Introduction

Coronary artery disease (CAD) is a primary cause of death worldwide, with an increasing prevalence1. The bigger challenge is the premature CAD affecting young populations, which is an unanticipated risk with a high recurrence rate and socio-economic impact2. Premature CAD is more prevalent among men who are obese, smokers, and those with dyslipidemia, a sedentary lifestyle, and drug abuse. Although they have a lower rate of hypertension and diabetes, their traditional cardiovascular risk factors are mostly uncontrolled, owing to their low adherence to risk factor modification measures3, 4.

Atherosclerotic cardiovascular disease is characterized by chronic inflammation, on top of being a lipid metabolic disorder. Inflammation heralds the initiation, progression, instability, and rupture of the atherosclerotic plaque5, 6. C-reactive protein (CRP), the homopentameric protein, is principally secreted from hepatocytes, macrophages, endothelial cells, lymphocytes, smooth muscle cells, and adipocytes. CRP is an acute systemic inflammatory biomarker that spikes in reaction to infection or inflammatory disorders, like cardiovascular disease, in asymptomatic people. CRP levels above 3 mg/L are pathogenic and associated with the severity and prognosis of atherosclerotic CAD7, 8.

Tumor necrosis factor (TNF)-α is a pro-inflammatory pleiotropic key regulatory cytokine implicated in the pathophysiological mechanisms that govern inflammation9. The TNF-α role is paramount in the initiation stage of the formation of the atherosclerotic plaque, as it prevents the clearance of damaged vascular cells and apoptotic bodies10. In contrast, the anti-inflammatory interleukin (IL)-10 is crucial in counteracting inflammation by controlling the homeostasis of inflammatory cells11. Moreover, oxidative stress is associated with coronary atherosclerosis and the reduced bioavailability of the angio-protective nitric oxide (NO) due to its conversion into the more damaging peroxynitrite radical12. Inflammation, gauged with CRP and governed by the interplay among oxidative stress, TNF-α, and IL-10, is implicated in coronary atherosclerosis and is related to its severity. They are being investigated as theranostic targets13, 14. Uneven or disrupted blood flow can lead to the development of atherosclerotic plaque. Low wall shear stress (WSS), produced by this flow pattern (which is a well-known mechanism of atherosclerosis), can cause arterial inflammation and propel the development of atherosclerosis and plaque15. Adhesion proteins and other inflammatory chemicals are produced in greater quantities when WSS is reduced, which attracts leucocytes and guides their migration into the artery wall15. This mechanism could elucidate how local inflammation events that are far from atherosclerotic lesions may trigger inflammatory activation and the advancement of coronary plaque15.

In this study, we planned to investigate how CRP, TNF-α, IL-10, and oxidative stress are implicated in the development of premature ST segment elevation myocardial infarction (STEMI), along with the correlation to disease severity in young Egyptian patients, based on their major role as pathogenic mediators and severity biomarkers.

Methods

Results

Baseline characteristics and routine serum biomarkers of the premature STEMI patients and healthy controls

The age comparison of our participants was not significantly different (38 ± 0.3 years for healthy controls versus 38 ± 0.4 years for patients). BMI was significantly higher among patients compared to controls (26 ± 0.4 versus 24 ± 0.2 kg/m²; P > 0.001). While the majority of our patients were smokers, smokers constituted a minority among controls (80% versus 10.9%; P > 0.001). Hypertensive patients constituted 16.5%, and for family history, 13% had hyperlipidemia and 16.5% had premature CAD in their families. Percutaneous coronary intervention (PCI) was performed on 9.5% of our patients (Table 1).

The patient group demonstrated non-significantly higher serum levels of total cholesterol (4.8 ± 0.1 mmol/L) and LDL-C (3.2 ± 0.08 mmol/L) compared to healthy controls (4.5 ± 0.1 mmol/L and 3.1 ± 0.1 mmol/L, respectively). Healthy controls had significantly greater HDL-C levels (1.5 ± 0.03 mmol/L versus 1.1 ± 0.02 mmol/L; P > 0.001) and lower triglyceride levels (3.0 ± 0.2 mmol/L versus 3.5 ± 0.3 mmol/L; P = 0.04) compared to the premature STEMI patients. Significantly high levels of CPK, CK-BM, and cTnT were observed in patients compared to healthy controls (P > 0.001) (Table 1).

	Patients (n = 115)	Control (n = 55)	P-value
CRP mg/L	38.7 ± 1.9	0.8 ± 0.04	> 0.001
TNF-α pg/mL	68.4 ± 4.7	10.8 ± 0.7	> 0.001
IL-10 pg/mL	11.9 ± 0.8	8.7 ± 0.4	=0.001
TNF-α/IL-10	6.8 ± 0.5	1.7 ± 0.2	> 0.001

Changes in serum inflammatory biomarkers in premature STEMI patients versus the control group

Serum CRP levels were significantly higher in premature STEMI patients than healthy controls (38.7 ± 1.9 versus 0.8 ± 0.04 mg/L; P > 0.001). Serum levels of TNF-α (68.4 ± 4.7 pg/mL versus 10.8 ± 0.7 pg/mL; P > 0.001) and IL-10 (11.9 ± 0.8 pg/mL versus 8.7 ± 0.4 pg/mL; P = 0.001) were significantly higher in patients compared to healthy controls. The TNF-α/IL-10 ratio was significantly higher in premature STEMI patients than controls (6.8 ± 0.5 versus 1.7 ± 0.2; P > 0.001) (Table 2). CRP levels positively correlated with TNF-α levels (r = 0.580 and P > 0.001) (Figure 1).

Parameters	Patients (n = 115)	Control (n = 55)	P-value
T-AOC, U/mL	25.5 ± 0.9	14.9 ± 0.4	> 0.001
NO, nmole/mL	10.5 ± 0.3	8.1 ± 0.3	> 0.001
GSH, µg/mL	3.8 ± 0.1	5.2 ± 0.2	> 0.001
L-ascorbic acid, µg/mL	7.8 ± 0.3	7.3 ± 0.3	0.172
LPO, µmole/L	15.8 ± 0.4	4.1 ± 0.2	> 0.001

Parameter	SS ≤ 12	12 < SS ≤ 21.5	SS > 21.5	p-value
CRP (mg/L)	30.6 ± 2.5	46.3 ± 2.9	53.7 ± 9	> 0.001
TNF-α (pg/mL)	49.3 ± 4.3	87.3 ± 8.6	88.6 ± 17.4	> 0.001
IL-10 (pg/mL)	11.9 ± 1.5	11.7 ± 0.5	14.9 ± 4.4	0.80
TNF-α/IL-10	5.9 ± 0.7	7.6 ± 0.7	6.5 ± 0.9	0.23
T-AOC (U/mL)	26.0 ± 1.2	25.8 ± 1.5	26.2 ± 6.3	0.99
NO (nmole/mL)	9.8 ± 0.5	11.0 ± 0.5	13.5 ± 1.1	=0.04
GSH (µg/mL)	3.8 ± 0.2	4.0 ± 0.2	4.3 ± 0.7	0.70
L-ascorbic acid (µg/mL)	7.3 ± 0.5	8.8 ± 0.6	6.2 ± 1.7	0.09
LPO (µmole/L)	15.7 ± 0.5	16.3 ± 0.7	16.5 ± 0.7	0.08

Biomarker	Cut off point	Sensitivity	Specificity	AUC
CRP	> 1.45pg/mL	97%	99.5%	0.98
TNF-α	> 22.31 pg/mL	83%	99.5%	0.97
IL-10	> 11.15 pg/mL	50%	85%	0.67
TNF-α/IL-10	> 3.55	77%	93%	0.91

Discussion

Hypertension, dyslipidemia, diabetes mellitus, and smoking are the standard modifiable cardiovascular risk factors. Although 12.9% of patients with STEMI have been demonstrated to be risk factor-free, they exhibit more aggressive disease and adverse outcomes24. Currently, atherosclerosis, as a chronic inflammatory process, incorporates inherited and environmental factors, metabolic disorders, and infections. These risk factors initiate and promote lesion development to the extent of acute thrombotic complications and serious clinical events25, 26. Since inflammation and oxidative burden are at the core of the clinical manifestations and complications of CAD, recent preventive and therapeutic trials have demonstrated the effectiveness of anti-inflammatory treatment for cardiovascular outcomes in patients with CAD. These interventions include changes in the serum CRP levels used as the primary endpoint, cytokine inhibitors and receptor antagonists, potentiating IL-10, and scavenging oxygen-free radicals27, 28, 29. Therefore, we planned this study to assess the role of CRP, TNF-α, IL-10, and oxidative stress in premature STEMI and its correlation to disease severity in Egyptian patients.

The systemic inflammatory biomarker, CRP, is extensively used as the primary endpoint and severity outcomes gold standard biomarker in the reported CAD studies28, 30. CRP binds the membrane of ischemic/hypoxic cells and activates their phagocytosis, without giving room for their recovery and regeneration31. In our patients, CRP revealed significantly higher levels in young STEMI patients than in controls and efficiently predicted premature STEMI at levels >1.45 pg/mL with 97% sensitivity, 99.5% specificity, and an AUC of 0.98. In accordance with our results, the studies of Han and Goswami . reported significantly elevated levels of CRP in CAD patients compared to healthy controls32, 33. Furthermore, our results presented an association between the severity of coronary artery lesions assessed by the SYNTAX score and the circulating levels of CRP, where the patient group with SS > 21.5 had the highest levels. Consistently, Liu suggested that hs-CRP levels can indicate the severity of CAD assessed by SS and aid in CAD risk stratification and prognosis 1. In the presence of classic risk factors, hs-CRP levels > 6 mg/L were independently associated with the risk of cardiovascular events in aged CAD patients presented with confirmed atherosclerotic occlusive disease. Additionally, a great reduction in the 2- and 5-year event-free survival was observed for patients with risk factors but lower hs-CRP levels34. Serum levels of hs-CRP in CAD patients independently predict the risk of rapid angiographic stenotic progression35.

Serum levels of TNF-α, a major pro-inflammatory cytokine, were found to be significantly and markedly elevated in our premature STEMI patients compared to healthy controls. TNF-α efficiently predicted premature STEMI at a cutoff level >22.31 pg/mL and displayed 99.5% specificity, 83% sensitivity, and an AUC of 0.970. Similarly, Kumari et al. and Goswami et al. reported significantly elevated levels of TNF-α in CAD patients compared to healthy controls32, 36. Inhibition of TNF-α signaling is one of the investigated new approaches for preventing CAD29, 34. Levels of TNF-α > 6 pg/mL were independently associated with cardiovascular event risk in aged CAD patients with confirmed atherosclerotic occlusive disease, greatly reducing 2- and 5-year event-free survival34. Our results also exhibited a significant association between the severity of coronary artery lesions assessed by the SYNTAX score and the circulating levels of TNF-α. TNF-α and CRP are major pathogenic players in STEMI patients, and their levels correlate positively with each other, but non-significantly negatively with IL-10. TNF-α levels increase with the increase in stent numbers30, 37. Patients with angiography-confirmed CAD presented significantly higher serum TNF-α levels compared to healthy controls38. Serum levels of TNF-α in CAD patients independently predict the risk of rapid angiographic stenotic progression35.

IL-10, a cytokine with anti-inflammatory properties, presented higher serum levels in our premature STEMI patients compared to the volunteers, with a cut-off point of >11.15 pg/mL, 50% sensitivity, 85% specificity, and an AUC of 0.67. Consistently, the study by Goswami exhibited higher levels of IL-10 in MI patients than controls32. In contrast, Kumari. presented lower levels of IL-10 in CAD patients than the controls36. Several previous studies on ACS patients demonstrated a significant association between elevated IL-10 levels and adverse cardiovascular events39. Furthermore, we observed a higher TNF-α/IL-10 level in premature STEMI patients than controls, which is supported by the studies of Kumari . and Goswami .32, 36. The findings of Karu indicated that the serum levels of IL-10 were significantly lower in CAD patients of both sexes compared to healthy controls40. The study by Tajfard e revealed a significant reduction in the levels of IL-10 between angiographically-confirmed CAD patients and healthy controls41. The serum levels of TNF-α and IL-10, among other cytokines, significantly positively correlated with the severity of CAD assessed by the Gensini score42. The cardioprotective effect of paeoniflorin glycoside was correlated with significant increases in IL-10 secretion by ox-LDL-treated Human coronary artery endothelial cells through inhibiting the Wnt/β-catenin pathway43. MicroRNA‐34a is implicated in atherosclerosis by increasing macrophage cholesterol, blood lipid, inflammatory cytokines, and cell adhesion molecules. Its plasma levels correlate positively with inflammatory cytokines such as TNF-α, but negatively with IL-1044.

The higher levels of antioxidant biomarkers observed in our patients compared to healthy controls are not surprising in light of previous publications with a high range of variance. Oxidative stress biomarkers have been extensively investigated in atherosclerosis and CAD, as oxidative stress and inflammation are pathogenically implicated in the disease and its risk factors. Oxidative stress is instrumental in the initiation with the deformed ox-LDL and endothelial dysfunction, progression, and complications of CAD, and may predict outcomes independently of traditional risk factors. Quenching reactive oxygen species (ROS) is among the new approaches to combating the disease45. Although a long list of oxidative stress biomarkers has been developed and used, they lack standardization and specificity for CAD, with significant limitations that hinder their integration into clinical practice45. In our patients, NO showed elevated levels in premature STEMI patients compared to healthy controls and exhibited increased levels with increasing SS, implying a correlation with disease severity. The study by Cheraghi . supports our findings, presenting increased levels of NO in CAD patients compared to healthy controls46. NO has complex involvement in myocardial infarction and CAD. It initially increases in response to ischemia and inflammation, but its bioavailability diminishes due to endothelial dysfunction and oxidative stress, leading to vascular dysfunction26. Moreover, serum levels of T-AOC and LPO were elevated, while GSH was reduced in our premature STEMI patients compared to the healthy controls. This is consistent with the findings of Cheraghi ., where levels of GSH in healthy individuals were higher than in CAD patients46. Lower GSH levels may contribute to the development or progression of CAD, as reduced antioxidant capacity may lead to increased oxidative stress and endothelial dysfunction, both of which are implicated in CAD pathogenesis26, 46. Variations in levels of the oxidative stress biomarkers used in our study compared to previous reports could be attributed to several plausible factors that are mainly dietary, as young CAD patients may be more vigilant about their diet: the prevailing Mediterranean diet, dietary supplements, and the high nitrate/nitrite contents in Egyptian foodstuffs. In middle-aged adults like our patients, the antioxidant capacities would be more vividly induced by reactive oxidative compounds47, 48. As NO was measured as nitrite, it is possible that it rather reflects peroxynitrite, a pro-oxidant marker, that would correlate with low NO bioavailability. Also, the apparent increase in T-AOC in our patients could be a reaction to the existing oxidative stress, as reflected by the increases in LPO and reduction in GSH.

Preclinical research and extensive epidemiological studies have demonstrated the key role inflammation plays in the pathophysiology of atherosclerosis. There are multiple mediators in the intricate pathophysiological relationship between inflammation and atherosclerosis. Previous studies have demonstrated that anti-inflammatory treatments can lower the risk of cardiovascular disease. However, the results of treatments specifically targeting inflammation remain debatable; further studies are needed to better understand this association in the future29.

The limitations of our case-control cross-sectional study included the single-center small sample size, without longitudinal patient follow-up and data collection. This limits its generalization and usefulness. Furthermore, as we targeted acute STEMI patients, the biomarkers' characteristics (cut-offs, sensitivity, specificity, and AUC) are neither generalized to non-acute cases nor to the general population. The longitudinal implications of our findings were difficult to assess due to the loss of follow-up for most of our patients.

Conclusions

Our findings indicate that the inflammatory markers—CRP, TNF-α, IL-10, TNF-α/IL-10 ratio, and oxidative stress—contribute to premature STEMI development in the Egyptian population, and they are correlated with the severity of coronary artery lesions. They revealed biomarker potential; strongest for CRP, followed by TNF-α, then TNF-α/IL-10 ratio, and weakest for IL-10. Future studies may target a deeper understanding of the underlying mechanisms of our observations and investigate other populations.

Abbreviations

ACS – Acute Coronary Syndrome, AUC – Area Under the Curve, ALP – Alkaline Phosphatase, ALT – Alanine Aminotransferase, AST – Aspartate Aminotransferase, BMI – Body Mass Index, BUN – Blood Urea Nitrogen, CAD – Coronary Artery Disease, CK-MB – Creatine Kinase-MB, CRP – C-reactive Protein, cTnT – Cardiac Troponin T, ECG – Electrocardiogram, GSH – Reduced Glutathione, HDL-C – High-Density Lipoprotein Cholesterol, hs-CRP – High-sensitivity C-reactive Protein, IL-10 – Interleukin-10, LDL-C – Low-Density Lipoprotein Cholesterol, LPO – Lipid Peroxidation, MDA – Malondialdehyde, MI – Myocardial Infarction, NO – Nitric Oxide, PCI – Percutaneous Coronary Intervention, ROC – Receiver Operating Characteristic, SPSS – Statistical Package for the Social Sciences, STEMI – ST-segment Elevation Myocardial Infarction, SYNTAX – Synergy Between PCI with Taxus and Cardiac Surgery, TAC – Total Anti-Oxidative Capacity, T-AOC – Total Antioxidant Capacity, TNF-α – Tumor Necrosis Factor-alpha, WSS – Wall Shear Stress

Acknowledgments

We highly appreciate the help in conducting this study and finalizing this manuscript, offered by Prof. Dr. Tarek H El-Metwally, Biochemistry Division, Department of Pathology, College of Medicine, Jouf University, Sakaka, Saudi Arabia, and, the Department of Medical Biochemistry, Faculty of Medicine, Assiut University, Assiut, Egypt, and, Prof. Dr. Hossam El-Din M Omar, Department of Zoology and Entomology, Faculty of Science, Assiut University, Assiut, Egypt, and, the Department of Basic Sciences, School of Biotechnology, Badr University at Assiut, Assiut, Egypt.

Author’s contributions

Omnia HM Omar and Marwa A Gaber designed the study protocol development, performed all lab investigations, and writing of the manuscript. Marwan S Mahmoud selected the participants, performed clinical assessments of all patients, interpretation of clinical findings, and participated in revising the manuscript. Abdel-Raheim MA Meki, Ahmed Y Nassar and Ayman KM Hassan revised the manuscript. All authors agreed to be accountable for every aspect of the study and accepted the final version of the article as submitted.

Funding

None.

Availability of data and materials

Data and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

The study was carried out in compliance with the Helsinki Declaration principles after being approved by the Institutional Review Board, Faculty of Medicine, Assiut University (Approval#: IRB17200689). Volunteering participants signed the informed consent form,after being briefed on the nature of the study.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.