European Journal of Cardiovascular Medicine

Cardiovascular disease (CVD) remains the predominant cause of morbidity and mortality globally, accounting for approximately 17.9 million deaths annually [1]. Atherosclerosis, the pathological process underlying most cardiovascular events, develops progressively over decades before clinical manifestation [2]. Early identification of individuals at elevated risk for atherosclerosis progression represents a critical opportunity for implementing preventive strategies and reducing cardiovascular burden.

The recognition of atherosclerosis as a chronic inflammatory disease has fundamentally transformed our understanding of its pathogenesis [3]. Inflammatory processes contribute to all stages of atherogenesis, from initial endothelial dysfunction through plaque development to eventual rupture and thrombotic complications [4]. This inflammatory paradigm has stimulated extensive research into biomarkers that might reflect underlying vascular inflammation and predict disease progression.

High-sensitivity C-reactive protein (hs-CRP) has emerged as the most extensively studied inflammatory biomarker in cardiovascular risk assessment [5]. Produced primarily by hepatocytes in response to interleukin-6 stimulation, CRP serves as a sensitive indicator of systemic inflammation. Large prospective studies have consistently demonstrated associations between elevated hs-CRP and increased cardiovascular event risk [6]. The JUPITER trial provided compelling evidence that hs-CRP-guided statin therapy reduces cardiovascular events even among individuals with low LDL cholesterol levels [7].

Beyond CRP, additional inflammatory mediators have been implicated in atherosclerosis pathophysiology. Interleukin-6 (IL-6) functions as a central cytokine coordinating inflammatory responses and directly influences atherogenic processes including endothelial activation, monocyte recruitment, and smooth muscle cell proliferation [8]. Tumor necrosis factor-alpha (TNF-α) promotes endothelial dysfunction and accelerates foam cell formation within developing plaques [9]. Fibrinogen, an acute-phase reactant and coagulation factor, contributes to both inflammatory and thrombotic aspects of atherosclerosis [10].

Carotid intima-media thickness (CIMT) has been established as a validated surrogate marker for subclinical atherosclerosis [11]. CIMT measurements correlate with cardiovascular risk factors, predict future cardiovascular events, and reflect the cumulative burden of atherosclerotic exposure [12]. Serial CIMT assessment enables quantification of atherosclerosis progression over time, providing insights into factors influencing disease trajectory.

While cross-sectional studies have demonstrated associations between inflammatory biomarkers and atherosclerosis burden, prospective data examining the relationship between baseline inflammation and subsequent atherosclerosis progression remain limited [13]. Furthermore, the relative contributions of different inflammatory markers to predicting progression independently of traditional risk factors require clarification. Understanding these relationships has implications for refining cardiovascular risk stratification and potentially guiding anti-inflammatory therapeutic approaches.

This prospective cohort study aimed to investigate the association between baseline inflammatory biomarkers and early atherosclerosis progression over a three-year follow-up period in individuals without established cardiovascular disease, with the hypothesis that elevated inflammatory markers would independently predict accelerated CIMT progression.

3.1 Study Design and Population

This prospective cohort study was conducted at the tertiary care hospital.

3.2 Sample Size Calculation

Sample size was calculated to detect a minimum correlation coefficient of 0.15 between inflammatory biomarkers and CIMT progression with 90% power at a 5% significance level. Accounting for an anticipated 15% dropout rate over the three-year follow-up period, a target enrollment of 450 participants was established.

3.3 Participant Selection

Adults aged 40-65 years were recruited from primary care clinics and community health screening programs.

Inclusion criteria: (1) Age 40-65 years; (2) Absence of known cardiovascular disease; (3) No current use of anti-inflammatory medications or statins; (4) Willingness to complete the three-year follow-up protocol; (5) Adequate acoustic window for carotid ultrasonography.

Exclusion criteria: (1) History of myocardial infarction, stroke, or peripheral arterial disease; (2) Known inflammatory or autoimmune conditions; (3) Active malignancy; (4) Chronic kidney disease (eGFR <60 mL/min/1.73m²); (5) Chronic liver disease; (6) Current infection or recent surgery within 4 weeks; (7) Use of immunosuppressive medications; (8) Pregnancy or lactation.

3.4 Baseline Assessment

All participants underwent comprehensive baseline evaluation including medical history, physical examination, and standardized questionnaires assessing lifestyle factors. Anthropometric measurements included height, weight, waist circumference, and body mass index (BMI). Blood pressure was measured using an automated oscillometric device following a 5-minute rest period, with the mean of three consecutive readings recorded.

3.5 Laboratory Measurements

Fasting venous blood samples were collected between 8:00 and 10:00 AM following overnight fasting. Samples were processed within two hours of collection and stored at -80°C for batch analysis of inflammatory markers.

Inflammatory biomarkers were measured using the following methods: hs-CRP by particle-enhanced immunonephelometry (detection limit: 0.1 mg/L); IL-6 by enzyme-linked immunosorbent assay (ELISA) with high sensitivity (detection limit: 0.5 pg/mL); TNF-α by ELISA (detection limit: 1.0 pg/mL); fibrinogen by Clauss method. Standard lipid panel, fasting glucose, and HbA1c were measured using automated clinical chemistry analyzers.

3.6 Carotid Ultrasonography

CIMT was measured using high-resolution B-mode ultrasonography with a 7.5-12 MHz linear array transducer by trained sonographers blinded to participant clinical and laboratory data. Measurements were obtained bilaterally at the far wall of the common carotid artery, 1 cm proximal to the carotid bulb. Three measurements were averaged for each side, and the mean of bilateral measurements was used for analysis. All examinations were performed following a standardized protocol, and quality control assessments demonstrated intraobserver and interobserver variability coefficients of <5%.

3.7 Follow-up Protocol

Participants were reassessed at 18 months and 36 months following baseline evaluation. Each follow-up visit included clinical assessment, laboratory testing, and carotid ultrasonography. CIMT progression was calculated as the difference between 36-month and baseline measurements.

3.8 Statistical Analysis

Statistical analyses were performed using SPSS version 27.0 (IBM Corporation) and R version 4.2.1. Continuous variables were expressed as mean ± standard deviation or median (interquartile range) as appropriate, and categorical variables as frequencies and percentages. Normality was assessed using the Shapiro-Wilk test.

Participants were stratified into tertiles based on baseline hs-CRP levels. Comparisons across tertiles were performed using one-way ANOVA with post-hoc Bonferroni correction for continuous variables and chi-square test for categorical variables. Correlations between inflammatory markers and CIMT progression were assessed using Pearson or Spearman correlation coefficients.

Multiple linear regression analysis was employed to examine associations between inflammatory biomarkers and CIMT progression, adjusting for traditional cardiovascular risk factors. Cox proportional hazards regression was used to assess risk of significant atherosclerosis progression (defined as CIMT increase ≥0.05 mm). A two-tailed p-value <0.05 was considered statistically significant

4.1 Study Population Characteristics

Of 450 participants initially enrolled, 428 (95.1%) completed the 36-month follow-up and were included in the final analysis. Dropout was due to relocation (n=8), withdrawal of consent (n=7), and loss to follow-up (n=7). The mean age was 52.4±7.8 years, with 48.6% male participants. Baseline characteristics stratified by hs-CRP tertiles are presented in Table 1.

Table 1: Baseline Characteristics Stratified by hs-CRP Tertiles

BMI: Body Mass Index; BP: Blood Pressure; LDL-C: Low-Density Lipoprotein Cholesterol; HDL-C: High-Density Lipoprotein Cholesterol; CIMT: Carotid Intima-Media Thickness

4.2 Inflammatory Biomarker Distribution

Baseline inflammatory biomarker concentrations demonstrated significant variation across the study population. Median hs-CRP was 1.8 mg/L (IQR: 0.8-3.6), mean IL-6 was 2.84±1.92 pg/mL, mean TNF-α was 4.68±2.34 pg/mL, and mean fibrinogen was 312±68 mg/dL. Strong positive correlations were observed between hs-CRP and IL-6 (r=0.58, p<0.001), hs-CRP and BMI (r=0.42, p<0.001), and hs-CRP and fibrinogen (r=0.46, p<0.001).

4.3 CIMT Progression and Inflammatory Biomarkers

Mean CIMT progression over 36 months was 0.048±0.026 mm. CIMT progression demonstrated a graded relationship with baseline hs-CRP tertiles. Significant differences in CIMT progression, inflammatory markers, and clinical outcomes across tertiles are presented in Table 2.

Table 2: CIMT Progression and Clinical Outcomes by hs-CRP Tertiles

4.4 Multivariate Analysis

Multiple linear regression analysis confirmed independent associations between inflammatory biomarkers and CIMT progression after adjusting for age, sex, BMI, blood pressure, lipid profile, glucose, and smoking status. Cox regression analysis identified independent predictors of significant atherosclerosis progression (Table 3).

Table 3: Multivariate Analysis of Predictors for CIMT Progression

HR: Hazard Ratio; CI: Confidence Interval; BP: Blood Pressure; LDL-C: Low-Density Lipoprotein Cholesterol

This prospective cohort study demonstrates that elevated baseline inflammatory biomarkers are independently associated with accelerated early atherosclerosis progression over a three-year follow-up period. Participants in the highest hs-CRP tertile exhibited more than double the CIMT progression rate compared to those in the lowest tertile, and this association persisted after comprehensive adjustment for traditional cardiovascular risk factors.

Our findings align with the growing body of evidence supporting inflammation as a central driver of atherosclerosis pathophysiology. The inflammatory hypothesis, pioneered by Ross and colleagues, posits that endothelial injury initiates an inflammatory cascade that promotes lipid accumulation, smooth muscle proliferation, and plaque development [14]. Our observation that baseline inflammatory status predicts subsequent structural arterial changes provides prospective validation of this conceptual framework.

The magnitude of association between hs-CRP and CIMT progression observed in our study is consistent with previous investigations. Lorenz and colleagues demonstrated in their meta-analysis that CRP elevation was associated with increased CIMT and predicted cardiovascular events [15]. The MESA study similarly reported associations between inflammatory markers and subclinical atherosclerosis burden across diverse populations [16]. Our data extend these observations by demonstrating temporal relationships between inflammation and disease progression.

Interleukin-6 emerged as a particularly strong predictor of atherosclerosis progression in our analysis. This finding has mechanistic plausibility, as IL-6 occupies a central position in the inflammatory signaling cascade and directly influences multiple atherogenic processes [17]. The IL6R Mendelian randomization consortium provided compelling genetic evidence supporting a causal role for IL-6 signaling in coronary heart disease risk [18]. Our results suggest that IL-6 measurement may provide prognostic information beyond that obtained from hs-CRP alone.

The independent associations observed between inflammatory markers and CIMT progression, after adjustment for traditional risk factors, have important clinical implications. Current cardiovascular risk assessment algorithms primarily incorporate conventional factors including age, blood pressure, cholesterol, diabetes, and smoking status [19]. The residual predictive value of inflammatory biomarkers suggests that inflammation captures aspects of cardiovascular risk not fully reflected by these traditional parameters.

The CANTOS trial represented a landmark demonstration that targeting inflammation directly can reduce cardiovascular events [20]. Treatment with canakinumab, an IL-1β inhibitor, significantly reduced major adverse cardiovascular events independent of lipid lowering, validating the inflammatory hypothesis at a therapeutic level. Our findings support the rationale for identifying individuals with elevated inflammatory burden who might derive particular benefit from anti-inflammatory interventions.

Several potential mechanisms may explain the observed associations. Inflammatory cytokines promote endothelial dysfunction, enhance LDL oxidation, stimulate vascular smooth muscle cell migration and proliferation, and destabilize existing atherosclerotic plaques [21]. Additionally, inflammation is closely linked to metabolic dysfunction, with adipose tissue serving as a significant source of pro-inflammatory mediators [22]. The correlation between BMI and inflammatory markers observed in our cohort reflects this interrelationship.

Study limitations warrant consideration. First, inflammatory biomarkers were measured at a single time point and may not fully capture temporal variations in inflammatory status. Serial biomarker assessment might provide more robust prognostic information. Second, while we adjusted for multiple confounders, residual confounding cannot be excluded in observational studies. Third, the relatively homogeneous study population may limit generalizability to more diverse populations. Fourth, CIMT represents a surrogate marker that, while validated, does not directly measure clinical cardiovascular events.

The clinical translation of these findings requires careful consideration. While inflammatory biomarker testing offers potential for improved risk stratification, questions remain regarding cost-effectiveness, optimal thresholds, and appropriate interventions for elevated levels [23]. Guidelines from major cardiovascular societies currently recommend hs-CRP measurement for intermediate-risk individuals where results might influence treatment decisions [24].

This prospective cohort study demonstrates that elevated baseline inflammatory biomarkers, particularly high-sensitivity C-reactive protein and interleukin-6, are independently associated with accelerated early atherosclerosis progression as measured by carotid intima-media thickness changes over three years. These associations persist after comprehensive adjustment for traditional cardiovascular risk factors, supporting the independent contribution of systemic inflammation to atherogenesis.

The findings reinforce the inflammatory paradigm of atherosclerosis and suggest that inflammatory biomarker assessment may enhance cardiovascular risk stratification in primary prevention settings. Individuals with elevated inflammatory burden represent a high-risk subgroup who may warrant intensified preventive interventions and closer surveillance. Future research should focus on determining whether inflammation-guided therapeutic strategies caeffectively reduce atherosclerosis progression and improve long-term cardiovascular outcomes.

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