In our prior studies, we explored the functional and structural characteristics of the arterial wall, some of which are also characteristic for arterial aging, and were particularly interested in the improvement of altered characteristics of the arterial wall by low doses of fluvastatin and valsartan [ 6 ]. We found significant improvement of arterial wall characteristics after 30 days of treatment in middle-aged males, with the beneficial effect slowly declining within nine months of treatment discontinuation [ 7 9 ]. Importantly, for age-related changes, the improvement of arterial wall characteristics was associated with increased telomerase expression and reduced inflammation as well as oxidative stress parameters [ 10 11 ]. Both decreased telomerase expression along with decreased telomeres’ length and increased activation of inflammation and oxidative stress are characteristic of aging, arterial aging, and aging-related disorders. Therefore, we hypothesized that “anti-aging” or “rejuvenation” effects could be achieved through treatment impacting the longevity genes. This impact should have the capacity to influence “aging-related” disorders. Considering our previous results, this approach could be particularly effective for decreasing aging-related changes of the arterial system.

An aging population, along with increased life expectancy and the prevalence of associated chronic diseases, has become an important medical and economic issue. Consequently, the burden of so-called “aging-related disorders,” such as associated cardiovascular diseases, degenerative diseases of the central nervous system, and malignant diseases, is also increasing [ 1 2 ]. These diseases represent one of the leading problems of the healthcare systems in developed countries around the globe. Effective strategies for their prevention are therefore needed. Aging-related disorders have been found to be causally associated with the altered expression of aging-related or so-called longevity genes [ 3 ]. In addition, telomere length per se [ 4 ], as well as telomerase expression [ 5 ], are also associated with aging-related disorders. In a narrower context, it seems logical that these genes represent mechanistic intracellular targets that could be altered to change the intracellular pro-aging milieu. It is expected that with the induction of expression of protective and suppression of expression of harmful genes, a new rejuvenated cellular phenotype could be reached that could influence the occurrence and course of aging-related disorders. In summary, so-called rejuvenating strategies, focused on modifying such genes’ expression, could possibly importantly impact the prevalence of aging-related disorders [ 3 ].

We analyzed the expression of longevity genes in the treatment and control groups. At the beginning of the study, there was no difference in longevity genes expression in the four different groups (low-dose fluvastatin, low-dose valsartan, low-dose fluvastatin and valsartan combination, and the control group). Differences between the groups were only observed after 30 days of treatment. Five months after treatment discontinuation, the expression of longevity genes in all treatment groups decreased almost to initial values. In the control group, the expression of longevity genes did not change during the study ( Figure 1 A–F).

3. Discussion

SIRT1, PRKAA, KLOTHO ). Moreover, these changes correlated with an improvement of functional and structural arterial wall characteristics as well as with telomerase activity, both assessed previously [8,9, In the present study, we showed that low-dose fluvastatin and valsartan in combination significantly increased the expression of several important longevity genes (). Moreover, these changes correlated with an improvement of functional and structural arterial wall characteristics as well as with telomerase activity, both assessed previously [ 7 10 ]. Overall, the results revealed increased expression of several longevity genes that seems to be causally associated with increased telomerase activity and improvement of arterial wall (initial aging-related) characteristics. The results are very promising and indicate that our relatively simple but innovative approach could have potential efficacy as a “rejuvenating agent,” particularly in the efforts to decrease the occurrence of aging-related disorders.

The present study was designed as a two-part study: The first part comprised the measurement of longevity genes in relation to treatment with low-dose fluvastatin and valsartan, and the second part comprised of a correlation analysis with previously measured relevant parameters (telomerase activity and functional/structural arterial wall characteristics). The studied population was a group of middle-aged males with already present aging-related changes. Since we found that a low-dose combination of fluvastatin and valsartan improved arterial wall characteristics, we aimed to further explore the mechanism behind these beneficial effects. Thus, from the previously obtained samples, we assessed the expression of longevity genes to explore the potential rejuvenating effect of our approach. We found that the low-dose fluvastatin and valsartan combination increased the expression of the SIRT1 (1.8-fold; p < 0.0001), PRKAA (1.5-fold; p = 0.0262) and KLOTHO (1.7-fold; p < 0.0001) genes after 30 days, whereas no differences in the expression of the NFE2L2 , mTOR , and NF-κB genes were observed. Fluvastatin or valsartan alone were less effective, increasing only the expression of the SIRT1 gene to a lesser extent. Moreover, the expression of the SIRT1 gene in the combination group positively correlated with telomerase activity and improvement of the arterial wall characteristics.

Importantly, the FDA recently approved the first interventional “anti-aging” study (MILES—Metformin in Longevity Study). Metformin, which, for this purpose, was repositioned from a solely antidiabetic drug to an “anti-aging” drug, is the interventional drug. This is based on a wealth of data indicating that metformin could influence the aging process, or more importantly, aging-related disorders. Interestingly, one of the most prominent of the several hypotheses underlying the “anti-aging” effects of metformin is the activation of longevity genes with consequent effects on energy metabolism, inflammation and oxidative stress. Several other currently ongoing studies with metformin are focusing on its “anti-aging” effects, such as VA-IMPACT, TAME and ePREDICE. In any event, a new period in which specific treatments of “aging-related disorders” are being studied, has already begun.

SIRT1 regulates DNA transcription and repair as well as cell survival, thus also inducing longevity. It was shown to have an important role in aging-related disorders of the cardiovascular [ SIRT1 expression, for example, simvastatin in endothelial progenitor cells [ SIRT1 expression has not been assessed. To the best of our knowledge and according to the literature, no studies have assessed the effect of valsartan on the SIRT1 gene in humans, either. A few studies were performed on rats or mice, in which valsartan and other sartans increased the expression of the SIRT1 gene [17, PRKAA gene encodes the catalytic subunit of the AMPK. AMPK is the primary regulator of cellular responses and acts as a sensor to maintain energy balance within the cell [21, KLOTHO decreased in aging-related disorders [ KLOTHO and consequently induced cardiorenal beneficial effects in patients with diabetes mellitus and diabetic kidney disease [ KLOTHO expression was only shown in animal studies [ NFE2L2 gene encodes a transcription factor, which regulates the proteins involved in responding to injury and inflammation. According to some studies, enhancing NFE2L2 activity may be beneficial in diabetic cardiomyopathy, mitochondrial dysfunction, and as an anti-aging agent, but further studies are needed [ NFE2L2 in the vascular smooth muscle cells [ mTOR , which was proven for fluvastatin in rats [ NF-κB gene expression mediated vascular and myocardial inflammation and was additionally associated with impaired endothelial function [ NF-κB gene activation in various animal and cell line models [ Sirtuins are a family of nicotinamide adenine dinucleotide (NAD)-dependent deacetylases, and according to some studies, are one of the key molecules involved in the regulation of aging and aging-related disorders [ 12 ].regulates DNA transcription and repair as well as cell survival, thus also inducing longevity. It was shown to have an important role in aging-related disorders of the cardiovascular [ 5 ] and nervous systems [ 13 ]. Some statins in therapeutic doses were shown to induceexpression, for example, simvastatin in endothelial progenitor cells [ 14 ]. On the other hand, atorvastatin and rosuvastatin reduced its expression in patients with coronary artery disease [ 15 ]. Until now, the effect of fluvastatin in therapeutic or low doses onexpression has not been assessed. To the best of our knowledge and according to the literature, no studies have assessed the effect of valsartan on thegene in humans, either. A few studies were performed on rats or mice, in which valsartan and other sartans increased the expression of thegene [ 16 18 ]. Thegene encodes the catalytic subunit of the AMPK. AMPK is the primary regulator of cellular responses and acts as a sensor to maintain energy balance within the cell [ 19 ]. In animal and cell culture studies, several statins were shown to induce the AMPK and eNOS pathway, acting in a vasoprotective manner [ 20 22 ]. Valsartan was also shown to act protectively through activation of the AMPK pathway in diabetic rats [ 23 ]; similar effects have been shown for telmisartan in human coronary artery cells [ 24 ]. The consequences of AMPK activations were divergent: In acute activation, it caused cell protection, whereas in chronic activation, it might have activated the pro-aging pathways and progressive degeneration during cellular senescence. There are various interactions between sirtuin and AMPK pathways [ 19 ]. The expression ofdecreased in aging-related disorders [ 25 ]. Valsartan in therapeutic doses increased the amount of plasma-solubleand consequently induced cardiorenal beneficial effects in patients with diabetes mellitus and diabetic kidney disease [ 26 ]. The effects of valsartan in low doses have not yet been studied in such a setting. The potential beneficial effects of statins onexpression was only shown in animal studies [ 27 ]. Thegene encodes a transcription factor, which regulates the proteins involved in responding to injury and inflammation. According to some studies, enhancingactivity may be beneficial in diabetic cardiomyopathy, mitochondrial dysfunction, and as an anti-aging agent, but further studies are needed [ 28 ]. Fluvastatin was shown to inducein the vascular smooth muscle cells [ 29 30 ]. Mammalian target of Rapamycin (mTOR) was shown to have an important role in cardiovascular diseases, oxidative stress and longevity [ 31 ]. In animal or cell studies, statins influenced the, which was proven for fluvastatin in rats [ 32 ] and for lovastatin in vascular smooth muscle cells [ 33 ]. In one study on rats, valsartan induced cardio protection against ischemic-reperfusion injury through the mTOR [ 34 ].gene expression mediated vascular and myocardial inflammation and was additionally associated with impaired endothelial function [ 35 ]. There is evidence that both statins and sartans could reduce thegene activation in various animal and cell line models [ 36 37 ].

8, To the best of our knowledge, no study like the present one, has been published. In our review of the literature, we found several different studies that assessed the effects of statins or sartans on longevity genes, but most of those studies were performed either on cell lines or animals. Therefore, the present study is the first to show that our new preventive cardiovascular approach, which was proven to induce the improvement of arterial wall functional and structural characteristics, and consequently decrease arterial age, additionally acted through the expression of several longevity genes. This could be one of the mechanisms lying behind the beneficial effects observed in our previous clinical studies [ 7 9 ]. Nevertheless, one of the limitations of the present study is that we used only qPCR, but validation by Western blotting would be of added value.

The results of the present study indicate that our innovative approach using short-term low-dose fluvastatin and valsartan has a potential in inducing the expression of certain longevity genes. These effects could be anti-aging or rejuvenating as well as act as a potential specific prevention/treatment for “aging-related” disorders. It can be speculated that using cycling, intermittent treatment with low-dose combination (every 6–12 years) starting at middle-age could postpone the occurrence of aging-related disorders. On the other hand, this approach could be used in the same population and with the same aim as metformin in the MILES trial. One of the major advantages of this approach is its cyclic, intermittent character, which, as previously described, could potentially activate the beneficial longevity genes for a time short enough not activate their contra regulatory mechanisms as well. With intermittently repeating cycles, this could lead to repetitive beneficial activations of the protective longevity genes. Thus, it could be speculated that the cumulative effect of these repeating cycles of treatment might eventually lead to successful specific prevention/treatment of “aging-related” disorders, most likely cardiovascular “aging-related” disorders.