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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 2  |  Issue : 2  |  Page : 136-141

Analysis of endothelial progenitor subpopulation cells, oxidative DNA damage, and their role in coronary artery disease


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

Date of Web Publication14-Jun-2018

Correspondence Address:
Dr. Mahaboob Vali Shaik
Laboratory of Genetics and Stem Cell Biology, Advanced Research Centre, Narayana Medical College and Hospital, Nellore - 524 001, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_41_18

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  Abstract 


Background: Endothelial dysfunction has been associated for the cause of atherosclerosis or cardiovascular diseases (CVDs). Endothelial progenitor cells (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. The current study was designed to analyze various subpopulations of EPCs and their DNA damage in CVDs. Methods: The study included 50 coronary artery disease (CAD) patients which was confirmed by angiography and 50 age-matched healthy controls without CAD. Flow cytometric analysis performed to measure subpopulations in EPCs in the peripheral blood using markers such as CD34, CD133, VEGFR2, and CD45. Oxidative DNA damage was analyzed in CD34+ cells. Mean EPC count was expressed as a percentage of total white blood cells. Three different subpopulations with CD45−/CD133+/VEGFR2+, CD45−/CD34+/VEGFR2+, and CD45−/CD34+/CD133+ coexpressions were measured with various percentages. Results: Subpopulation of CD45−/CD34+/VEGFR2+ cells had shown significant (P = 0.001) decrease in CAD patients in comparison with the healthy controls. There was no significant difference in the subpopulations of CD45−/CD34+/CD133+ cells (P = 0.005) and CD45−/CD133+/VEGFR2+ cells (P = 0.005) in CAD and healthy controls. The CD45−/CD34+/VEGFR2+ subpopulation EPC showed positive correlation with the severity of coronary stenosis (r = 0.35, P = 0.026), while other EPC subpopulation count did not show any correlation. Oxidative DNA damage was higher in CAD compared with controls. The number of EPC subpopulation CD45−/CD34+/VEGFR2+ was inversely correlated with oxidative DNA damage (P = 0.001), hypertension (P = 0.001), and diabetes mellitus (P = 0.004). Conclusion: We observed an association between CD45−/CD34+/VEGFR2 subpopulation EPCs and DNA damage in CAD condition. These findings support a cell biologist in searching the role of EPC populations in the pathophysiology or diagnosis of the disease by a clinician.

Keywords: Atherosclerosis, cardiovascular risk factors, coronary artery disease, endothelial progenitor cells, pathophysiology


How to cite this article:
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;2:136-41

How to cite this URL:
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 [serial online] 2018 [cited 2019 Jul 21];2:136-41. Available from: http://www.bmbtrj.org/text.asp?2018/2/2/136/234459




  Introduction Top


Cardiovascular diseases (CVDs) remain one-third of all deaths covering all regions of the world, with estimated 17.29 million deaths globally. Recently published on 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 was estimated 442.7 million prevalent present worldwide.[1] Ischemic heart disease was the leading cause of CVD in each world region, followed by stroke.

Endothelial dysfunction (ED) crucially contributes to the development of impaired coronary and systemic perfusion with fundamental impact on morbidity and mortality. ED is considered an integral state in the development of heart failure, regardless its phenotypes.[2]

Abundant formation of reactive oxygen species and unavailability of nitric oxide within the vascular wall are the key determinants in ED.[3] The integrity and functional activity of the endothelial monolayer are essential for protection against the initiation of atherosclerosis.[4] It is continually renewed and repaired in response to aggression. Various circulating cells are involved in this regeneration process: mature endothelial cells, monocytes capable of phenotypic and functional differentiation into endothelial cells, and endothelial progenitor cells (EPCs). Endothelial progenitors contribute to vascular homeostasis; thus, their reduction or dysfunction could be involved in the development of ED and CVD.[5],[6],[7],[8]

The balance between endothelial injury and recovery is important for reducing cardiovascular events.[9] EPCs mobbed from bone marrow and peripheral tissue residences and are involved reparative processes through differentiation into mature endothelial cells which are promising biomarkers of ED with possible predictive value.[10],[11] EPCs exist as heterogeneous population which originated from multiple precursors of the bone marrow and different stages of differentiation in the peripheral blood. Hence, EPCs show positive to hematopoietic stem cell markers CD34, CD133 and an endothelial marker VEGFR2.[12] In support, animal studies proved that EPCs with CD34+ are rapidly mobilized after vascular injury, in response to increased circulating VEGF levels, and contribute to neovascularization of injured tissues.[13] Increased EPC deterioration due to stress, diabetes, and hypertension has been shown to be associated with higher risk for cardiovascular events in individuals with coronary artery disease (CAD).[14],[15],[16] EPC dysfunction was associated with decreased number and poor function of circulating EPCs. Clinical trials have shown that EPC count was an independent predictor of CV mortality, newly diagnosed heart failure, and their related outcomes.[17],[18],[19] However, several controversies such as immune phenotypes of EPC population, their dysfunction, and its related risk factor remain to be clear to identify the prediction of CVD mortality and its outcome and its molecular mechanism.

Our previous study indicates that there is a progressive decrease in EPCs levels in CAD patients. Patients with low EPC counts had a higher incidence of cardiovascular events. The level of circulating EPCs with CD34+CD45−KDR+ expression predicts the occurrence of cardiovascular events and may help identify patients at increased cardiovascular risk.[20] However, there were less/no data related to identifying the role of these cell populations to predict the risk/severity of CAD. The reversal of EPC population dysfunction may potentially prevent the progression of CVD. It was also shown that increased oxidative DNA damage leads to increased senescence in EPCs in CVD. Hence, the current study was designed to understand the cumulative role of DNA damage in the regulating EPC subpopulation number in relationship with oxidative stress in CAD to prove EPCs as markers of severity in CAD.


  Methods Top


Study population

A simple random prospective study on 50 CAD patients as confirmed by a cardiologist through angiography was conducted between October 2015 and February 2016. Cardiovascular risk factors and previous events were evaluated. Fifty normal age-matched healthy persons were taken to analyze the same marker as a control group.

Analysis of endothelial progenitor cell subpopulation using flow cytometry

A 2 ml aliquot of the peripheral blood in 5 ml sodium citrate tube from 50 CAD patients and 50 healthy controls was stained with fluorescence or phycoerythrin (PE)-conjugated mouse anti-human monoclonal antibodies. Cell populations were analyzed by FACScanto II System (Becton Dickinson, US) as previously reported.[21],[22],[23] 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 FACS tube, 10 μl of FITC-conjugated anti-human CD34 mAb (BD Biosciences, US) and 10 μl of PE-conjugated anti-human VEGFR2 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 scatter-ffluorescence dot-plot analysis after appropriate gating. Here, we gated CD34+ cells and then examined the resulting population for dual expression of VEGFR2 marker. Data were processed using the FACS Diva software (Becton Dickinson). The same immunofluorescent cell staining was also performed with fluorescent-conjugated antibody to CD45 and CD133. In our experiment, three subpopulations of EPCs are named: (1) CD45−/CD133+/VEGFR2+ cells; (2) CD45−/CD34+/VEGFR2+ cells; and (3) CD45−/CD34+/CD133+ triple cells. For each analysis, corresponding negative control with IgG–FITC antibody was used. The number of EPCs as a percentage of the total live cells was calculated for each sample and was further normalized by subtracting the percentage of the relevant isotype control.

Oxidative DNA damage

Five-milliliter peripheral blood subjected to histopaque-density gradient centrifugation to collect mononuclear cells and CD34+ cells was isolated by positive selection using Magnetic Associated Cell Sorting System (Miltenyi Biotec). A Biotrin OxyDNA Test Kit (Biotrin, Dublin, Ireland) was used to evaluate the oxidative DNA damage in CD34+ cells following the manufacturer's recommendations. Immediately, 1 × 106 CD34+ cells were incubated for 1 h at 37°C with 50 μl of Biotrin blocking buffer, washed twice, and then incubated for 1 h at room temperature in the dark with 100 μl of FITC-labeled 8-oxoguanine probe. The cells were washed twice and analyzed by FACScanto II System (Becton Dickinson, US), and the mean fluorescent intensity of the different cell populations was recorded.

Statistical analysis

Continuous variables were expressed as means and standard deviations. Results from fflow cytometry are expressed as the percentage mean cell number per one million events. Comparisons between the CAD and control groups were performed using a two-sided independent sample Student's t-test. Associations between EPC subpopulation and DNA damage, diabetes mellitus, age, and hypertension were assessed individually by the Pearson's correlation coefficient. Statistical analysis was performed by Statistical Package for the Social Sciences (IBM SPSS Statistics 20.0; Chicago, IL, USA).


  Results Top


The mean age of the cases and control was 42.5 ± 7.3 years (range, 32–68) and 43.7 ± 8.2 years (range, 35–65), respectively. There is no significant difference (P = 0.085) observed between cases and control group in age parameter. Oxidative DNA damage (8-oxoguanine levels) in CD34+ and T-cell populations was higher in the CAD group than in healthy subjects, but it was not shown significant difference (P = 0.058) between the two groups.

Subpopulation of endothelial progenitor cell counts

The mean leukocyte count was 6.52 ± 2.42 in CAD group and 6.86 ± 2.72 in control group. The mean CD34 count in CAD and control group was 256.5 ± 98.5 and 352.6 ± 110.5/106 white blood cells (WBCs), respectively. There was no statistical significance (P = 0.071) observed in mean CD34 count between two groups. The mean CD133 count in CAD and control group was 41.5 ± 15.8 and 46.8 ± 17.2/106 WBC, respectively. There was no statistical significance (P = 0.09) observed in mean CD133 count between two groups [Table 1].
Table 1: Comparison of endothelial progenitor cell subpopulation levels and risk factors in coronary artery disease and healthy groups

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The CD45−/CD34+/VEGFR2+ subpopulation cells were observed as 2.86% ± 0.9% and 5.96% ± 3.7% in CAD and control with statistical significance, P < 0.001. CD45−/CD133+/VEGFR2+ subpopulation cells were observed as mean of 2.62% and 4.85% in CAD and control, with P = 0.005. CD45−/CD133+/CD34+ subpopulation cells were observed as 0.92% ± 0.4% and 1.38% ± 0.7% in CAD and control, with P = 0.005 [Table 1] and [Figure 1].
Figure 1: (a) Comparison of various sub-population of endothelial progenitor cells in CAD and healthy subjects. Number of CD45-/CD34+/VEGFR2+cells, number of CD45-/CD133+/VEGFR2+ cells, and number of CD45-D133+/CD34+ cell; (b) Number of CD34+cells; (c) Number of CD133+ cells; (d) Oxidative DNA damage of CD34+ cells. Bars represent mean values

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A higher significant change was observed in CD45−/CD34+/VEGFR2+ subpopulation cells among all other subpopulation cells of endothelial cells between CAD and control group.

The relations of various circulating EPC population counts in the peripheral blood to oxidative DNA damage, diabetes mellitus, and age factors are presented in [Table 2].
Table 2: Correlation between various endothelial progenitor cell counts in the peripheral blood and oxidative DNA damage, age, diabetes mellitus, and hypertension risk factors in coronary artery disease subjects (n=50) and age-matched control subjects (n=50)

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CD45−/CD34+/VEGFR2+ levels in the peripheral blood tended to be negatively related to oxidative DNA damage (r = −0.423) with significant difference, P = 0.001 in CAD, but this count in control subject showed r = 0.382 without significance, P = 0.067. CD45−/CD133+/VEGFR2+ levels in CAD subjects showed positive correlation with oxidative DNA damage with statistical significance, but there was no significance observed in control subjects. CD45−/CD133+/CD34+ levels also showed the same pattern of correlation. CD45−/CD133+/CD34+ and CD45−/CD133+/VEGFR2+ population levels had not significantly related to age both in CAD and control subjects. The number of CD45−/CD34+/VEGFR2+ was inversely correlated age (P = 0.001) in CAD subjects. CD45−/CD34+/VEGFR2+ levels showed statistical relationship with diabetic mellitus risk factor. The number of CD45−/CD34+/VEGFR2+ was inversely correlated with age hypertension (P = 0.001) in CAD subjects [Table 2].


  Discussion Top


This study to investigate circulating EPCs in patients with CAD as expressed by three distinct subpopulations: CD34+/133+, CD34+/133+/VEGFR2+, and CD34+/VEGFR2+ cells. As a control, age-matched healthy individual, their EPC subpopulation were estimated in peripheral blood. CD34+/133+, CD34+/133+/VEGFR2+, and CD34+/VEGFR2 markers have been studied and established to check the severity of congestive heart failure and hypertrophic cardiomyopathy.[24],[25] In the previous studies, circulating EPCs are determined by a culture method, but results vary on the basis of culture conditions.[16],[21],[26],[27] In our study, EPCs have been analyzed by flow cytometry, the gold standard method for the quantification of EPCs in the peripheral blood on the basis of the expression of surface markers.[28]

In our study, individual CD34+ cell population and CD133+ cell population count did not show significant difference between CAD patients as compared to patients without CAD. A study by Werner et al. showed the number of EPC with CD34+ and VEGFR2+ count, with a mean of 86.3 ± 71.9. This study demonstrated that an individual measurement of CD34+ and VEGFR2+ EPCs is a useful tool to predict cardiovascular outcomes in patients with CAD.

In our study, coexpression of phenotypic markers by flow cytometric analysis showed that CD45−/CD34+/VEGFR2+ subpopulation EPC count observed to be lower in CAD and showed significant change as compared with individuals without CAD. CD45−/CD133+/VEGFR2+ subpopulation EPC count also observed to be lower in CAD and showed significant change as compared with individuals without CAD. CD45−/CD133+/CD34+ subpopulation EPC count also observed to be lower in CAD and showed significant change as compared with individuals without CAD.

Different reports have discussed EPC subpopulation levels and their pattern in different cardiac disease conditions.

In the condition of acute myocardial infarction (AMI), it has been shown that EPC count starts increasing up to subsequent peak at day 5 and the rapid decline thereafter, normalizing within 2 months.[29],[30]

In ischemic condition, the circulating CD34+ and CD133+ VEGFR2+ EPCs are upregulated.[13],[31] CD45−/CD34+/VEGFR2+ EPC subpopulation was increased in hypertrophic cardiomyopathy (HCM) patients compared with healthy individuals. However, CD45−/CD34+/CD133+ subpopulation levels did not show significant alterations between HCM and healthy subjects.[25]

Hence, it needs to correlate with the cardiac risk factors with EPC levels to demonstrate their coexpression pattern to set up the diagnostic marker to screen CAD.

In our study, there were decreased levels of EPC observed in CAD than age-matched non-CAD healthy individual, but no correlation was observed between age and EPC count. A study by Vasa et al. demonstrated that the number of EPCs was significantly reduced by 40% compared with age-matched healthy volunteers. Age did not show correlation with the number of EPCs.[21]

In our study, systolic and diastolic blood pressure value does not show any significant correlation with EPC number. However, Werner et al. observed that the correlation between low EPC count and high blood pressure values disappears by the multivariate analysis.[11] The reason may be due to less sample size. The present study showed that circulating EPC level was associated with negatively correlated to lymphocyte count without significant difference.

The interplay between inflammation and oxidative stress affects the initiation, progression, and complications of CVD and inflammation/oxidative stress that modulate EPC bioactivity. Hence, in the present study, we performed DNA damage analysis in EPC subpopulation, which is caused by inflammation/oxidative stress to identify their correlation with CAD.

In our present study, DNA damage marker 8-oxo-2'- deoxyguanosine was 252.6 ± 102.6 in EPC of CAD patients than 162.5 ± 55.2 in controls, which showed increased levels. In a study by Bhat and Gandhi, they showed that DNA damage in the peripheral blood leukocytes of patients with CAD and AMI was significantly (P < 0.001) increased than the control subjects.[32] The increased DNA damage in CAD patients may be due to the consequence of disease and/or drug therapy. It may be assumed that accelerated aging/risk factor-associated oxidative stress on circulating endothelial progenitors leads to DNA damage associated with CAD and/or AMI.

Reports in the literature have also documented same observations. Botto et al. reported increased levels of DNA damage levels in patients with CAD, and Demirbag et al., Rajesh et al., and Bhat et al. in their previous studies also observed significantly increased levels of DNA damage in patients with AMI compared to patients with unstable angina, implying that the levels of DNA damage increase with the severity of CAD.[20],[33],[34],[35],[36],[37]

In total subpopulation EPC, oxidative DNA damage showed positive correlation with CD45−/CD34+/VEGFR2+ EPC population with significant difference in our study. However, other population in the peripheral blood did not show any significant difference. Due to DNA damage observed majorly in CD45−/CD34+/VEGFR2+ population, hence, it may be kept as a standard marker of EPC among other population in the peripheral blood.

The results of our study suggest that peripheral blood contains various subpopulation EPCs, in which CD45−/CD34+/VEGFR2+ population showed lower in CAD patients than CD133+/VEGFR2+ and CD45−/CD133+/CD34+ subpopulation. DNA damage, especially in CD34+/VEGFR2+ population, was identified to be as risk factor which may lead to CAD. These findings support the notion that EPC subpopulation with CD45−/CD34+/VEGFR2 coexpression, and their correlation with oxidative DNA damage may play an important role in pathogenesis of CAD. This study has shown that the circulating level of EPCs not only is associated with cumulative cardiovascular risks, but it is also predictive of future cardiovascular events and the progression of atherosclerosis in patients with CAD.


  Conclusion Top


CAD patients with low numbers of CD45−/CD34+/VEGFR2+ subpopulation cell count had higher risk of endothelial damage. Although some correlations were found between circulating EPC subpopulation levels and risk factors, namely, oxidative DNA damage, diabetes, and smoking (inverse relation), those relations were found to be too weak for EPC prediction in a CAD. CD45−/CD34+/VEGFR2+ expression had shown high significance, which may be useful to estimate EPCs to predict the endothelial regeneration capacity in CAD. Further, extensive studies need to exemplify the circulating endothelial progenitor subpopulation cells in other cardiac disease condition; those may lead to the development of a treatment strategy to replenish the endothelial monolayer after injury.

Acknowledgment

Authors acknowledge to Narayana Medical College & Hospitals for providing reagents and infrastructure. Thanks are extended to PG students/Staff of Cardiology Department involved in sample/data collection.

Financial support and sponsorship

This research does not receive any fund from public or private organization. It is a intramural project.

Conflicts of interest

There are no conflicts of interest.



 
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