Plasma levels of sirtuin 1 and sirtuin 2 in ascending aortic aneurysm patients - a comparative study.

Aortic aneurysm (AA) is the second most prevalent aortic disease that may affect the thoracic or abdominal aorta. In thoracic AA, dilated lesions occur mainly in the ascending aorta [ 1 ]. The risk of life-threatening aortic dissection or rupture increases with an increase in the aortic diameter. Current treatment options are limited to blood pressure control and surgery. However, surgery is associated with considerable mortality [ 1 – 3 ]. Moreover, patients with AA, especially asymptomatic ones, are at increased risk of cardiovascular disease [ 1 ]. As effective nonsurgical interventions in AA are lacking, there has been an ongoing search for biological markers that might be useful in the diagnosis and treatment of the disease [ 4 ]. Candidate markers include sirtuins, the mammalian homologs of yeast silent information regulator-2 protein (SIR2) mediating longevity in unicellular yeast under calorie restriction [ 5 , 6 ]. The mammalian sirtuin family comprises seven members, SIRT1–7, classified into four types depending on localization, activity, and function [ 7 ]. The first two members of the sirtuin family, sirtuin 1 (SIRT1) and sirtuin 2 (SIRT2), participate in various processes from cell proliferation and apoptosis, through DNA repair, to metabolism [ 7 ]. Experimental studies showed that, among the many areas of activity, SIRT1 is essential for maintaining vascular wall integrity and contributes to the prevention of AA [ 8 – 11 ]. However, to date, there have been no studies assessing plasma or serum sirtuin levels in human plasma or serum. Considering these gaps in knowledge, the aim of this study was to evaluate plasma SIRT1 and SIRT2 levels in patients with and without ascending AA. Another objective was to assess associations between sirtuin levels and the clinical and laboratory variables to improve the current understanding of how sirtuins affect AA. This may have important implications for management of the disease.

The study included patients hospitalized at the University Teaching Hospital in Lodz, Poland, and were treated at an outpatient cardiology clinic of the same hospital between March 2019 and March 2020. The research was a continuation of preliminary data published previously [ 12 ]. Patients with a diagnosis of ascending AA were enrolled. Individuals without AA served as controls. Plasma SIRT1 and SIRT2 levels were measured in each participant, and differences between patients with ascending AA and controls were assessed. The demographic and clinical data of patients were retrieved from medical records, and their associations with sirtuin levels were evaluated. The study population included consecutive patients with ascending AA and controls without AA. The inclusion criteria for cases were as follows: age > 18 years, diagnosis of ascending AA according to the 2014 European Society of Cardiology guidelines on the diagnosis and treatment of aortic diseases [ 1 ], written informed consent to participate in the study, and availability of relevant data in hospital medical records. The inclusion criteria for controls were the same as for those with a diagnosis of ascending AA. Data extracted from medical records included demographic data, anthropometric measurements, and clinical data such as comorbidities, laboratory tests, and imaging results. Blood samples were collected from patients in a fasting state in the morning and before treatment implementation. The plasma samples were frozen and thawed on the day of sirtuin measurement. Sirtuin 1 and sirtuin 2 levels were assessed using the human SIRT1 and SIRT2 enzyme-linked immunosorbent assay (ELISA) laboratory kit (Shanghai Sunredbio [SRB] Technology Co., Ltd, China). Optical density was determined at 450 nm using a microplate reader (SYNERGY HT, BioTek Instruments Inc., USA). The study was approved by the Ethics Committee of the Medical University of Lodz (RNN/139/16KE; as of 10 May 2016). Written informed consent to participate in the study was obtained from all patients. The study conformed to the principles outlined in the Declaration of Helsinki. Statistical analysis was performed using the R statistical software version 4.1.3. The Fisher exact test and the Pearson χ 2 test were used to compare the distribution of categorical variables between cases and controls. The Shapiro–Wilk test was used to assess the normality of distribution for continuous variables. Variables with normal distribution were compared between groups using the Student t-test, and those with nonnormal distribution — using the Wilcoxon rank sum test (Mann–Whitney test). In most cases, continuous variables did not show a normal distribution; therefore, medians and 25th (Q1) and 75th (Q3) percentile values were presented in the descriptive statistics for continuous variables. The Spearman rank correlation was used to assess correlations between sirtuin levels and other continuous variables. The receiver operating characteristic curve (ROC) was used to determine the cutoff values for SIRT1 and SIRT2 levels to differentiate between patients with and without AA.

The study group included 32 patients (87.5% of men) with ascending AA with a median age of 67 years (IQR: 59.50–75.25). About one-third of patients (31.2%) had symptomatic AA. The most common comorbidities included hypertension (100%), atrial fibrillation (AF, 46.9%), coronary artery disease (CAD, 37.5%) chronic kidney disease (CKD, 34.4%), and chronic obstructive pulmonary disease (COPD, 18.8%). The control group included 37 adequately matched (sex-, age- and disease-matched) patients without AA (median age: 68 years, IQR: 64.00, 74.00). There were no differences in demographic data, comorbidities, and laboratory results between cases and controls ( Tab. 1 ). Plasma SIRT1 levels were slightly higher in patients with ascending AA than in controls (median IQR: 9.40 [7.42, 22.70] vs. 7.70 [7.00, 11.00]) and the same result was noted with regard to plasma SIRT2 levels (median IQR: 8.00 [6.52, 18.12] vs. 7.70 [6.20, 11.90]); however, the differences did not reach statistical significance ( Fig. 1 ). Sirtuin 1 levels correlated with SIRT2 levels both in patients with ascending AA (R = 0.867, p < 0.001) and in controls (R = 0.825, p 0.05). There were also no correlations between sirtuin levels and age or body mass index, except for a weak correlation of SIRT1 with age in the control group (R = 0.354, p = 0.032). Sirtuin 2 levels were inversely correlated with the levels of total cholesterol (R = −0.354, p = 0.032) and low-density lipoprotein (LDL) cholesterol (R = −0.359, p = 0.030) in the control group. However, no correlations between sirtuin levels and laboratory test results were found in patients with ascending AA. The comparison of SIRT1 levels between cases and controls depending on existing comorbidities indicated higher SIRT1 levels in patients with ascending AA who did not have the diagnosis of CAD (p = 0.012), AF (p = 0.030), or COPD (p = 0.026) ( Tab. 2 ). Moreover, patients with ascending AA who did not have specific comorbidities (such as diabetes, CKD, smoking, heart failure, hyperlipidemia, cancer, and previous cardiac surgery) had generally higher median SIRT1 levels than disease-matched controls, but the difference was not significant. Thus, the presence of comorbidities had possibly evened out the differences in SIRT1 levels (especially in patients with AF, diabetes, hyperlipidemia, and smoking) or reversed those differences (in patients with CAD, COPD, CKD, or previous cardiac surgery). Differences in SIRT2 levels between cases and controls showed borderline significance in the case of COPD, with higher levels in controls than in patients with AA (p = 0.054). Sirtuin 2 levels were generally higher in patients with ascending AA without comorbidities than in disease-matched controls, except for previous cardiac surgery and heart failure. However, the differences were not significant. The analysis of sirtuin levels in patients with ascending AA revealed higher SIRT2 levels in patients without COPD vs. those with COPD (p = 0.04) ( Tab. 2 ). The remaining differences included higher median SIRT1 and SIRT2 levels in patients with ascending AA with a history of cardiac surgery and no diagnosis of CAD, diabetes, CKD, hyperlipidemia, cancer, or smoking as well as in patients with heart failure (New York Heart Association class III/IV) or AF. However, these differences were not significant. In the control group, SIRT1 levels were higher in patients with a history of cardiac surgery (p = 0.035). Median SIRT1 and SIRT2 levels were also higher in patients with any comorbidity except for cancer, but the differences were not significant ( Tab. 2 ). The receiver operating characteristic curve analysis of the diagnostic ability of sirtuins to predict ascending AA revealed that a cut-off value of 8.05 ng/mL for SIRT1 levels differentiated between patients with and without AA. For sirtuin 2 levels, the cut-off value was 7.65 ng/mL ( Fig. 2 ).

Sirtuins, the proteins involved in numerous physiological processes, have emerged as a possible target in the treatment of various pathological conditions such as neurodegenerative, cardiovascular, and metabolic diseases or cancer. However, before becoming a treatment target, the contribution of sirtuins to a pathophysiological process has to be confirmed in research. The significance of sirtuins in AA was described in numerous studies conducted mainly on mouse models [ 9 – 11 , 13 , 14 ]. Wang et al. [ 10 ] reported that SIRT1 overexpression in transgenic mice prevented the development of an induced aneurysm. While the availability of human samples is limited, it was demonstrated that in specimens collected intraoperatively from patients with abdominal AA, the expression and activity of SIRT1 in smooth muscle cells was significantly reduced [ 11 ]. In clinical practice, it would be valuable to measure sirtuins in more available biological material such as plasma or serum. However, according to available research, no previous studies assessed serum or plasma sirtuin levels in patients with AA. This prompted the current investigation into plasma levels of sirtuins in patients with ascending AA and to evaluate whether SIRT1 and SIRT2 levels are associated with the presence of AA. Also under assessment were correlations between SIRT1 and SIRT2 levels and various clinical factors. The study group was comprised of 32 patients with ascending AA. Most patients were male, which confirms that AA is more likely to occur in men than in women [ 15 ]. The clinical characteristics of patients reflected the common risk factors for AA, such as advanced age, high blood pressure, high cholesterol levels, and CAD [ 16 ]. To identify the possible relationship between sirtuin levels and the presence of AA, sirtuin levels were compared between patients with ascending AA and controls. Patients with ascending AA had slightly higher SIRT1 and SIRT2 levels than controls, but the difference was not significant. This finding may seem surprising for two reasons. First, most previous studies indicated that the development of AA is associated with reduced SIRT1 expression [ 10 , 11 , 13 ], however, these studies were conducted on mouse tissue samples, which may account for differences compared to the present findings based on human plasma samples. Second, other studies showed lower SIRT1 levels in patients with cerebrovascular stroke [ 17 ], diabetes [ 18 ], or primary open-angle glaucoma [ 19 ], as compared with control groups without those diseases. Although these studies also measured sirtuin levels using the ELISA method — suggesting that methodological differences are unlikely to explain the discrepancy in results — their study groups were larger, which may have contributed to achieving statistical significance, unlike in the current study. Additionally, the control groups in those studies were described as “healthy” subjects. However, it was not clarified whether “healthy” meant free of the specific condition under investigation or free of any comorbidities. In contrast, the control group in this study included individuals without AA but with other comorbidities, which may have masked potential differences in sirtuin levels between patients with and without AA. As shown, the discrepancies between the present results and those of other studies may stem from differences in study design. The available data on the association of sirtuins with AA come from in-vitro studies of tissue samples, obtained mainly from mice, which limits a direct comparison with the current results. Moreover, the discrepancies may be — at least partially — caused by the characteristics of controls. Herein, the control group included patients without AA but with multiple comorbidities (similar to patients with ascending AA). In contrast, other studies assessing the differences in blood sirtuin levels included healthy controls, which is the typical approach in research studies. However, considering that most elderly patients have numerous diseases, it was assumed that — from a clinical perspective — it would be more useful to compare sirtuin levels between patients with and without AA, but with similar comorbidities. In such a cohort, SIRT1 and SIRT2 levels were comparable between patients with and without AA, with a trend toward higher values in patients with ascending AA, especially for SIRT1. The above findings differ from most published reports, but there were also studies that showed no significant differences in sirtuin levels between cases and controls or higher sirtuin levels in patients with some diseases. Charążka et al. [ 20 ] revealed that sirtuin levels did not differ significantly between patients with polycystic ovary syndrome and controls. Sansone et al. [ 21 ] reported that SIRT1 levels increased with the severity of endometriosis. In a study by Zheng et al. [ 22 ], elevated plasma SIRT2 levels predicted heart failure after acute myocardial infarction. Also, in a previous study [ 12 ], SIRT1 and SIRT2 levels were higher in patients with ascending AA than in those with ascending aortic dissection. The lack of significant differences in sirtuin levels between patients with ascending AA and controls in the present study may be partially explained by the size of the study group and the presence of comorbidities that may affect AA. Nevertheless, sirtuin levels in this study were not lower in patients with ascending AA than in controls, which is in contrast to some other reports that described either reduced sirtuin levels in patients with specific diseases vs. healthy controls [ 17 , 18 ] or decreasing sirtuin levels along with an increase in disease severity [ 23 ]. However, it is important to note again that these studies included larger sample sizes and defined their control groups as healthy individuals, which may account for the observed differences. To date, no reference ranges for plasma or serum sirtuin levels have been established in humans, which further complicates the interpretation of the present results. In this study, patients with ascending AA had a median SIRT1 level of 9.40 (IQR: 7.42, 22.70) ng/mL and a median SIRT2 level of 8.00 (IQR: 6.52, 18.12) ng/mL. It is difficult to determine if these levels are elevated, normal, or reduced. For now, the only conclusion is that these levels are slightly higher than in patients without AA; however, normal values have not been determined. Available studies assessing sirtuin levels reported the mean or median SIRT1 levels to range from 0.4 ng/mL [ 24 ] to 1.7 ng/mL in healthy controls [ 25 ]. In all these cases, sirtuin levels were measured with the ELISA. The levels reported for healthy individuals are considerably lower than those observed in the current study in patients with or without AA. Therefore, it was hypothesized that existing comorbidities may affect sirtuin levels or sirtuin levels are increased in patients with ascending AA. To investigate deeper regarding the effect of comorbidities on sirtuin levels, sirtuin levels were compared between patients with and without AA in the subgroups of patients classified according to comorbidities. In the subgroup of patients without CAD, AF, and COPD, SIRT1 levels were higher in patients with ascending AA than in controls. A similar trend, although nonsignificant, was observed for SIRT1 and SIRT2 levels in other subgroups including patients with diabetes, CKD, hyperlipidemia, smoking, and cancer. On the other hand, in the control group, SIRT1 and SIRT2 levels were generally higher for all comorbidities except cancer. Additionally, in patients with ascending AA, the presence of COPD was associated with lower SIRT2 levels. The analysis of laboratory test results revealed that SIRT2 levels were inversely correlated with total cholesterol and LDL-cholesterol levels in the control group. Similar results were reported for LDL-cholesterol in patients with polycystic ovary syndrome [ 20 ]. In the present study, cut-off values were also searched for sirtuin levels differentiating patients with and without AA, which might be useful in clinical practice. The cut-off levels were 8.05 ng/mL and 7.65 ng/mL for SIRT1 and SIRT2, respectively. As mentioned above, no data on plasma sirtuin levels in patients with ascending AA are available; therefore, further studies are needed to corroborate these results. The main limitation of this study is the small sample size, which may have reduced statistical power and limited the ability to detect subtle differences between groups. As a result, the findings should be interpreted with caution. Larger studies are needed to confirm and extend these results. Another limitation concerns the selection of the control group, which included individuals with similar comorbidities. While this reflects the clinical reality, it may have obscured differences in sirtuin levels. Additionally, unmeasured factors such as medication use, lifestyle, and environmental exposures may have influenced the results and this shoud be evaluated in further studies. However, the present findings, though preliminary and not statistically significant, may support future investigations. As mentioned, sirtuins are involved in key cellular processes that are highly relevant in the pathophysiology of AA. Therefore, sirtuin levels may serve as potential biomarkers for early detection, risk stratification, or monitoring of disease progression in patients with AA. Furthermore, growing interest in the pharmacological modulation of sirtuins suggests their potential as therapeutic targets. Agents that activate SIRT1 have shown promise in preclinical studies involving cardiovascular and metabolic disorders. While the clinical translation of these findings in the context of AA remains speculative, the current results support the rationale for further research into the diagnostic and therapeutic relevance of sirtuins in vascular disease.

In conclusion, according to available research, this study was the first to assess SIRT1 and SIRT2 levels in patients with and without AA. It did not show significant differences in sirtuin levels between patients with and without AA; however, there was a trend toward higher SIRT1 levels in patients with ascending AA. This is a relatively surprising finding, considering that other studies indicated a protective effect of sirtuins against the development of AA, and that reduced sirtuin levels were usually reported in patients with different diseases. Therefore, the present findings warrant further research to determine a link between sirtuin levels and AA and to investigate if elevated sirtuin levels could serve as a risk factor for life-threatening ascending AA.