addition to mitochondrial pathology, the presence of various factors which induce permanent inflammation were equally highlighted and detected as potential causes regarding aging. With this, inflammatory senescence (immunosenescence) became a considered marker and hallmark of aging biology [ 16 , 17 ]. Finally, to fully maintain well-functioning healthy cells and protein networks, an extensive machinery of molecular chaperones, proteases, and many other additional factors are equally required [ 18 , 19 ]. In observing all the enlisted factors, one wonders how our morphological, physiological, and molecular knowledge regarding aging will be translated into actual functional therapies? Preventing disease conditions is overly critical in this concept, but reversing the already developed malfunctions is the challenge clinicians face in their daily practice. To support this concept, geroscience, an emerging cross-disciplinary field was established in the last decade with a goal to unravel the molecular and cellular mechanisms responsible for aging as a major risk factor and driver of age-related chronic diseases [ 20 , 21 , 22 ]. By reviewing the enlisted hallmarks, we observed that most investigations and analyses primarily reconnoiter the journey from the day of our birth, thus leaving the power and knowledge regarding the most dynamic period of our physical development unaccounted and in the dark. To overcome this discrepancy, we suggest the conceptual and therapeutic approach of aging could significantly benefit from observing, extending, and harnessing our knowledge regarding embryonic development, and that utilizing these findings throughout our adult life may significantly re-enhance the regenerative potential of the human body. In the next chapters of this review, we intend to demonstrate and instantiate the viability of this alternative approach in the context of hypoxic heart injury. Naturally, the goal is to employ all future discoveries in the daily routine beside therapy, to increase lifespan and quality of living during old age.

The beta thymosins are a family of highly conserved acidic 5 kDa peptides, originally thought to be thymic hormones and first described in 1966 by A.L. Goldstein and A. White [ 65 ]. Currently, sixteen beta thymosins are known, of which, only three are present in humans: Thymosin β4 (TB4), Thymosin β10 (TB10), and Thymosin β15 (TB15) ( Figure 2 ). While they possess a highly conserved and homologous structure (43–44 amino-acid residues), they are expressed from from variant gene products, and thus they are biochemically and functionally distinct molecules [ 66 ]. Beta thymosins differ from alpha thymosins upon their isoelectric point: below 5 are termed α and between 5 and 7 are termed β [ 67 ]. TB4 is the main peptide, representing nearly 70–80% of the total beta thymosin content [ 68 ]. Structurally, TB4 contains a central actin-binding domain surrounded by two alpha helices and an amino (N) and carboxy (C) terminal variable region ( Figure 2 ). It is present in high concentrations (up to 0.4 mM) in various adult tissues, especially in the spleen, lungs, thymus, brain, and heart [ 69 ], as well as in macrophages, tumor and human blood cells, while serum contains less than 1% of the TB4 amount present in the entire blood system. Apart from blood, TB4 may be equally found in wound fluid or in additional body fluids, such as saliva or tears [ 67 , 70 , 71 , 72 ].

Identification of the hallmarks of senescence defined numerous potential target points in reversing or influencing age-related degenerative processes [ 1 , 7 , 8 , 10 , 16 , 17 , 95 , 96 ]. To translate these findings regarding the hypoxic heart, it is equally critical to precisely define the targets of the human infarction pathology to successfully inhibit or reverse functional loss. These factors include inhibition of cardiac cell death, inflammation, and scar formation ( Figure 1 ) [ 97 ]. In focusing on TB4′s influence upon inflammatory processes, earlier data revealed monocytes produce TB4 sulfoxide in response to glucocorticoid administration. TB4 sulfoxide was discovered to block neutrophil chemotaxis in vitro and to possess a potent anti-inflammatory activity in vivo [ 98 ]. Moreover, immunomodulatory effects of TB4 prevented apoptosis by decreasing cytochrome c release from mitochondria by increasing BCL-2 expression and decreasing caspase activation [ 99 ]. We know TB4 also plays a role during sepsis, a dysregulated host response to infection resulting in life-threatening organ damage. A sustained F-actinemia may create endothelial injury and microthrombi characteristic of sepsis pathology. While F-actinemia is present in sepsis, TB4, as an actin-binding protein, inhibited polymerization of F-actin. Currently, ongoing studies utilize the peptide’s regenerative power in the infected or injured eye (Pseudomonas aeruginosa-induced keratitis [ 100 ], corneal wound healing [ 101 ]) or, as a doping agent, to enhance performance and skeletal muscle regeneration (TB500) [ 102 ]. In dermal phase II trials, TB4 promoted wound healing by accelerating the rate of repair. Patients with pressure ulcers, stasis ulcers, and epidermolysis bullosa benefited from TB4 therapy. The results concluded TB4 is safe, well-tolerated and possessed additional use for skin regeneration [ 103 , 104 ]. The significance of TB4 regarding cardiac regeneration and repair was first indicated in a publication by members of our research group, affiliating a leading role for the peptide in aiding cardiac function following hypoxia [ 87 ]. We discovered that external administration of TB4 promotes myocardial cell migration and survival in embryonic tissue in vitro and retains this property following birth. After coronary artery ligation in mice, the peptide enhanced myocyte survival and improved cardiac function, suggesting TB4 may be a novel therapeutic target in the setting of acute myocardial damage from heart attacks and other myocardial diseases among children and adults. Remarkably, the degree of improvement when TB4 was administered systemically through intraperitoneal injections or only locally within the cardiac infarct was not statistically different, suggesting the beneficial effects of TB4 likely occurred through a direct effect on cardiac cells rather than through an extracardiac source [ 87 ]. While investigating the potential mechanisms and interactors through which TB4 may be influencing myocardial cell migration and survival events via phage display, we proved ILK-mediated Akt activation to inhibit myocadial cell death may be one of the role players in the process. The observations in vivo were consistent with the effects of TB4 on cell migration and survival demonstrated in vitro and suggest activation of ILK and subsequent stimulation of Akt may, in part, explain the enhanced cardiomyocyte survival induced by TB4, although it was clearly unlikely that only this single mechanism is responsible for the full repertoire of TB4′s cellular effects [ 87 ]. Moreover, multimorbid analyses of diabetic and dyslipidemic porcine ischemic heart models equally revealed an attractive therapeutic potential of adeno-associated virus encoding TB4 in ischemic heart disease [ 105 ], supporting the described benevolent effects of TB4′s administration following cardiac injury. Since the original discoveries, numerous supporting reports have been published regarding the positive impact of TB4 or its N- or C-terminal domains on cardiac regeneration and repair, suggesting the molecule has a real potential to positively influence age-initiated alterations in the adult human heart and vessels [ 93 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 ].

Reversing age-related alterations of the body is a long-desired goal of humankind. To achieve this goal, it is critical to first precisely understand, analyze and define the macroscopic, cellular and molecular processes of age-related alterations of the body. In this review, our focus highlights aspects of the heart, in which we outlined some of the alternatives scientists utilize to avoid cellular death and to restore, as yet without much success, the physiological conditions. Due to the complexity of the picture, however, reversing the negative processes of organ damage or aging is nearly impossible. To overcome complications and technical hurdles, we propose instead of stopping or fixing aging related processes, we redirect the adult organ towards its earlier developmental state with the hope this approach may also overcome the significant impact of environmental influence and genetic variations, since early development of the human organo- and embryogenesis seems to be more universal ( Figure 5 ). Despite the extravagant premise, the dream of returning to our youth may not be so far-fetched. Nature actually provides an enormous list of molecules, some of which are silenced after birth but could serve as a potential treatment to reverse age, at least at the level of a single yet complicated organ such as the heart. The question remains: can postnatal increase of developmentally relevant proteins and peptides reverse organ ageing or damage in a beneficial way? We believe the answer to this question is yes. In our earlier research, we introduced a naturally secreted small molecule, Thymosin beta-4, which is capable of such miracles not only regarding the heart but in the brain and kidneys [ 69 , 130 ]. The protein was first purified from the thymus and binds and alters the cytoskeletal actin filaments by sequestering actin monomers and in doing so, influences actin filament assembly and regulates migration of various cell types such as endothelial cells, myocardial cells or epicardial progenitors [ 115 ]. It equally inhibits cellular death by activating and binding numerous role players of the focal adhesion complex, which, eventually, results in the activation of Akt, a proven substrate of ILK with wide-ranging signalling functions that affect growth, survival and motility [ 87 ]. Naturally, all the other mechanisms by which TB4 initiates the increase of cardiac function are still under investigation by a host of scientists worldwide. Undoubtedly, its capability in activating the adult epicardium and its ability to resemble its embryonic function without risking injury suggests grounds for hope that the molecule does achieve the same in other organs. Reminding adult cells of their highly proliferative state is not without risk, as the applied treatment may easily result in unwanted malignancies. Although TB4 was reported to be a prognostic marker for highly metastatic cancer states and its tumor promo-ting properties were equally demonstrated [ 131 , 132 ], its true nature regarding tumorigenesis is controversial. In our hands, the molecule significantly inhibited the progression of pancreatic cancer following systemic administration in mice in vivo (unpublished results). An additional factor supporting this result is that TB4 was equally introduced as a candidate tumor suppressor in male breast cancer [ 133 ] and was demonstrated to have a tumor suppressive function in myeloma development by other research teams [ 134 , 135 ]. In addition, the high safety profile observed in Phase I and Phase II clinical trials this far strongly anticipate that TB4 will be safe and efficacious at the applied local or systemic dose for broad clinical applications in the future [ 136 ]. We still do not know all the details regarding this special molecule; however, we genuinely believe there are more like it concealed in the human body, awaiting discovery. With their help, we may be capable of fulfilling our dream of reversing age, and as brilliantly foreseen by Professor Schneider [ 137 ], perhaps we will be able to create an alternative for the legend, and Prometheus will soon be unleashed and no longer bound.