Atherosclerosis remains the leading pathological basis for cardiovascular diseases, including coronary artery disease, stroke, and peripheral arterial disease, which collectively account for the highest global mortality burden. Once regarded primarily as a lipid storage disorder, atherosclerosis is now firmly established as a chronic inflammatory disease of the arterial wall, involving complex interactions between lipids, immune cells, endothelial cells, and inflammatory mediators.

The vascular endothelium plays a critical role in maintaining vascular homeostasis by regulating vascular tone, inhibiting platelet aggregation, and suppressing inflammatory cell adhesion. Endothelial dysfunction represents one of the earliest detectable abnormalities in atherogenesis and precedes overt plaque formation. This dysfunctional state is characterized by impaired nitric oxide (NO) bioavailability, increased permeability, and enhanced expression of endothelial adhesion molecules.

Among the molecular regulators of vascular inflammation, nuclear factor-κB (NF-κB) has emerged as a master transcription factor controlling the expression of pro-inflammatory cytokines, chemokines, and adhesion molecules. In endothelial cells, NF-κB activation occurs in response to oxidized low-density lipoprotein (oxLDL), reactive oxygen species, shear stress, and pro-inflammatory cytokines, leading to sustained inflammatory signaling.

NF-κB regulates the transcription of key cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), both of which play critical roles in endothelial activation, leukocyte recruitment, and plaque progression. Additionally, NF-κB induces endothelial adhesion molecules including ICAM-1 and VCAM-1, facilitating monocyte adhesion and transmigration into the vascular intima—an essential step in foam cell formation and plaque development.

Despite extensive experimental evidence supporting NF-κB’s role in atherosclerosis, clinical data linking circulating NF-κB levels with endothelial dysfunction markers in human atherosclerosis remain limited, particularly in the Indian population. Understanding these relationships is essential for identifying novel biomarkers and therapeutic targets.

The present study was therefore designed to investigate NF-κB-mediated inflammatory signaling and its association with cytokines, adhesion molecules, and nitric oxide in patients with atherosclerosis.

This comparative, cross-sectional observational study was conducted in the Department of Biochemistry, Index Medical College, from January 2022 to December 2024. A total of 220 participants were enrolled and divided into two groups:

• Group I (Cases): 110 patients aged 40–60 years with clinically and/or radiologically confirmed atherosclerosis
• Group II (Controls): 110 age- and sex-matched healthy individuals with no history of cardiovascular or inflammatory disease

Inclusion and Exclusion Criteria

Patients with acute infections, autoimmune diseases, malignancy, chronic renal or hepatic disease, recent myocardial infarction, or those receiving anti-inflammatory or antioxidant therapy were excluded. Smokers, chronic alcoholics, pregnant, and lactating women were also excluded.

Sample Collection

After overnight fasting, 5 mL of venous blood was collected under aseptic conditions. Serum and plasma samples were separated and stored at −80°C until analysis.

Biochemical Analysis

• NF-κB: Measured using sandwich ELISA (p65 subunit)
• IL-6 and TNF-α: Quantified using high-sensitivity ELISA kits
• ICAM-1 and VCAM-1: Determined by sandwich ELISA
• Nitric Oxide: Estimated by Griess reaction
• Lipid Profile: Measured using automated enzymatic methods

Statistical Analysis

Data were analyzed using SPSS version 25. Results were expressed as mean ± SD. Independent Student’s t-test compared groups. Pearson’s correlation assessed relationships among variables. Multiple linear regression identified independent predictors of endothelial dysfunction. A p-value < 0.05 was considered statistically significant.

Both groups were comparable in age and sex distribution. However, BMI and blood pressure were significantly higher in the atherosclerosis group (p < 0.001), indicating greater cardiometabolic risk.

Inflammatory Biomarkers

NF-κB levels were significantly elevated in atherosclerosis patients (6.72 ± 1.34 ng/mL) compared to controls (3.01 ± 1.12 ng/mL; p < 0.001). Similarly, IL-6 and TNF-α levels were markedly higher in cases (p < 0.001), reflecting a heightened inflammatory state.

Endothelial Adhesion Molecules

ICAM-1 and VCAM-1 levels were significantly increased in the atherosclerosis group (p < 0.001), indicating endothelial activation and enhanced leukocyte adhesion.

Nitric Oxide Levels

NO levels were significantly reduced in patients with atherosclerosis (14.2 ± 3.6 µmol/L) compared to controls (27.8 ± 4.1 µmol/L; p < 0.001), reflecting impaired endothelial function.

Correlation and Regression Analysis

NF-κB demonstrated strong positive correlations with IL-6 (r = 0.72) and VCAM-1 (r = 0.68). NO showed a significant inverse correlation with IL-6 (r = −0.60). Regression analysis identified NF-κB as the strongest independent predictor of endothelial dysfunction (β = 0.42, p < 0.001)

Table 1. Demographic and Baseline Characteristics of Study Participants

Values expressed as mean ± SD.

*Statistically significant (p < 0.05)

Table 2. Comparison of Inflammatory Biomarkers Between Groups

Patients with atherosclerosis show a significantly heightened inflammatory state.

Table 3. Endothelial Adhesion Molecules in Study Groups

Elevated ICAM-1 and VCAM-1 indicate endothelial activation and leukocyte recruitment.

Table 4. Serum Nitric Oxide Levels

Significant reduction in NO reflects endothelial dysfunction.

Table 5. Oxidative Stress Marker (Malondialdehyde)

Elevated lipid peroxidation in atherosclerosis.

Table 6. Lipid Profile Comparison

Pro-atherogenic dyslipidemia in patients.

Table 7. Pearson Correlation Analysis Between Key Biomarkers

Strong inter-relationships between inflammation, oxidative stress, and endothelial dysfunction.

Table 8. Multiple Linear Regression Analysis: Predictors of Endothelial Dysfunction

NF-κB is the strongest independent predictor; NO is protective.

This study provides compelling clinical evidence supporting the central role of NF-κB-mediated inflammation in atherosclerosis. Elevated NF-κB levels were strongly associated with increased cytokine production, endothelial activation, and reduced NO bioavailability, collectively contributing to endothelial dysfunction.

The strong association between NF-κB and IL-6 underscores NF-κB’s role as an upstream regulator of inflammatory cytokine cascades. IL-6, in turn, contributes to hepatic acute-phase responses and vascular inflammation, promoting plaque progression. Similarly, elevated TNF-α amplifies endothelial dysfunction by inducing oxidative stress and adhesion molecule expression.

The significant elevation of ICAM-1 and VCAM-1 reflects NF-κB-driven endothelial activation, facilitating leukocyte recruitment into the arterial wall. Reduced NO levels further exacerbate this process by impairing vasodilation and promoting a pro-thrombotic state.

Collectively, these findings reinforce the concept that atherosclerosis is driven by a self-perpetuating inflammatory loop, with NF-κB at its core.

NF-κB-mediated inflammatory signaling plays a pivotal role in endothelial dysfunction and atherogenesis. Its strong associations with cytokines, adhesion molecules, and NO depletion highlight NF-κB as a key biomarker and potential therapeutic target in atherosclerosis.

Acknowledgments

The authors acknowledge the Department of Biochemistry, Index Medical College, for laboratory support.

Conflict of Interest

None declared.

Funding

No external funding was received.

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