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A Review of the Literature and a Working Hypothesis by Dmitry Dzhagarov (Jagarovi), Ph.D.
Abstract
The purpose of this review is to substantiate a new (yethypothetical) approach to the treatment of age-dependent diseases, which consists in cleansing the body of aged cells with the help of senolytics and prevention of fibrosis by stimulating the replenishment of the formed “cleared” of old cells space with rejuvenated cells using a combination of ROCK inhibitor with 5-LOX inhibitor and palmitoylethanolamide. It is assumed that this combination of drugs will work synergistically, causing conditional reprogramming of cells in situ and, as a consequence, proliferation of progenitor cells necessary to make up for losses.
“Inflammageing”, the chronic low-grade inflammation that develops with advanced age [1, 2] is a very significant risk factor for morbidity and mortality in older people.The most likely cause of this inflammation is the aged cells [3], which accumulate in the tissues of the body during its aging [4]. These cells, that for a number of reasons have lost the ability to proliferate, pass into a state of permanent rest, called cellular senescence and begin to secrete a secret consisting of a broad set of substances that cause inflammation: cytokines, chemokines, as well as growth factors and proteases that cause matrix remodeling. This paracrine secretion known as SASP (senescence-associated secretory phenotype), acts on the surrounding cells, causing their degradation and premature aging [5, 6].
While the body is young, its immune system is quite effective in cleansing of aged cells, so they do not interfere with tissue regeneration. In old age, however, the effectiveness of this process can be compromised, as evidenced by the tendency to the accumulation of aging cells in the tissues of the elderly [4, 7, 8]. Senolytics are medicines that allow removing aged cells from the body, and help to counteract the development of age-related pathology in the elderly [9, 10, 11].
It is assumed that, ridding the body of aged cells, the senolytic should activate regeneration processes that will replace the “released space” occupied by old cells with new cells. However, in practice, especially in the case of certain tissues of the old body, such replenishment does not always occur. For example, Pax7-expressing satellite cells, which are formed at the embryo stage, are indispensable for the regeneration of skeletal muscle in adults, and the depletion of their pool leads to myopathy [12, 13]. Similarly, the loss of skin fibroblasts is usually not accompanied by replenishment with new cells – fibroblast membranes extend to fill the empty space of lost neighboring fibroblasts instead of proliferation [14]. Moreover, in some cases, senolytics can even reduce the functionality of tissues. In particular, the use of one of the senolytics, dasatinib, caused endothelial dysfunction and pulmonary hypertension [15], which could be corrected using ROCK inhibitors.
Consequently, I think that senolytics should not be used as monotherapy, but in combination with methods of replacing the loss of old cells by increasing the number of younger progenitor cells.
Attempts to carry out replacement therapy by transplantation of mesenchymal stem cells are, as a rule, not very successful due to the observed mass death of cells after transplantation, caused by a hostile environment [16]. A better approach would be to activate the filling of the "released space" by stimulating endogenous replacement mechanisms. But in the adult body, such a replacementis usually fibrous scar tissue, instead of functional tissue. In industrialized countries, chronic fibrotic diseases account for more than 45% of all deaths [17].
Therefore, methods for activating the division of progenitor cells while suppressing the formation of fibrous tissue are necessary. A difficulty here is that the progenitor cells are closely bound
to the old matrix on which they are sitting, and to each other, and this impedes their division and renewal.
This feature of a cell to attach to neighboring cells and to the intercellular matrix, as well as its inability to multiply by division and susceptibility to apoptosis/anoikis, largely depends on the activity of the RHO-associated protein kinase ROCK, which is akey regulator of the cell cytoskeleton [18].
A study of wound healing processes showed that wound treatment with ROCK inhibitors sharply increased the number of so-called leader cells that behave “independently” from other cells [19]. Moreover, it turned out that somatic cell cultures under the influence of the ROCK inhibitor acquired the character traits of stem cells and the ability to proliferate indefinitely, if they are affected by soluble factors released by co-cultivated feeder cells during apoptosis of that feeder cells caused by radiation [20].Unlike ordinary cells, the number of divisions of which, obeying the Hayflick phenomenon [21], is limited due to the shortening of telomeres, such "immortalized" cells could divide an infinite number of times [22].
As a matter of fact, “immortalized” cells were known before (for example, HeLa tumor cells), but the peculiarity of these was that removal of the ROCK inhibitor and/or apoptotic products of feeder cells allows the cells to differentiate normally [23, 24].Since this “immortalization” turned out to be reversible, these cells were called Conditionally Reprogrammed Cells (CRC). It is important to note that at the same time, these cells retain anormal karyotype and do not become tumor [25]. Moreover, insome cases, CRC culture method fails to propagate precisely cancer cells [26, 27, 28].
Despite the fact that CRC culture method allows in vitro propagation of adult cell culture without any genetic manipulations [29], it nevertheless affects cell youth indicators. By restoring of two functional characteristics that decrease with cell aging – the functional state of mitochondria and cellular metabolism – inhibition of ROCK reverses the aging of fibroblasts with Hutchinson-Guildford syndrome (HGPS), as well as normal but aged fibroblasts [30].
Treatment of old tendon stem and progenitor cells by ROCK inhibitor leads to their rejuvenation, they become similar to younger cells. Namely, they become softer, the volume of cells decreases, and their cytoskeleton consists of more freely packed stress actin fibers, similar to what is observed in young cells [31].
It is known that increased ROCK activity precedes a number of vascular pathologies, including atherosclerosis, stroke, diabetes,endothelial dysfunction and hypertension [32, 33, 34, 35, 36]. Therefore, ROCK inhibitors can be used to treat various forms of cardiovascular diseases, including stroke, coronary heart disease and heart failure, hypertension, pulmonary hypertension, angina pectoris, and a number of other diseases characteristic of elderly people [37, 38].
It is important to note that conditional immortalization of cells – gaining the ability to proliferate for a long time – is closely associated with activation of the expression of one of the natural isoforms of the p53 protein, namely Δ133p53α, which is known to suppress the p53 protein-induced aging genes, displacing p53 from the promoter regions of these genes [39].
Under in vitro conditions, the Δ133p53α isoform, in combination with the ROCK inhibitor, successfully replaced the soluble factors released during apoptosis of feeding cells under the influence of radiation. Δ133p53α added to the medium together with the ROCK inhibitor was sufficient for the formation of CRC cells. At the same time, knockdown of endogenous Δ133p53 synthesis inhibited cell proliferation, which was shown by the authors to be associated with a loss of expression of hTERT mRNA (a catalytic subunit of the telomerase enzyme necessary for lengthening telomeres) [40]. Nevertheless, the Δ133p53 isoform alone is not a sufficient condition for telomerase activation, since it, by suppressing of miR-34a, increases only the duration of replicative cell life, although telomere lengths continue to decrease [41]. Obviously, the telomerase activation mechanism turns on only after reaching a critically short telomere length [42] or while suppressing ROCK. Since overexpression of Δ133p53 dominates the expression of proinflammatory cytokines associated with aging (SASP) and also promotes the repair of DNA damage [43], it was proposed to useΔ133p53 to combat premature aging in children with progeria (Hutchinson-Guildford syndrome) [44].
Considering the fact that Δ133p53α is destroyed by autophagy during replicative aging [45], reconstitution of the Δ133p53α poolmay be a useful remedy for replenishing and rejuvenating the stemcells pool.
The inhibition of 5-lipoxygenase (5-LOX) enzyme under theinfluence of YWCS (Tyr-Trp-Cys-Ser) peptide [46] allows toinduce the formation of Δ133p53α, and at the same time factors p73and Δ160p53 involved in DNA repair [47, 48]. However peptides such as Δ133p53α or YWCS are not stable enough for long storage and also are readily broken down by proteases under physiological conditions, which prevents their widespread use [49]. It is logical, therefore, to replace them with more stable small molecules.
In addition to YWCS peptide, other substances, such as Balkaleincan inhibit 5-LOX lipoxygenase and one of the cell aging pathways induced by it [50, 51]. Baicalein, a flavonoid derived from the rootsof Scutellariae baicalensis Georgi, has shown health benefits for an array of human diseases due to its broad biological activity, such as antifibrotic, antiviral and anticancer properties [52, 53]. It can be used successfully as hepatoprotective [54], radioprotective [55], cardioprotective against ischemia reperfusion injury [56] and geroprotective [57, 58, 59] medicine. Moreover, baicalein possesses potent ROCK inhibitory activity, and such ROCK inhibition suppresses actin stress fiber formation by inhibiting the phosphorylations of myosin light chain 2 and myosin phosphatase [60].
Another 5-LOX inhibitor Zileuton (zileuton, commercial nameZyflo) convincingly proved its therapeutic properties in asthma anda number of other diseases [61, 62, 63].
Famous for its potent anti-inflammatory, anti-oxidant,and anti-cancer properties curcumin (a polyphenoliccompound in turmeric that extends life span in Drosophila melanogaster [64], Caenorhabditis elegans [65], but have no sizable effect on old mouse lifespan [66, 67]) also reduces the expression of 5-LOX gene [68] and, to a certain extent, at the post translational level the catalytic activities of 5-LOX [69].
It is logical to assume from Shekhar et al. results that 5-LOXinhibitors will be able to replace Δ133p53 as an activator of CRC cell formation in a mixture with a ROCK inhibitor (or even without it). The use of 5-LOX inhibitors instead of Δ133p53 or irradiated feeder cells in long-term cell culture technology will simplify CRCmethod and standardize cultivation conditions by switching to chemical culture media.
In addition, such a simplification of the technology of conditionally reprogrammed cells opens up the possibility of transferring this technology from a test tube to the body, affecting cell proliferation in situ. Moreover, the medicines that are needed for this have long been used individually for thetreatment of a number of human diseases.
In particular, ROCK inhibitors, which have proven their ability to activate prolonged proliferation of cells in vitro – fasudil (tradename Eril®) [70] and ripasudil (Glanatec®) [71] – have long been approved in Japan and in China for use in the clinic for the safe treatment of cerebral vasospasm [72], as well as glaucoma, where the ROCK inhibitor acts in two ways: on blood pressure and as a neuroprotector [73, 74].
Fasudil helps fight the loss of intelligence and memory in the elderly [75, 76]. In addition, fasudil has the ability to prevent the differentiation of lung fibroblasts into myofibroblasts of scar tissue [77], which makes it a powerful therapeutic tool in the treatment of pulmonary fibrosis [78]. In animal experiments, it prevented fibrosis of the heart muscle and also improved coronary blood flow [79]. Another ROCK inhibitor Y-27632 improves the results of glucocorticoid treatment of aging dystrophy of muscles, since in this case stem cell pool was replenished [80].
The above indicates that after treatment of age-dependent diseases with senolytics, an additional positive effect should be expected in the treatment with ROCK inhibitors approved for clinical use (fasudil or ripasudil) together with 5-LOX inhibitors (baicalein, berberine or zileuton).
Since, despite clear evidence of potential benefits, no one has yet studied the effect of a combination of ROCK inhibitors with 5-LOX inhibitors on the lifespan and age-related diseases of old mice, this gap needs to be filled.
It is unlikely that such a combination of drugs that obviously should stimulate proliferation of progenitor cells, will promote oncogenesis, since ROCK inhibitors [81] and 5-LOX inhibitors [82] are commonly used to prevent it.
By placing a combination of ROCK and 5-LOX inhibitors in microspheres from a lactic and glycolic acid copolymer (PLGA) for controlled prolonged release [83], it should be possible to initiate the formation of CRC cells in situ directly in an animal. The powder from such microspheres can be used to treat diseases of various organs (lungs, liver, heart, eyes, skeletal muscles) leading to dystrophy or fibrosis, as well as to combat a number of age-related and oncological diseases. In the form of a suspension or in the form of a powder for inhalation, it can be administered directly to the desired area of the body.
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