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 Table of Contents  
Year : 2018  |  Volume : 2  |  Issue : 4  |  Page : 290-294

Role of essential trace elements and telomere length in endothelial cell senescence in patients with coronary artery disease

1 Advanced Research Centre, Laboratory of Genetics and Stem Cell Biology, Narayana Medical College and Hospital, Nellore, Andhra Pradesh, India
2 Department of Cardiology, Narayana Medical College, Nellore, Andhra Pradesh, India

Date of Submission06-Aug-2018
Date of Decision09-Sep-2018
Date of Acceptance07-Oct-2018
Date of Web Publication11-Dec-2018

Correspondence Address:
Dr. Mahaboob V Shaik
Advanced Research Centre, Laboratory of Genetics and Stem Cell Biology, Narayana Medical College, Nellore - 524 003, Andhra Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_117_18

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Introduction: Vascular endothelial cell senescence has been involved in endothelial dysfunction and inflammation and promotes atherosclerosis; moreover, vascular endothelial cells with senescent phenotype have been found in the human atherosclerotic plaques. The mechanism of senescence of endothelial cell through telomere length (TL) shortening has been explained but, with reference to micronutrient status, was not fully clarified. To our knowledge, there is no study on the level of telomere/telomerase with the combination of micronutrient levels in coronary artery disease (CAD). Methods: Twenty patients of angiographically diagnosed CAD and twenty age-matched controls (≤40 years) without CAD were studied for their circulatory endothelial progenitor cells (EPCs) by flow cytometry. EPC-TL was studied by real-time quantitative polymerase chain reaction. Copper (Cu), zinc (Zn), and selenium levels in serum were analyzed by atomic absorption spectrophotometry. The association across the serum trace elements and TL was made and correlated with disease condition. Results: EPC count in CAD patients was observed to be lower than that compared with controls (0.18% vs. 0.049%) (P < 0.0001). Cellular TL in CAD statistically decreased compared to that of normal controls (4.21 vs. 5.32) kb/genome (P = 0.008). Serum Zn and Cu levels were significantly low in CAD compared with the controls (P < 0.001). TL and Cu and Zn levels were found positively correlated in the CAD patients and controls (P < 0.001). Conclusion: Reduced levels of telomerase may be due to lower concentration of Cu and Zn, which leads to decreased antioxidant capacity. Establishing a standardized method of evaluating TL and trace elements involved in cholesterol metabolism is essential before its routine use in preventive cardiology.

Keywords: Atherosclerosis, endothelial progenitor cells, telomerase, trace elements

How to cite this article:
Shaik M, Shaik MV, Gangapatnam S. Role of essential trace elements and telomere length in endothelial cell senescence in patients with coronary artery disease. Biomed Biotechnol Res J 2018;2:290-4

How to cite this URL:
Shaik M, Shaik MV, Gangapatnam S. Role of essential trace elements and telomere length in endothelial cell senescence in patients with coronary artery disease. Biomed Biotechnol Res J [serial online] 2018 [cited 2023 Jan 28];2:290-4. Available from: https://www.bmbtrj.org/text.asp?2018/2/4/290/247239

  Introduction Top

Coronary artery disease (CAD) is a major problem worldwide. In a report published on the Journal of the American College of Cardiology, Roth et al. looked at the global burden of disease and mortality over the course of 25 years; it's estimate 442.7 million prevalent present worldwide.[1] Atherosclerosis and thrombosis underlying CAD involve multiple cell types. The integrity and functional activity of the endothelial monolayer are essential for protection against the initiation of atherosclerosis.[2] Trace elements' imbalance may be influencing the underlying secondary metabolic changes resulting in clinical manifestation of CAD.[3]

Increased endothelial progenitor cell (EPC) deterioration due to stress, diabetes, and hypertension has been shown to be associated with higher risk for cardiovascular events in individuals with CAD.[4],[5],[6] EPCs are exposed to oxidative stress during vascular injury as residents of blood vessel walls or as circulating cells, homing to the sites of neovascularization.

Although notable achievements have been made in the medical, interventional, and surgical management of CAD, the need for its prevention is more important. Among other modalities, this calls for defining evidence-based new biomarkers, which on their own or in combination with other known biomarkers may predict the risk of CAD to enable the institution of appropriate preventive strategies. Vascular endothelial cell senescence has been involved in endothelial dysfunction and inflammation which promotes the process of atherogenesis; moreover, vascular endothelial cells with senescent phenotype have been found in the human atherosclerotic plaques. However, the underlying mechanisms of aging-induced attenuation of endothelial functions with reference to micronutrient status were not fully clarified.

As telomere length (TL) by its nature is a marker of cell senescence, it is of particular interest when studying the lifespan and fate of endothelial cells and cardiomyocytes, especially so because TL seems to be regulated by various factors, notably certain cardiovascular risk factors, such as smoking, hypertension, hyperlipidemia, and obesity that are associated with high levels of oxidative stress. To gain insights into the links between TL and cardiovascular disease (CVD) and to assess the usefulness of TL as a new marker of cardiovascular risk, it seems essential to study on TL measurement and its correlated risk factors. Various nutrients influence TL potentially through mechanisms that reflect their role in cellular functions including inflammation, oxidative stress, DNA integrity, DNA methylation, and activity of telomerase, the enzyme that adds the telomeric repeats to the ends of the newly synthesized DNA.[7] Zinc (Zn)-dependent enzymes in the cell include DNA polymerases, RNA polymerases, and reverse transcriptases.[8],[9] Zn also has a protective role in oxidative stress.[10] Zn supplementation also reduces the incidence of infection,[11],[12] which is another factor that leads to telomere attrition by higher turnover of cells. Thus, it is possible that Zn may affect TL by influencing telomerase activity, DNA integrity, oxidative stress, and susceptibility to infection.

There is no study to identify the role of Zn, copper (Cu), and selenium (Se) in telomere shortening in patients with CAD. Establishing a well-standardized and accurate method of evaluating TL and micronutrient levels, which were involved in cholesterol metabolism, is essential before its routine use in preventive cardiology. In this study, we explore the role of micronutrients (Cu, Zn, and Se) and telomere genetic involvement in vascular senescence in CAD by comparing the numerical status and senescence of circulating EPCs in CAD patients comparing with controls.

  Methods Top

Twenty patients with CAD and twenty without CAD were studied for their circulatory EPC-TL and the corresponding serum Cu, Zn, and Se levels. EPCs were measured by flow cytometry in all patients at Advanced Research Center, Narayana Medical College and Hospital, Nellore, Andhra Pradesh, India. CAD was diagnosed based on the documented history, physical examination, and electrocardiogram findings namely 1-1, 4-1, 5-9, 5-2, or 9-2 (Minnesota codes). Body mass index (BMI) was calculated using the following formula: weight (kg)/height (m2) and an individual with BMI >27 kg/m2 was considered as obese. Blood pressure was recorded using sphygmomanometer and patients with systolic blood pressure (SBP) >140 and diastolic blood pressure (DBP) >90 mmHg were termed hypertensive as per Joint National Committee-7. The proposal was reviewed and approved by Research and Ethical committee of Cardiology, Narayana Medical College, Nellore, Andhra Pradesh, India. The Ethics Committee Approval number was Andhra Pradesh, India 2017 and date was 2017-2018.

Quantification of endothelial progenitor cells

Mononuclear cells (MNCs) were isolated from blood samples by HISTOPAQUE density gradient centrifugation. EPCs were isolated using magnetic Activated Cell Sorting using CD34 multisort kit. Flow cytometry carried out to identify EPCs was followed by previous protocol.[13],[14] 2 ml of the peripheral blood in 5 ml sodium citrate tube from all participants were stained with fluorescence or phycoerythrin (PE)-conjugated mouse anti-human monoclonal antibodies. Cell populations were analyzed by FACSCanto™ II System (Becton Dickinson, US). Staining was performed according to the manufacturer's instructions. Red blood cells (RBCs) were deprived from whole blood using RBC lysis solution (Sigma Chemicals, US). In a fluorescence-activated cell sorting (FACS) tube, 10 μl of fluorescein isothiocyanate-conjugated anti-human CD34 mAb (BD Biosciences, US) and 10 μl of PE-conjugated anti-human KDR mAb (BD Biosciences, US) and 1 × 108 cells were mixed and kept for incubation at 4°C for 30 min in the dark. Cells were washed twice with FACS buffer. The percentage of positivity against each antibody was determined using side scatterfluorescence dotplot analysis after appropriate gating. Here, we gated CD34+ cells and then examined the resulting population for dual expression of KDR marker. Data were processed using the FACSDiva™ software (BD Biosciences, San Jose, CA, USA). The same immunofluorescent cell staining was also performed with fluorescent-conjugated antibody to CD45, CD34, and KDR. In our experiment, EPCs are named: CD45−/CD34+/KDR+.

Real-time polymerase chain reaction

Total DNA was extracted using the QIAmp DNA mini kit (Qiagen, Hilden, Germany) and quantified using NanoDrop™ (Thermo Fisher Scientific, Wilmington, Delaware USA). EPC senescence as determined by EPC-TL measurement by real-time quantitative polymerase chain reaction.

Micronutrient analysis

Serum levels of micronutrient (Cu, Zn and Se) were determined by atomic absorption spectrophotometer (SHIMDAZU, Japan) and a comparative analysis of the findings were made. The correlation among TL and micronutrient levels with clinical condition was also measured.

Statistical analysis

Mean and standard deviation were calculated for all study parameters. Means were compared using Student's t-test. Discrete variables were compared using Chi-square test. Data obtained were analyzed by the Statistical Package for Social Sciences (IBM SPSS) 17.0 version. United States; 2010. Student's t-test was used to compare the means of EPC number and EPC-TL in cases and controls. Values were considered statistically significant at P < 0.05.

  Results Top

The mean percent of EPC count (% of total white blood cell [WBC]) was significantly (P = 0.001) lower in CAD patients than that of controls (0.049 [0.058–0.25] vs. 0.18 [0.041–0.22]). The mean EPC-TL (kb/genome) was also markedly lower in CAD patients compared to that of controls (cases: 4.21 [3.29–4.65]; control: 5.32 [4.27–6.28]) [Table 1].
Table 1: Analysis of serum copper, zinc, and selenium levels in coronary artery disease patients and controls and their significant association

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Cu, Zn, and Se levels observed were lower in CAD than controls. The mean ± standard deviation (SD) of serum Zn levels was found to be 58.22 ± 30.5 and 73.52 ± 32.22 μg/dL in CAD and control groups, respectively, which was found to be statistically significant (P = 0.001).

The mean ± SD of serum copper levels in CAD cases and control groups were also found to be statistically significant (P = 0.04). Significant association of serum Cu was observed with CAD even after adjustment for total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL), and very LDL cholesterol and Zn. The mean ± SD) serum selenium levels in cases and control groups were found to be 56.2 ± 11.1 μg/dL and 65.2 ± 4.2 μg/dL, respectively [Table 2] and [Table 3].
Table 2: Serum copper, zinc, and selenium levels in coronary artery disease patients and controls classified by age group

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Table 3: Serum copper, zinc, and selenium levels in coronary artery disease patients and controls classified by sex group

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EPC-TL positively correlated with serum level of zinc in CAD patients. EPC-TL positively correlated with the serum level of Cu in CAD patients. Other risk factors and their correlation mentioned in [Table 4].
Table 4: Correlation between telomere length, endothelial progenitor cell number, and micronutrients in coronary artery disease

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When patients with lower serum Cu concentration were compared with those having normal serum copper concentration, prevalence of hypertriglyceridemia in low serum Cu patients was noticed.

In the present study, serum Cu was positively associated with SBP, BMI, HDL cholesterol, and inversely associated with age and DBP.

  Discussion Top

This study comprised twenty patients having CAD and 20 controls without CAD. The mean percentage of EPCs (percentage of total WBC) was significantly (P = 0.001) lower in CAD patients compared to controls. The observation was consistently correlated with previous studies.[15],[16],[17]

Statistically significant (P = 0.04) lower serum Cu concentration was observed in patients with CAD than controls without CAD. This observation was mostly close to the previous study.[18] When patients with lower serum Cu concentration were compared with those having normal Cu concentration, the prevalence of low EPC number (P = 0.0001) in low serum Cu patients was noticed. The beneficial effect of Cu may occur via increased gene expressions of superoxide dismutase (SOD) and vascular endothelial growth factor,[19] suggesting a role of Cu in angiogenesis (i.e., endothelial regeneration through EPC), whose reduction is involved in endothelial cell senescence.[20]

Serum Zn concentration was found to be significantly lower (P = 0.001) in CAD (low Cu patients) than controls (normal serum Cu concentration).

Shortened TL was observed in CAD patients compared to controls (P = 0.007). Patients with lower TL were strongly associated/correlated with patients with lower Cu and Zn levels. EPC-TL positively correlated with Cu (r = 0.501) and Zn (r = 0.562) levels in CAD,

whereas Se levels in CAD did not show any correlation with CAD patients' low EPC number.

Both Cu and Zn show high correlation with EPC's TL between 4.11 and 4.26 kb/genome. Hence, the current study describes that telomere shortening due to lower Cu and Zn levels may induce the senescence of EPCs, which triggers the CAD.

The integrity and functional activity of the endothelial monolayer are essential for protection against the initiation of atherosclerosis. Endothelial progenitors contribute to vascular homeostasis; thus, their reduction or dysfunction could be involved in the development of endothelial dysfunction (ED) and CVD. In this study, EPC dysfunction was associated with decreased number and poor function of circulating EPCs. Lower EPCs in CAD patients in the present study suggest impaired repair mechanism predisposing to ED at middle age. Data indicate that the loss of circulating EPCs in the circulation of CAD patients may be involved in the pathogenesis of ED. A shorter EPC-TL and a reduced activity of telomerase in the CAD patients induce accelerated senescence of EPCs, which results in lowering EPCs. It may play an important mechanistic role in CAD epidemic in India, which is having CAD higher burden. Telomere shortening and its risk factors need to be exemplified to understand the CAD disease pathogenesis. Normally, healthy functioning of telomeres requires adequate methylation. The important point to understand is that an adequate supply of methyl donors is needed for telomeres to work properly. Chronic stress and depression typically indicate a lack of methyl donors, meaning telomeres are undernourished and prone to accelerated aging. Many nutrients help protect and enhance our DNA's repair capacity, including that of telomeres. A lack of antioxidants leads to increased free radical damage and more risk for damage to telomeres. Lack of Zn causes an excessive amount of DNA strand breakage and excessive numbers of short telomeres. A novel antioxidant that contains Zn is carnosine, which has been shown to slow the rate of telomere depletion in human fibroblast cells, while extending their longevity. In vitro studies and animal experiments provided evidence that Zn could potentially protect against telomere damage by reducing oxidative stress and inflammation.[7],[21] A strong genetic component in TL regulation was found in twin studies.[22],[23] The responsible genes have not been identified yet. However, first data revealed a coordinated increase of the abundance of glutathione peroxidase-1 and CuZnSOD mRNA in human fibroblast strains with low telomere shortening rate,[24] implying two major antioxidative enzymes in TL regulation. A study by Cipriano et al. (2009) demonstrated that short telomeres were associated with impaired zinc homeostasis and inflammation in old hypertensive participants.[25] Hence, micronutrient deficiencies and aging process can lead to telomere genetic instability.

  Conclusion Top

Low levels of Cu and Zn with mean TL of 4.21 kb/genome were suitable for discriminating CAD patients from patients without CAD. The reason why the patients had reduced levels of Cu and Zn is likely to be oxidative stress, which can be defined as an increased exposure to oxidants and/or decreased antioxidant capacities. However, further studies are needed to investigate their translational potential in the real clinical practice.

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Conflicts of interest

There are no conflicts of interest.

  References Top

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Subramanyam G, Vijaya J. Trace Elements in Cardiovascular Disease. Tirupati, India: S.V. Medical College; 1997. p. 15.  Back to cited text no. 3
Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med 2003;348:593-600.  Back to cited text no. 4
Ghani U, Shuaib A, Salam A, Nasir A, Shuaib U, Jeerakathil T, et al. Endothelial progenitor cells during cerebrovascular disease. Stroke 2005;36:151-3.  Back to cited text no. 5
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Terhune MW, Sandstead HH. Decreased RNA polymerase activity in mammalian zinc deficiency. Science 1972;177:68-9.  Back to cited text no. 8
Springgate CF, Mildvan AS, Abramson R, Engle JL, Loeb LA. Escherichia coli deoxyribonucleic acid polymerase I, a zinc metalloenzyme. Nuclear quadrupolar relaxation studies of the role of bound zinc. J Biol Chem 1973;248:5987-93.  Back to cited text no. 9
Bray TM, Bettger WJ. The physiological role of zinc as an antioxidant. Free Radic Biol Med 1990;8:281-91.  Back to cited text no. 10
Bao B, Prasad AS, Beck FW, Snell D, Suneja A, Sarkar FH, et al. Zinc supplementation decreases oxidative stress, incidence of infection, and generation of inflammatory cytokines in sickle cell disease patients. Transl Res 2008;152:67-80.  Back to cited text no. 11
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Shaik MV, Shaik M, Gangapatnam S. Analysis of endothelial progenitor subpopulation cells, oxidative DNA damage, and their role in coronary artery disease. Biomed Biotechnol Res J 2018;2:136.  Back to cited text no. 13
  [Full text]  
Shaik MV, Gangapatnam S, Edwin R. A study of analysis of endothelial progenitor cells in peripheral blood in patients with coronary artery disease. J Cardiovasc Dis Res 2016;7:27.  Back to cited text no. 14
Fadini GP, Miorin M, Facco M, Bonamico S, Baesso I, Grego F, et al. Circulating endothelial progenitor cells are reduced in peripheral vascular complications of type 2 diabetes mellitus. J Am Coll Cardiol 2005;45:1449-57.  Back to cited text no. 15
Hristov M, Fach C, Becker C, Heussen N, Liehn EA, Blindt R, et al. Reduced numbers of circulating endothelial progenitor cells in patients with coronary artery disease associated with long-term statin treatment. Atherosclerosis 2007;192:413-20.  Back to cited text no. 16
Vemparala K, Roy A, Bahl VK, Prabhakaran D, Nath N, Sinha S, et al. Early accelerated senescence of circulating endothelial progenitor cells in premature coronary artery disease patients in a developing country – A case control study. BMC Cardiovasc Disord 2013;13:104.  Back to cited text no. 17
Latheef SA, Subramanyam G, Reddy KN. Association of serum copper and coronary artery disease in elderly population of South India. Trace Elem Electrolytes 2006;23:29.  Back to cited text no. 18
Jiang Y, Reynolds C, Xiao C, Feng W, Zhou Z, Rodriguez W, et al. Dietary copper supplementation reverses hypertrophic cardiomyopathy induced by chronic pressure overload in mice. J Exp Med 2007;204:657-66.  Back to cited text no. 19
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Frenck RW Jr., Blackburn EH, Shannon KM. The rate of telomere sequence loss in human leukocytes varies with age. Proc Natl Acad Sci U S A 1998;95:5607-10.  Back to cited text no. 22
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Serra V, von Zglinicki T, Lorenz M, Saretzki G. Extracellular superoxide dismutase is a major antioxidant in human fibroblasts and slows telomere shortening. J Biol Chem 2003;278:6824-30.  Back to cited text no. 24
Cipriano C, Tesei S, Malavolta M, Giacconi R, Muti E, Costarelli L, et al. Accumulation of cells with short telomeres is associated with impaired zinc homeostasis and inflammation in old hypertensive participants. J Gerontol A Biol Sci Med Sci 2009;64:745-51.  Back to cited text no. 25


  [Table 1], [Table 2], [Table 3], [Table 4]


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