Precipitation of acute coronary syndrome in an interactive association of oxidative stress biomarkers with soluble receptor for advanced glycation end-products

Abstract

Background: As a repercussion of oxidative stress, reactive oxygen species augment the biochemical reaction of amino and carbohydrate groups of proteins, thus forming oxidative products known as Advanced Glycation End-products (AGEs), associated with acute coronary syndrome (ACS). Together, this milieu provokes oxidative stress and promotes the development of ACS.

Methods: The studied cohort had 76 ACS individuals and 76 healthy controls. Blood samples of both groups were collected to measure the concentration of AGEs, Malondialdehyde (MDA), and the activity of antioxidants. Levels of lipid profile and inflammatory biomarkers were accessed in the study. The spectrophotometric ELISA kit was used to determine the levels of parameters.

Result: The results indicated a significant increase in AGEs, MDA, LDL, and total cholesterol levels. Increased inflammatory markers, Intercellular adhesion molecule 1, and vascular cell adhesion proteins-1 in ACS patients were compared with the control subjects. A decrease is recorded in superoxide dismutase, HDL, and glutathione levels in ACS individuals.

Conclusion: Our study demonstrated increased serum concentration of AGEs and several other factors, such as increased oxidative markers and, conversely, reduction in the antioxidant in ACS individuals. Therefore, we conclude these variables can be a potential biomarker in ACS individuals.

KEYWORDS:

• Acute coronary syndrome
• advanced glycation end products
• intercellular adhesion molecule 1
• oxidative stress
• reactive nitrogen species
• receptor for advanced glycation end products

1. Introduction

Acute Coronary Syndrome (ACS) is a term used to describe a group of clinical conditions that result from a sudden decrease or complete blockage of blood flow to the heart muscle. Variably, ACS may include the symptoms of unstable angina and myocardial infarction, evidently resulting in a heart attack. The clinical presentations of these symptoms may also range from those for ST-segment elevation myocardial infarction (STEMI) to presentations found in non–ST-segment elevation myocardial infarction (NSTEMI).

Lipid plaques formation and thrombosis of coronary vessels are the wide spectrum of ACS. The rupture of an atherosclerotic plaque and thrombus embolism are the primary underlying factors behind this pathology. However, secondary plaque rupture or its erosion subsequently produces thrombogenic substrates into the bloodstream, potentiating the activation of thrombose formation and leading to acute pathogenesis of ACS [1, 2], and eventually giving the symptoms of ACS [3]. Additionally, the thin fibrous cap of the over-lying lipid plaques can be the causative agent in inflammation [4]. Previous studies demonstrated the putative mechanisms of collaborating cascades of lymphocytes, macrophages, interleukins, and adhesion molecules potentiate the thrombogenicity of atherosclerotic plaque [5].

The cardiovascular ailment has been the focus of various groups, and several potential biomarkers have been identified in ACS individuals. Nevertheless, inflammatory biomarkers have been a significant target because the inflammatory mechanisms’ sequel eventually causes atherosclerotic plaque rupture and can result in erosion [6].

Eroded plaque exposes sub-endothelial space to platelets and can further enhance proinflammatory and pro-thrombotic status. Studies of plaque show that lymphocytes and macrophages are abundant in the micro-environments of the rupture site in patients with ACS [7]. Macrophages incite the vascular cell adhesion proteins (VCAMs) and regulate leukocyte attachment and their trans-endothelial relocation. Various other stimuli, including interleukins and lipopolysaccharide (LPS) [8, 9], invariably contribute to the pathogenesis of atherogenesis. Dislocation of plaque is reported to be associated with apoptosis in the carotid arteries of patients with ACS [10]. Nevertheless, an eroded plaque has a fragile, thin cap of apoptotic smooth muscle cells, which produces collagen [11].

Oxidative stress (OS) is associated with developing reactive oxygen species (ROS) damage to cells and tissue. Nevertheless, the association of ROS with ACS is exceedingly credible. ROS can produce deleterious effects on lipids, proteins, lipoproteins, and nucleic acids [12]. In cardiovascular diseases, lipid peroxidation is reported to be a substantial factor. Lipid peroxidation biomarker malondialdehyde (MDA) arises when polyunsaturated fatty acids get attacked by ROS [13]. Also, advanced oxidation protein products (AOPPs) result from OS-derived ROS, inevitably induce oxidation of proteins, and may act as inflammatory mediators, thus leading to excessive stimulation of inflammation and upregulation of immune cells, which may account for immune system impairment in ACS [14].

These glycation products further undergo rearrangement, dehydration, and condensation to become advanced glycation end-products (AGEs). The interaction of AGEs with their specific RAGE leads to alteration in gene expression, cell proliferation, cell migration, and activation of the different signalling mechanisms involved in the pathophysiology of atherosclerosis and vascular complications [15]. Protein oxidation significantly oxidized low-density lipoprotein (oLDL), which is pro-inflammatory and cytotoxic, upregulating various cytokines and chemokines [6]. Furthermore, macrophages uptake these oLDL and form foam cells and proinflammatory cytokine amplification, further triggering endothelial cell dysfunction and subsequent plaque destabilization [16]. The narrative of the current study was to demonstrate the role of biomarkers in ACS and their association with oxidative and anti-oxidative status.

2. Materials and methods

The study had 152 subjects in two groups; individuals having ACS, 76 subjects, and healthy individuals as control; 76 subjects. ACS individuals were selected from the hospital attached to the Institute of Cardiology, Lahore. After their investigation with; stress echocardiography, thallium scan, ECG, and the presentation of their acute symptoms, the consultant had already labelled these patients as ACS. Written, informed, and signed consent forms were obtained, approving their participation in the study. Healthy controls were recruited from the general population randomly. None of the subjects and the controls had a history of chronic Illness, and neither was under any treatment. The study was conducted following sanction by the Institutional Research and Ethics Committee at the University of Lahore under the number IRB # UOL/1335/04/19 dated 15th April 2019.

A rigorous power analysis determined the number of participants in our study. In addition, the power analysis allowed us to estimate the sample size needed to detect the anticipated effect size with a desired level of statistical power of P = 0.05 and the desired power level of 25%.

After informed consent, blood samples were taken in serum separating gel and clot activator tube. It was spun for ten minutes at 3000 RPM, and the separated serum was stored at −70°C. All samples were subjected to analysis in one batch after calibration of the microplate spectrophotometers “SpectraMax Plus 384”. The desired wavelength was used, as mentioned in the leaflets of the commercial ELISA kit. To minimize the variations of test results, the intra-assay coefficients of variation were used, and replicates of all the tests were run within the same plate to measure the variance between data points of all the parameters.

2.1. Assessment of oxidative stress variables

The analytical analysis of different variables was performed using commercially available standard kits, which were carefully selected to have high sensitivity in the measurements of variables. Essentially the protocols mentioned in the respective leaflets were observed. QuantiChrom^TM kits measured Malondialdehyde (MDA); (DTAC-100). The AGEs were assessed with (ab273298). The levels of sRAGE were quantified with the help of BioVendor sRAGE human ELISA kit. (RD191116200R). QuantiChrom^TM kits measured nitric oxide (NO); (D2NO-100).

2.2. Determination of lipid profile biomarkers

Determination of LDL and HDL was done by using ELISA Kit ABCAM (ab65390). In addition, MyBioSource was used to assess total cholesterol (MBS168179) and Triglyceride ELISA Kit MBS729616.

2.3. Assessment of inflammatory biomarkers

Human sVCAM-1 ELISA Kit (MBS175995) and Human sICAM-1 ELISA Kit, Thermofisher Scientific. BMS201.

2.4. Assessment of antioxidants

QuantiChrom^TM kits were used for the measurements of Malondialdehyde (MDA); (DTAC-100), Glutathione (GSH); (DIGT-250). Abcam kits measured Glutathione reductase (GSH-R); (ab102530). EnzyChromTM kits measured Superoxide dismutase (SOD); (ESOC-100).

2.5. Statistical analysis

SPSS, version 17.0, was engaged in analyzing and managing the data. In the analysis between ACS and the control group, an independent t-test was applied. Statistically, significant differences were considered when the p-value was <0.05. In addition, the correlation between variables was assessed using Pearson correlations; the r-value was determined, of which a positive value indicates a strong correlation and a negative value shows a weak correlation between the variables.

3. Results

The demographic data in Table 1 is impeccable, with lots of valuable information. Fair distribution of subjects is seen as far as socioeconomic status is concerned. However, much of ACS prevalence is noticed in the worker with jobs, which may be their sedentary style in their offices, which is also evident from the routine exercising individuals. Furthermore, increased BMI also confirms this finding. The increased number of smokers and diabetic individuals in the group is another hallmark for the increase in the production of ROS and dysfunctional lipid profile due to diabetic prevalence.

Table 1. Shows the demographic data of the participant of both groups.

Parameters	Number of Patients (n) Total no. 76	Number of Control (n) Total no. 76
Mean Age – years ± SD	62 ± 5	57 ± 6
Socio-economic status (n)	Economic class = 21	Economic class = 24
	Lower class = 24	Lower class = 14
	Upper class = 31	Upper class = 36
Occupation (n)	Labor = 21	Labor = 32
	Worker – Job = 41	Worker – Job = 24
	Businessman = 14	Businessman = 20
Exercise Routine (n)	Daily = 14	Daily = 16
	Occasional = 26	Occasional = 44
	No routine = 34	No routine = 16
Family history ACS (n)	31 Answered Yes	14 Answered Yes
	Other does not know	Other does not know
Addiction	None	None
Marital Status	All Married	All Married
BMI	28 ± 0.27	23 ± 0.20
Smoker (n)	Yes = 55	Yes = 45
	No = 21	No = 31
Diabetes (n)	Yes = 65	Yes = 31
	No = 11	No = 45
Systolic BP (mmHg) (Measured at the time of induction)	129 ± 4	118 ± 4
Diastolic BP (mmHg) (Measured at the time of induction)	87 ± 3	78 ± 4
Hypertension (n)	57	21
Previous history of hospitalization because of the cardiac-related issue (n)	56	11
Other serious Illness (n)	None	None

Table 2 consolidates both groups’ stress, inflammatory, and antioxidant concentrations. The mean serum MDA, NO, AGEs, and sRAGE levels primarily implicate the increase in the concentration of the variables in the patients. Significantly, elevated value in the ACS reveals the occurrence of OS in response to elevated ROS. Albeit, all these parameters have a noticeably significant p-value, indicating their role in the ACS.

Table 2. Comparison of stress, inflammatory, and antioxidant levels between ACS patients and control subjects. The P-value of <0.05 was significantly correlated with positive findings.

Variables	Control (n = 76) Mean ± SD	Patients (n = 76) Mean ± SD	P-value Control vs. Subject
Stress Biomarkers
MDA (nmol/ml)	0.817 ± 0.046	3.99 ± 0.12	0.024
NO (ng/ml)	19.21 ± 3.24	69.22 ± 10.15	0.003
AGEs (ng/ml)	0.89 ± 0.091	4.027 ± 0.54	0.022
sRAGE (pg/ml)	86.54 ± 15.35	578.21 ± 10.20	0.037
Inflammatory biomarkers
sICAM-1 (ng/ml)	75.177 ± 12.29	238.64 ± 21.29	0.000
sVCAM-1 (ng/ml)	1012.115 ± 35.29	2107.648 ± 16.35	0.019
Antioxidants levels
SOD (µg/ml)	0.562 ± 0.008	0.190 ± 0.001	0.025
Glutathione (µg/ml)	8.290 ± 1.89	5.350 ± 0.45	0.036
GSH-R (µmol/ml)	8.596 ± 2.15	4.59 ± 1.59	0.014

Similarly, the inflammatory biomarkers were analyzed in ACS patients and healthy controls. An increased level is observed in ACS patients than in healthy controls of sICAM-1 (238.64 ± 21.29 vs. 75.177 ± 12.29 ng/ml) and sVCAM (2107.648 ± 16.35 vs. 1012.115 ± 35.29 ng/ml) is observed with P = 0.000 and P = 0.019 respectively. Increased inflammatory biomarkers in the ACS individual indicate the imbalance created by the OS, thus causing pathological changes in the endothelium of the microvasculature system. However, an increase in the ICAM and VCAM is not an isolated phenomenon; other inflammatory biomarkers, such as interleukins and chemokines, also integrate into these pathways.

The antioxidant levels that overcome the OS are measured in patients and healthy controls. The levels of SOD, GSH, and GSH-R were as follows: patients with ACS (0.190 ± 0.001 µg/ml), (5.350 ± 0.045 µg/ml), and (4.59 ± 1.59 µmol/ml) and healthy controls (0.562 ± 0.008 µg/ml), (8.290 ± 1.89 µg/ml), and (8.596 ± 2.15 µmol/ml) respectively. These results indicate that the concentrations of SOD, GSH, and GSH-R in ACS patients were significantly decreased than in healthy subjects (P = 0.025), (P = 0.036), and (P = 0.014), respectively. Depletion of the antioxidants is inevitable under the two to three-fold increase in the stress biomarkers. Therefore, if this depletion is not replenished, the orchestration of the proinflammatory process continues to prevail under the increased inflammatory biomarkers.

Figure 1 describes the lipid profile biomarkers in both groups. The levels of total cholesterol (579 ± 1.310 nmol/L), triglyceride (2.14 ± 0.193 nmol/L), and LDL (1.89 ± 0.078 nmol/L) were markedly increased in patients with ACS; total cholesterol (9.01 ± 1.200 nmol/L), triglyceride (4.27 ± 0.215 nmol/L) and LDL (3.87 ± 0.090 nmol/L). Variable P-values were reported in these parameters. However, all values were less than 0.05, suggesting significant changes in both study groups. In addition, HDL level had a statistically significant decrease (P = 0.001) in ACS patients compared to healthy controls (0.98 ± 0.011 vs. 2.87 ± 0.010 nmol/L).

Figure 1. Comparison of lipid profile markers between ACS patients and control subjects. An increase in cholesterol, triglycerides, and LDL gives prominent peaks in the graph bar chart. *P = 0.013, **P = 0.022, ***P = 0.001, ****P = 0.001.

Table 3 shows the correlation of serum AGE with OS markers and antioxidants. There is a significant positive relationship between sICAM-1 and AGEs (r = 0.714), sVCAM-1 and AGEs (r = 0.625), whereas a significant inverse relationship between SOD and AGEs (r = −0.610). Needless to mention, that increase in the inflammatory markers will directly influence AGE production through the non-enzymatic biochemical reaction with proteins and antioxidants trying to overcome this phenomenon, thus producing an inverse relationship.

Table 3. Correlation of AGE with OS markers. A positive r-value of more than 0.500 indicates a strong correlation between the two variables. Similarly, the negative r-value shows a weak correlation between two variables, as in SOD and AGE, in this table.

Variables	r-value
NO vs. AGEs	0.789
LDL vs. AGE	0.723
sICAM-1 vs. AGE	0.714
sVCAM vs. AGE	0.625
SOD vs. AGE	−0.610

4. Discussion

Numerous studies have documented the essential role of inflammation in accelerating ACS. AGEs, the ligand for RAGE and its downstream signalling cascade, is a well-documented phenomenon in the literature and is proposed to have an essential role of AGE and RAGE to cause inflammation in the micro-vasculature, particularly in ACS [17, 18]. In our study group, we found a significant association between AGE levels with the inflammatory markers (Table 2); these impeccable changes thus directly cause the pathogenesis of ACS [19]. AGE and RAGE interaction alters the downstream signalling cascade inside the cell and releases diverse inflammatory mediators, aggravating the generation of OS. All of these can promote the pathophysiology of ACS [20]. Consistent with the finding of previous studies, our findings have shown an increase in the levels of sRAGE in patients with ACA compared to healthy individuals, subsequently suggesting that the sRAGE could be associated with the pathogenesis of ACS [21].

Albeit the pathological mechanism of ACS is still under discussion, there are loads of documented confirmations that inflammation and OS accompany the prevalence of ACS. In addition to inflammation, oxidative stress is another crucial factor in the implication of ACS pathogenesis. ROS are produced as by-products in small amounts, and various enzymes and pathways contribute to this autoxidation process [22, 23]. Excessive ROS production disrupts the delicate balance between antioxidants and ROS, leading to cellular damage through attacks on DNA, proteins, and lipids. Calcium dysregulation, triggered by increased cytosolic calcium levels, further contributes to cell disruption and damage [24]. Excessive calcium in mitochondria also affects ATP assembly and utilization.

Moreover, ROS stimulates the production of proinflammatory markers, such as adhesion molecules VCAM-1 and ICAM-1, which promote the recruitment of inflammatory cells, exacerbating the inflammatory response [25]. fibroblasts, lymphocytes, and smooth muscle cells generate ROS. The continuous activation and recruitment of leukocytes contribute to the development of thrombi in micro-vessels and further compromise ischemia-reperfusion, aggravating the condition [26, 27].

The progression of ACS pathogenesis involves oxidative stress, inflammation, and oxidation of LDL, ultimately resulting in the formation of macrophage-derived foam cells, smooth muscle cell proliferation, activation of matrix metalloproteinases, and breakdown of the extracellular matrix within the affected area, leading to plaque rupture [28]. The present study showed a progressive increase in LDL and AGE serum levels in patients with ACS. Consistent with previously documented studies [29], our research revealed a progressive increase in LDL and AGE in serum levels of ACS patients, and increased LDL shows a strong positive correlation with AGE (r = 0.723). Furthermore, the levels of another oxidative damage biomarker MDA were significantly higher in ACS patients than in healthy individuals. Albeit, the present study shows that an increase in the LDL can produce oxidized LDL production under OS conditions, which can trigger the inflammation associated with the pathogenesis of ACS. The sequential occurrence of pathological phenomena is divided into three phases (Figure 2).

Figure 2. This figure illustrates the sequence for the formation of plaque. Each of these phases has its relationship with each other. However, the progression of these phases can become unavoidable on the severity of the preceding phase.

Preceding similar studies suggested antioxidant consumption during myocardial injury [30]. Different antioxidants, GSH, and SOD were markedly dropped in ACS patients [31]. The current study elaborated on the antioxidant system in patients with ACS compared to normal controls. Decreased serum levels of SOD, GSH, and GSH-R were recorded in the current study, which, however, is inevitable under the prevailing conditions of increased inflammatory biomarkers. Some research workers have reported that OS-induced production of ROS and Reactive Nitrogen Species is the primary cause of apprehension in ACS.

Studies have consistently demonstrated that the role of OS and the associated lipid peroxidation were significantly increased in ACS patients compared with the normal controls. The results of the current study are in accordance with the study by Bastani et al., reporting elevated MDA levels in ACS patients compared to healthy controls [32]. Moreover, our study demonstrated elevated levels of NO, indicating endothelial dysfunction in ACS patients and its detrimental effects. We found a significant positive correlation between NO and AGEs (r = 0.789), further supporting the involvement of endothelial dysfunction in ACS pathogenesis.

Our study provides evidence supporting the role of inflammation, oxidative stress, and lipid peroxidation in the pathogenesis of ACS. Our findings contribute to the growing body of knowledge regarding these mechanisms and their potential implications for developing targeted therapeutic interventions.

5. Conclusion

The current study suggested that elevated concentrations of AGEs and sRAGE, excessive lipid peroxidation, increased inflammatory markers, and antioxidant depletion can directly and actively exaggerate the inflammatory action or surrogate the inflammatory mediators in ACS. Our research group suggests that these variables have a fundamental role in the onset and progression of ACS. Thus their assessment may be a notion to assess the risk, diagnosis, and prevention of ACS. However, limitations and other confounding factors could be evaluated in studying the large cohorts. Furthermore, there is a need to assess individuals suffering from DM and metabolic syndrome in two cohorts or simultaneously in one study to make a comparison.

Authors’ contributions

Prof. Dr Aamenah Malik = AAM; Dr. Maira Mahmood = MAM; Dr. Mazhar Mushtaq = MZM; Dr. Hira Sohail = HIS; Dr. Masooma Talib = MAT; Dr. Farhat Ijaz = FAI.

Table

	AAM	MAM	MZM	HIS	MAT	FAI
Concepts	✓					✓
Design	✓	✓
Definition of intellectual content			✓	✓	✓	✓
Literature search	✓	✓	✓	✓		✓
Data acquisition	✓	✓		✓	✓
Manuscript preparation	✓	✓	✓	✓		✓
Manuscript editing	✓	✓	✓		✓
Manuscript review	✓	✓	✓		✓	✓

Consent for publication

All authors approved and consented to publication.

Ethical approval and consent to participate

Experimental protocols were approved, and ethical approval was taken from the Research Ethical Committee of The University of Lahore with reference No UOL/1335/04/19. Informed written consent was acquired from patients and healthy participants before including them in the study.

Availability of data and materials

Data are available on request.

Disclosure statement

No potential conflict of interest was reported by the author(s).