Low-Dose Fluvastatin and Valsartan Rejuvenate the Arterial Wall Through Telomerase Activity Increase in Middle-Aged Men

Abstract

Background and Aim:

Previously, we have shown that slightly to moderately aged arteries in middle-aged males can be rejuvenated functionally by sub-therapeutic, low-dose fluvastatin and valsartan treatment. Here, we explore whether this treatment could also increase telomerase activity. We hypothesized that telomerase activity might be associated with (1) an improvement of arterial wall properties and (2) a reduction of inflammatory/oxidative stress parameters (both observed in our previous studies).

Methods:

The stored blood samples from 130 apparently healthy middle-aged males treated with fluvastatin (10 mg daily), valsartan (20 mg daily), fluvastatin and valsartan combination (10 and 20 mg), respectively, and placebo (control), were analyzed. The samples were taken before and after treatment lasting 30 days, and 5 months after treatment discontinuation. Telomerase activity was measured in blood leukocytes by a TaqMan Gene Expression Assay.

Results:

Low-dose fluvastatin or valsartan increased telomerase activity (106.9% and 59.5% respectively; both p < 0.05, vs. control), whereas their combination was even more effective (an increase of 228.0%; p < 0.001, vs. control). No change was noted in the control group. Importantly, increased telomerase activity obtained in the combination group significantly correlated with arterial function, measured by flow-mediated dilation (FMD) (r = 0.79; p < 0.001) and C-reactive protein concentration (r = −0.54; p = 0.02) and total anti-oxidative status (r = 0.50; p = 0.03).

Conclusion:

We found that a low-dose combination of fluvastatin and valsartan substantially increased telomerase activity, which significantly correlated with an improvement of endothelial function and a decrease of inflammation/oxidative stress. These findings could lead to a new innovative approach to arterial rejuvenation.

Introduction

Telomeres are the ends of linear chromosomes that consist of multiple repetitions of short DNA sequences of six nitrogenous bases rich in guanidine (TTAGGG). These sequences are repeated over several thousand base pairs.^1,2 Each cell division shortens the telomeres. Telomerase is an enzyme complex that synthesizes multiple tandem repeats of DNA at the chromosomes' ends during cell division and consequently compensates for the erosion of the DNA ends during replication. It is one of the key elements of maintaining telomere integrity.^1
–3 The telomerase enzyme complex contains a reverse transcriptase (human telomerase reverse transcriptase [hTERT]) protein and a template-containing RNA component (TR).³ Telomere length is considered to be one potential designator of biologic aging.^2,4

Cellular senescence has been proven to have a significant role in cardiovascular diseases and cardiovascular aging. Cellular senescence is marked by telomerase activity diminution and consequent telomere shortening. In the cardiovascular system, these processes are the most prominent in the endothelial progenitor cells, endothelial cells, and vascular smooth muscle cells.^1,2,5 When the telomere length in the aforementioned cells shortens to a critical point, the cells enter the process of apoptosis and cellular senescence, which leads to vascular system aging.¹ In general, cellular senescence is associated with many phenotypic changes suggestive of aging.⁵ This is further accelerated by oxidative stress, inflammation, cardiovascular risk factors (unhealthy lifestyle, smoking, etc.), and other stimuli.⁴ Therefore, treatment that activates telomerase activity could be an effective anti-aging agent that induces arterial rejuvenation.⁶ Statins have been shown to act on telomere biology in therapeutic^7
–9 as well as in low doses.¹⁰ On the other hand, the effects of sartans or a combination of low-dose fluvastatin and valsartan on telomere biology has not been studied yet.

In our previous studies, we found that short-term (1-month) treatment with low-dose fluvastatin, valsartan, and especially their low-dose combination improved functional and structural arterial wall characteristics through anti-oxidative and anti-inflammatory activity.¹¹ These observations, at least partially, point to arterial rejuvenation. Rejuvenation of the arteries has long been the goal of rejuvenation medicine. The aim of the present study was to investigate whether selected drugs in particularly low doses could also increase telomerase activity in the same population. We further hypothesized that telomerase activity might be associated with an improvement of arterial wall properties and inflammatory/oxidative stress parameters (both measured and determined in our previous studies).

Methods

Study design and participants

This study was designed as a further analysis of stored blood samples from our three previous studies that investigated the effects of low-dose fluvastatin, valsartan, or their low-dose combination on arterial wall properties in apparently healthy, middle-aged males.^12
–14 The treatment period was 30 days in all three studies; blood samples were collected, and ultrasound measurements of the arterial wall properties were performed at the beginning and at the end of the treatment period (day 0 and day 30). The measurements were repeated 5 months after treatment discontinuation.

Overall, 130 middle-aged, apparently healthy male participants were recruited: 25 were treated with fluvastatin (10 mg daily), 20 with valsartan (20 mg daily), and 20 with a low-dose combination of fluvastatin (10 mg daily) and valsartan (20 mg daily). Accordingly, the same number of participants received a placebo. All of the participants were randomized blindly into these groups.^12
–14 The National Medical Ethics Committee of Slovenia approved the studies, and informed consent was obtained from all participants. Inclusion criteria were age between 30 and 50 years, non-smoking, normal blood pressure values, body mass index values below 30 kg/m², no clinical cardiovascular disease, no history of any other chronic disease, or any regular medication therapy. The subject characteristics have already been extensively described in our previous publications.^12
–14

Blood sampling

Three samples of peripheral whole blood were collected from the cubital vein of each participant before starting the treatment (day 0), after the treatment (day 30, i.e., on the day when the therapy was concluded), and 5 months after treatment discontinuation (follow-up). The whole blood samples were collected in 10-mL EDTA tubes and stored at −80°C. Prior to RNA extraction, the samples were centrifuged at 4000 rpm for 25 min to obtain pellets for RNA extraction.

RNA isolation

Total RNA was isolated from blood using an miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA was quantified using a NanoDrop spectrophotometer, and cDNA was synthesized from 300 ng of the total RNA using the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Foster City, CA) according to the manufacturer's protocols.

Quantitative real-time PCR for hTERT expression

The expression of hTERT in the tested samples was performed using TaqMan Gene Expression Assay Hs00972656_m1 (Applied Biosystems) according to the manufacturer's instructions. The housekeeping gene glyceraldehyde 3-phoshate dehydrogenase (GAPDH) was used as an endogenous control. Briefly, quantitative real-time PCR (qPCR) was performed using the ABI 7900 Fast Real-Time PCR System (Applied Biosystems). Individual qPCR reactions were carried out in a 10-μL reaction mix with 2× TaqMan Universal PCR Master Mix (Applied Biosystems), 1× TaqMan Gene Expression Assay (Applied Biosystems), and 200 ng of complementary DNA (cDNA). Each sample was analyzed in triplicate. RNA isolated from human MCF7 breast adenocarcinoma cells (American Type Culture Collection [ATCC], Rockville, MD) was used as a positive control for hTERT expression. The data were analyzed by the SDS2.4 software and Ct values were extracted. Fold-differences in hTERT expression were calculated using the comparative cycle threshold (Ct) method as described previously,¹⁵ where data were normalized to day 0 for each participant.

Data analysis

All values were expressed as mean ± standard error of the mean (SEM). The differences between values were assessed by one-way analysis of variance (ANOVA). When a significant interaction was present, the Bonferroni post-test was performed. Correlations among arterial wall properties and inflammation/oxidative parameters, described previously,^11

–14 and telomerase activity assessed in the present study were calculated using Pearson correlation coefficients. A p value of less than 0.05 was considered significant. All statistical analyses were performed using the Graph Pad Prism 5.0 software.

Results

Telomerase activity

We performed an analysis of telomerase activity between treatment groups (treated vs. control groups) and an analysis within each group (before vs. after treatment). At the beginning of the study, there was no difference in telomerase activity among four different groups (low-dose fluvastatin, low-dose valsartan, low-dose combination of fluvastatin and valsartan vs. control). After 1 month of treatment with low-dose fluvastatin and low-dose valsartan, telomerase activity increased significantly compared to the slight insignificant increase in the control group, by 106.9% and 59.5%, respectively (both p < 0.05). Telomerase activity increased maximally after 1 month of treatment with a low-dose fluvastatin and valsartan combination, i.e., by 228.0% (p < 0.001) compared to the control group (Fig. 1C).

FIG. 1.

Telomerase activity in peripheral leukocytes in low-dose fluvastatin group (10 mg daily) (a), low-dose valsartan group (20 mg daily) (b), low-dose combination of fluvastatin and valsartan (10 mg and 20 mg daily, respectively) (c), and control group (d) before the treatment (0), at the end of treatment after 30 days (30), and 5 months after treatment discontinuation (FU). Values are presented as means ± standard error of the mean (SEM). (*) p < 0.05 and (***) p < 0.001, versus initial values in the separate groups. (+) p < 0.05 and (+++) p < 0.001, versus day 30 in the control group.

Treatment with low-dose fluvastatin, valsartan, or their low-dose combination significantly increased telomerase activity after 1 month of treatment compared to the initial values measured within the separate groups before treatment. Telomerase activity increased by 140.7% with low-dose fluvastatin (p < 0.05), by 85.5% with low-dose valsartan (p < 0.05), and by 280.5% with the low-dose combination (p < 0.001) treatment. Five months after discontinuation of treatment, telomerase activity decreased to almost initial values in all treatment groups. In the control group, telomerase activity did not change significantly during the course of the study (Fig. 1A–D).

Five months after discontinuation of treatment, the telomerase activity decreased to almost initial values in groups treated with either low-dose of fluvastatin or valsartan. In the low-dose combination group, the telomerase activity 5 months after discontinuation of treatment was much lower than immediately after treatment, but still higher compared to the initial values; however, the difference was not significant.

Correlations between baseline characteristics of the study participants and telomerase activity

There were no significant correlations between baseline characteristics of the study participants and telomerase activity at the beginning of the study, except for age (β = −0.236; p < 0.05), as presented in Table 1.

Table 1.

Linear Regression Analysis Between the Baseline Characteristics of the Study Participants and Telomerase Activity

Parameter	β	p
Age (years)	−0.236	0.049^*
BMI (kg/m²)	−0.029	0.820
LDL-C (mmol/liter)	−0.103	0.426
Plasma glucose (mmol/liter)	−0.030	0.816
SBP (mmHg)	−0.070	0.589
DBP (mmHg)	−0.043	0.738
Heart rate (bpm)	−0.014	0.916

p < 0.05.

BMI, body mass index; LDL-C, low-density lipoprotein cholesterol; SBP, systolic blood pressure; DBP, diastolic blood pressure; bpm, beats per minute.

Correlations between telomerase activity, arterial wall properties, and inflammation/oxidative stress parameters

The correlations between telomerase activity, arterial wall properties, and inflammation/oxidative stress parameters, described previously,^11

–14 were calculated for all treatment groups. In the low-dose fluvastatin group, telomerase activity positively correlated with flow-mediated dilation (FMD) of the brachial artery (r = 0.49; p = 0.03) and negatively with vascular cell adhesion molecule-1 (VCAM-1) concentration (r = −0.44; p = 0.05). In the low-dose valsartan group, telomerase activity positively correlated with FMD (r = 0.46; p = 0.02) and negatively with high-sensitivity C-reactive protein (hsCRP) (r = −0.70; p < 0.001). In the low-dose combination group, telomerase activity correlated positively with FMD (r = 0.79; p < 0.001) and with total anti-oxidative status (TAS) (r = 0.50; p = 0.03) and negatively with hsCRP concentration (r = −0.54; p = 0.02).

Discussion

In the present study, we found that 1 month of treatment with low-dose fluvastatin, valsartan, and especially their combination substantially increased telomerase activity. Furthermore, increased telomerase activity was significantly associated with endothelial function improvement (measured as FMD) and a decrease in inflammation and oxidative stress. Taking all current and previous results together, it seems that cyclic, short-term, and particularly combination treatment with low-dose fluvastatin and valsartan could represent an effective arterial rejuvenation approach.

Apparently healthy middle-aged participants were recruited for the present study. Although not having risk factors for cardiovascular diseases, their endothelial function was significantly diminished. The endothelial dysfunction observed in the studied population was attributed to age-associated changes of arterial wall functioning and was the main target of our new innovative vascular rejuvenation approach. Blood samples were taken before and after treatment with low-dose fluvastatin, valsartan, or their low-dose combination. Telomerase activity measurement in the present study was performed in blood leukocytes, because a significant relationship between telomere length in leukocytes and vascular tissues for humans had been shown previously.¹⁶

Accordingly, we propose the same phenomenon for the telomerase activity because the two are strongly connected. We found that telomerase activity increased in all treatment groups after 1 month of treatment compared to the level in the control group (day 30)—in fluvastatin by 106.9%, valsartan by 85.5%, and maximally in the combination group, i.e., by 228.5%. In all treatment groups, telomerase activity increased after a short period of 1 month of treatment, compared to the initial values in each observed group (fluvastatin by 140.7%, valsartan 85.5%, and the combination by 280.5%). The increase in telomerase activity was associated with improvement in FMD and/or the anti-oxidative/anti-inflammatory action of the low-dose drugs used. These associations might be causal, parallel, or coincidental. Considering the complexity of the arterial wall biology, it seems likely that these relations are causal.

We speculate that the treatment increased telomerase activity in endothelial cells, allowing for their better functioning, which manifested as an improvement in FMD. Furthermore, the decrease in inflammation/oxidative stress additionally protected telomerase activity. The telomerase activity decreased after treatment discontinuation almost reaching initial values in both the low-dose fluvastatin and low-dose valsartan treatment groups. Increased telomerase activity was still observed in the group treated with a low-dose combination of fluvastatin and valsartan, although the difference did not reach the level of statistical significance. However, on the basis of this observation, we can further speculate that the combination is a much more potent activator of telomerase activity than each drug separately. The mechanism of this prolonged effect is not known, but it is in line with our previous observations that a low-dose combination still has significant protective effects on the arterial wall months after treatment discontinuation.¹³

To the best of our knowledge, this is the first study assessing the effect of low-dose statin, valsartan, or their low-dose combination on telomerase activity. In previous studies, the beneficial effects of statins on telomerase activity had been observed, and in most studies, statins were used in therapeutic doses.^7
–9 We found only one study evaluating the effect of low-dose statin treatment on telomerase activity.¹⁰ The effects of sartans or their combinations with statins had not been tested yet. Low-dose atorvastatin was shown to delay the onset of replicative senescence of endothelial cells by counteracting the increased reactive oxygen species (ROS) production linked to endothelial cell aging and through their effect on hTERT.¹⁰ Boccardi et al. found that treatment with statins at therapeutic doses was associated with higher telomerase activity, longer telomere length in leukocytes, and significantly lower telomere shortening with aging.⁷ Statin treatment was associated with longer leukocyte telomere length in patients after myocardial infarction.⁹

It is known that endothelial cells undergo only a limited number of cell divisions, and then stop dividing, entering a state that is designated replicative senescence. Shortening of telomeres in endothelial cells is believed to be a molecular clock that triggers this senescence. It has been shown that increasing telomerase activity might modulate or supress shortening of telomeres. Even more, it had been shown in vitro that nitric oxide (NO) increased telomerase activity in endothelial cells, thereby protecting endothelial cells from senescence. NO might decrease oxidative stress and protect telomerase or might directly up-regulate telomerase activity via transcriptional and/or post-transcriptional mechanisms. The demonstration that NO affects telomerase activity and consequently delays endothelial cells' senescence establishes its endothelial rejuvenation function.¹⁷

We could speculate that treatment with low-dose fluvastatin and valsartan increases telomerase activity in endothelial cells by increasing synthesis of NO in the endothelium (as shown in our previous studies, i.e., indirectly by increasing NO synthase activity and improving NO dependent vasorelaxation).^18
–20 Additionally, it has been established that there was a biphasic dose-dependent effect of atorvastatin and cerivastatin on angiogenesis in vitro and in vivo. It turned out that at low therapeutic concentrations, these drugs posed a pro-angiogenic effect, while acting angiostatic at high concentrations.²¹ Taking these facts together, we could further speculate that low doses of the drugs used could be more potent in rejuvenating the arterial wall than the therapeutic doses used in everyday clinical practice.

We could additionally speculate about some insights into the mechanism behind the telomerase activity increase when treating with a low-dose combination of fluvastatin and valsartan. Because telomerase activity correlates with both the FMD (basically influenced by NO) and inflammation/oxidative stress parameters, it could be deduced that the underlying mechanism lies in the increase in NO and decrease of the inflammation/oxidative stress. This assumption is also supported by the observations from the literature, where NO increase leads to increase in telomerase activity.¹⁷

Determination of telomerase activity only rather than the telomere length per se represents a limitation of the present study. Nevertheless, this was done intentionally because there were no significant changes in telomere length in our pilot study. Therefore, we believe that determination of telomere length is a less sensible method, because it is difficult to detect small changes in telomere length that would be expected during short time course studies (this study was intentionally designed this way²²) as in our study (1–5 months). Another limitation of our study was the measurement of telomerase activity in peripheral blood leukocytes and not directly in the arterial wall. The present study was clinical, thus it was impossible to obtain such samples. However, the strong correlation between telomere length in peripheral blood leukocytes and telomere length in the arterial wall has already been shown.¹⁶

Our inability to distinguish whether the observed telomerase activity result was the consequence of increased telomerase activity in the endothelial cells already present in the vascular system or an increased number of endothelial progenitor cells or both is another partial limitation resulting from the design of the present study. In any case, increased telomerase activity was observed and was a clear sign of arterial rejuvenation.

In conclusion, we found that 1 month of treatment with low-dose fluvastatin, valsartan, and especially their low-dose combination substantially increased telomerase activity in apparently healthy middle-aged males. Additionally, these findings correlated with improvement of functional arterial properties, particularly FMD improvement, and also with the diminishment of inflammation and oxidative stress levels. Therefore, it is logical to deduce that the approach tested could lead to a new innovative approach for arterial rejuvenation. Obviously, larger-scale human studies in this emerging promising field are desired and needed.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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