Yegor E. Yegorov

Telomere Length is a Biomarker of Cumulative Oxidative Stress, Biologic Age, and an Independent Predictor of Survival and Therapeutic Treatment Requirement Associated With Smoking Behavior

Globally, tobacco use is associated with 5 million deaths per annum and is regarded as one of the leading causes of premature death. Major chronic disorders associated with smoking include cardiovascular diseases, several types of cancer, and chronic obstructive pulmonary disease (lung problems). Cigarette smoking (CS) generates a cumulative oxidative stress, which may contribute to the pathogenesis of chronic diseases. Mainstream and side stream gas-phase smoke each have about the same concentration of reactive free radical species, about 1 × 1016 radicals per cigarette (or 5 × 1014 per puff). This effect is critical in understanding the biologic effects of smoke. Several lines of evidence suggest that cigarette smoke constituents can directly activate vascular reactive oxygen species production. In this work we present multiple evidence that CS provide the important risk factors in many age-related diseases, and is associated with increased cumulative and systemic oxidative stress and inflammation. The cited processes are marked by increased white blood cell (leucocytes, WBCs) turnover. The data suggest an alteration of the circulating WBCs by CS, resulting in increased adherence to endothelial cells. Telomeres are complex DNA–protein structures located at the end of eukaryotic chromosomes. Telomere length shortens with biologic age in all replicating somatic cells. It has been shown that tobacco smoking enhances telomere shortening in circulating human WBCs. Telomere attrition (expressed in WBCs) can serve as a biomarker of the cumulative oxidative stress and inflammation induced by smoking and, consequently, show the pace of biologic aging. We originally propose that patented specific oral formulations of nonhydrolized carnosine and carcinine provide a powerful tool for targeted therapeutic inhibition of cumulative oxidative stress and inflammation and protection of telomere attrition associated with smoking. The longitudinal studies of the clinical population groups described in this study including elderly support the hypothesis that telomere length is a predictor of survival and therapeutic treatment requirement associated with smoking behavior.

Reverse transcriptase inhibitors suppress telomerase function and induce senescence-like processes in cultured mouse fibroblasts

Spontaneous transformation of mouse embryonic fibroblasts in the presence of the reverse transcriptase inhibitors azidothymidine and carbovir led to the formation of telomerase‐free clones. After prolonged cultivation of fibroblasts in the presence of carbovir, resistant cells with a very high level of telomerase activity were obtained. Azidothymidine and carbovir, but not dideoxycytidine, induced senescence‐like processes in cultures of immortal mouse fibroblasts. After long‐term incubation, cell proliferation gradually decreased, their morphology becoming similar to that of the senescent ones. The process was reversible: after inhibitor removal, the cells, including the giant ones, entered mitoses. All these data suggest that reverse transcriptase inhibitors block telomerase function in mouse cells.

Smoking and health: association between telomere length and factors impacting on human disease, quality of life and life span in a large population-based cohort under the effect of smoking duration.

Reactive oxygen species (ROS) are of primary importance as they cause damage to lipids, proteins, and DNA either endogenously by cellular mechanism, or through exogenous exposure to environmental injury factors, including oxidation insult factors, such as tobacco smoke. Currently 46.3 million adults (25.7 percent of the population) are smokers. This includes 24 million men (28.1 percent of the total) and more than 22 million women (23.5 percent). The prevalence is highest among persons 25–44 years of age. Cigarette smokers have a higher risk of developing several chronic disorders. These include fatty buildups in arteries, several types of cancer and chronic obstructive pulmonary disease (lung problems). As peripheral leukocytes have been the main target of human telomere research, most of what is known about human telomere dynamics in vivo is based on these cells. Leukocyte telomere length (TL) is a complex trait that is shaped by genetic, epigenetic, and environmental determinants. In this article, we consider that smoking modifies leukocyte TL in humans and contributes to its variability among individuals, although the smoking effect on TL and its relation with other metabolic indices may accelerate biological aging and development of smoking‐induced chronic diseases in a large human population‐based cohorts with smoking behavior. Recent studies confirmed that individuals with shorter telomeres present a higher prevalence of arterial lesions and higher risk of cardiovascular disease mortality. This study originally suggests that efficient therapeutic protection of TL and structure in response to stresses that are known to reduce TL, such as oxidative damage or inflammation associated with tobacco smoking, would lead to better telomere maintenance. Recently, we have discovered the potential use of telomere‐restorative imidazole‐containing dipeptide (non‐hydrolized carnosine, carcinine) based therapy for better survival of smokers. We conclude that a better therapeutic or nutritional maintenance of TL may confer healthy aging in smokers and exceptional longevity in regularly ROS‐exposed human survivors.

Duration of senescent cell survival in vitro as a characteristic of organism longevity, an additional to the proliferative potential of fibroblasts.

More than 40 years have passed since the original publication by Hayflick and Moorhead led to the concept of the ‘Hayflick limit’ of the maximum number of divisions which somatic cells undergo in vitro. This concept is still regarded as a fundamental characteristic of species longevity. Here we want to emphasize another characteristic of somatic cells, namely, the duration of their survival in vitro in the non‐dividing state after cessation of proliferation. This is suggested on the basis of results of recent experiments with so‐called Japanese accelerated senescent mice. Results of these experiments reveal a good correlation between the longevity of the mice, the number of duplications of their fibroblasts in vitro, and the survival time of these cells in the non‐dividing state. In routine culture conditions, cell survival time may be very long, as much as a few years. However, when the cells are grown under conditions of oxidative stress, cellular longevity is markedly shortened. This new test may serve as an additional marker of organismic longevity. The comparative value of both tests, the classical ‘Hayflick limit’ and the new test, is discussed.

Telomere-dependent senescent phenotype of lens epithelial cells as a biological marker of aging and cataractogenesis: the role of oxidative stress intensity and specific mechanism of phospholipid hydroperoxide toxicity in lens and aqueous

Cataract formation represents a serious problem in the elderly and has a large impact on healthcare budget. Aging and cataract formation are relatively complex phenomena, both in vivo and in vitro. Telomeres are special structures at the end of chromosomes. They shorten during each round of replication, and this has been characterized as a mitotic counting mechanism. Our review analysis in this work shows that the rate of telomere shortening in human lens epithelial cells during aging and cataract formation is modulated by oxidative stress as well as by differences in antioxidative defense capacity of the normal and cataractous crystalline lenses. Presented in this review studies suggest that telomere shortening in human lens cells and increased oxidative stress are the result of the peroxidative damage to the lens cell membranes and biomolecules induced in the lack of reductive detoxification of phospholipid hydroperoxides as the triggering mechanism of cataractogenesis. Lipid peroxidation (LPO) is a causative factor of cataract. The increased concentrations of primary molecular LPO products (diene conjugates, lipid hydroperoxides) and end fluorescent LPO products were detected in the lipid moieties of the aqueous humor samples obtained from patients with senile and complicated cataracts when compared to normal donors. The progressive accumulation of oxidative damage may act as an important mechanism for organism aging and cataractogenesis. The oxidative stress form and intensity might determine the lens senescence rate and cataract type, making efforts in the cataract prevention challenge more complex. The analyzed challenge in this work is that the reduction in telomere shortening rate and damages in telomeric DNA make an important contribution to the anticataract and life‐extension effect of carnosine administered systemically in the formulations stabilizing a dipeptide from the enzymatic hydrolysis with carnosinase, or topically administered to the eye with carnosine ophthalmic prodrug N‐acetylcarnosine and lubricant formulations thereof including corneal absorption promoters. Telomere length in the human crystalline lens cells is a reflection of aging, cataractogenesis, and lifespan in biogerontological studies.

Advanced Drug Delivery of N-Acetylcarnosine (N-Acetyl-beta-alanyl-Lhistidine),Carcinine (Beta-alanylhistamine) and L-carnosine (Beta-alanyl-L-histidine) in Targeting Peptide Compounds as Pharmacological Chaperones for Use in Tissue Engineering, Human Disease Management and Therapy: From in vitro to the Clinic

A pharmacological chaperone is a relatively new concept in the treatment of certain chronic disabling diseases. Cells maintain a complete set of functionally competent proteins normally and in the face of injury or environmental stress with the use of various mechanisms, including systems of proteins called molecular chaperones. Proteins that are denatured by any form of proteotoxic stress are cooperatively recognized by heat shock proteins (HSP) and directed for refolding or degradation. Under non-denaturing conditions HSP have important functions in cell physiology such as in transmembrane protein transport and in enabling assembly and folding of newly synthesized polypeptides. Besides cellular molecular chaperones, which are stress-induced proteins, there have been recently reported chemical, or so-called pharmacological chaperones with demonstrated ability to be effective in preventing misfolding of different disease causing proteins, specifically in the therapeutic management of sight-threatening eye diseases, essentially reducing the severity of several neurodegenerative disorders (such as age-related macular degeneration), cataract and many other protein-misfolding diseases. This work reviews the biological and therapeutic activities protected with the patents of the family of imidazole-containing peptidomimetics Carcinine (β-alanylhistamine), N-acetylcarnosine (N-acetyl-β-alanylhistidine) and Carnosine (β-alanyl-L-histidine) which are essential constituents possessing diverse biological and pharmacological chaperone properties in human tissues.

Diabetes Mellitus: Novel Insights, Analysis and Interpretation of Pathophysiology and Complications Management with Imidazole-Containing Peptidomimetic Antioxidants

Patients suffering from the severe complications associated with both insulin- (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM): nephropathy, retinopathy, neuropathy, and atherosclerosis are still largely left without a prospect of an efficient treatment. Chronic hyperglycaemia, the primary clinical manifestation of diabetes, is associated with development of certain of the diabetic complications. The accelerated formation of advanced glycation end-products (AGEs) due to elevated glycemia has repeatedly been reported as a central pathogenic factor in the development of diabetic microvascular complications. Glucose and α-dicarbonyl compounds chemically attach to proteins and nucleic acids without the aid of enzymes. Initially, chemically reversible Schiff base and Amadori product adducts form in proportion to glucose concentration. The major biological effects of excessive nonenzymatic glycosylation are leading to increased free radical production and compromised free radical inhibitory and scavenger systems, inactivation of enzymes; inhibition of regulatory molecule binding; crosslinking of glycosylated proteins and trapping of soluble proteins by glycosylated extracellular matrix (both may progress in the absence of glucose); decreased susceptibility to proteolysis; abnormalities of nucleic acid function; altered macromolecular recognition and endocytosis; and increased immunogenicity. The discovery of chemical agents that can inhibit deleterious glycation reactions is potentially of great therapeutic benefit to all diabetes-associated pathologies. This study demonstrates the progress in development of patented carnosine mimetics resistant in formulations to enzymatic hydrolysis with human carnosinases that are acting as a universal form of antioxidant, deglycating and transglycating agents that inhibit sugar-mediated protein cross-linking, chelate or inactivate a number of transition metal ions (including ferrous and copper ions), possess lipid peroxidase type of activity and protection of antioxidant enzymes from inactivation (such as in a case of superoxide dismutase). Carnosine biological mimetics react with methylglyoxal and they are described in this study as a glyoxalase mimetics. The imidazole-containing carnosine biological mimetics can react with a number of deleterious aldehydic products of lipid peroxidation and thereby suppress their toxicity. Carnosine and carcinine can also react with glycated proteins and inhibit advanced glycation end product formation. These studies indicate a therapeutic role for imidazole-containing antioxidants (non-hydrolized carnosine, carcinine, D-carnosine, ophthalmic prodrug N-acetylcarnosine, leucyl-histidylhidrazide and patented formulations thereof) in therapeutic management strategies for Type 2 Diabetes.

Oxidative damage impact on aging and age-related diseases: drug targeting of telomere attrition and dynamic telomerase activity flirting with imidazole-containing dipeptides.

It has been documented that telomere-associated cellular senescence may contribute to certain age-related disorders, including an increase in cancer incidence, wrinkling and diminished skin elasticity, atherosclerosis, osteoporosis, weight loss, age-related cataract, glaucoma and others. Shorter telomere length in leukocytes was associated crosssectionally with cardiovascular disorders and their risk factors, including pulse pressure and vascular aging, obesity, vascular dementia, diabetes, coronary artery disease, myocardial infarction (although not in all studies), cellular turnover and exposure to oxidative and inflammatory damage in chronic obstructive pulmonary disease. It has been proposed that telomere length may not be a strong biomarker of survival in older individuals, but it may be an informative biomarker of healthy aging. The data reveal that telomere dynamics and changes in telomerase activity are consistent elements of cellular alterations associated with changes in proliferative state and in this article these processes are consequently considered as the new therapeutic drug targets for physiological control with advanced drug delivery and nutritional formulations. In particular, the presence of highly specific correlations and early causal relationships between telomere loss in the absence of telomerase activity and replicative senescence or crisis, and from the other side, telomerase reactivation and cell immortality, point to new and important treatment strategies or the therapeutic manipulation during treatment of age related disorders and cancer. Once better controls and therapeutic treatments for aging and age-related disorders are achieved, cellular rejuvenation by manipulating telomeres and enzyme telomerase activity may reduce some of the physiological declines that accompany aging. In this work, we raise and support a therapeutic concept of using non-hydrolyzed forms of naturally occurring imidazoledipeptide based compounds carnosine and carcinine, making it clinically possible that slowing down the rate of telomere shortening could slow down the human aging process in specific tissues where proliferative senescence is known to occur with the demonstrated evidence of telomere shortening appeared to be a hallmark of oxidative stress and disease. The preliminary longitudinal studies of elderly individuals suggest that longer telomeres are associated with better survival and an advanced oral nutritional support with non-hydrolyzed carnosine (or carcinine and patented compositions thereof) and patented N-acetylcarnosine lubricant eye drops are useful therapeutic tools of a critical telomere length maintenance that may fundamentally be applied in the treatment of age-related sight-threatening eye disorders, prolong life expectancy, increase survival and chronological age of an organism in health control, smoking behavior and disease.

Enhanced control of proliferation in telomerized cells

Clones of telomerized fibroblasts of adult human skin have earlier been obtained. It was shown that despite their fast growth in mass cultures, these cells poorly form colonies. Conditioned medium, antioxidants, and reduced partial oxygen pressure enhanced their colony formation, but not to the level characteristic of the initial cells. The conditioned medium of telomerized cells enhanced colony formation to a much greater extent than that of the initial cells. A study of proteome of the telomerized fibroblasts has revealed changes in the activities of tens of genes. A general trend consists in weakening and increased lability of the cytoskeleton and in activation of the mechanisms controlling protein degradation. However, these changes are not very pronounced. During the formation of immortal telomerized cells, selection takes place, which appears to determine changes in the expression of some genes. It was proposed that a decrease in the capacity of telomerized cells for colony formation is due to increased requirements of these cells to cell-cell contacts. The rate of cell growth reached that characteristic of mass cultures only in the largest colonies. In this respect, the telomerized fibroblasts resembled stem cells: they are capable of self-maintenance, but “escape” to differentiation in the absence of the corresponding microenvironment (niche), which is represented by other fibroblasts. Nondividing cells in the test of colony formation should be regarded as differentiated cells, since they have no features of degradation, preserve their viability, actively move, grow, phagocytize debris, etc. It was also shown that telomerization did not prevent differentiation of myoblasts and human neural stem cells. Thus, the results obtained suggest the existence of normal mechanisms underlying the regulation of proliferation in the telomerized cells, which opens possibilities of their use in cell therapy, especially in the case of autotransplantation to senior people, when the cell proliferative potential is markedly reduced and accessibility of stem cells is significantly restricted.

Sparse plating increases the heterogeneity of proliferative potential of fibroblasts

High heterogeneity of proliferative potential in the cultures of diploid human fibroblasts was reported in many studies. It was generally believed that the heterogeneity of proliferative potential of human fibroblasts reflects the unevenness of their senescence. However we show here that immortalized (telomerized) human fibroblasts obey the same rule. Up to 50% of these cells rapidly ceased to proliferate when plated at low density in contrast to usual conditions of mass culture where at least 98% of these cells keep on proliferating. Initially, we proposed that the appearance of non-dividing or slow-dividing cells in low-density cell culture experiments could be caused by cell damage due to the experimental setup. Indeed, lowering of oxygen level and addition of conditioned medium improved colony formation, but there were a large number of non-proliferating cells (13-20%). When we sparsely plated cells on a feeder layer of cells of certain density, the portion of non-proliferating cells decreased to 2%, i.e. became the same as in mass culture. Thus, the heterogeneity of proliferative potential is partially a result of the adverse effect of low cell density.