Circulating biomarkers of cardiovascular disease are related to aneurysm volume in abdominal aortic aneurysm

Background: Surveillance programs in abdominal aortic aneurysms (AAA) are mainly based on imaging and leave room for improvement to timely identify patients at risk for AAA growth. Many biomarkers are dysregulated in patients with AAA, which fuels interest in biomarkers as indicators of disease progression. We examined associations of 92 cardiovascular disease (CVD)-related circulating biomarkers with AAA and sac volume. Methods: In a cross-sectional analysis, we separately investigated (1) 110 watchful waiting (WW) patients (undergoing periodic surveillance imaging without planned intervention) and (2) 203 patients after endovascular aneurysm repair (EVAR). The Cardiovascular Panel III (Olink Proteomics AB, Sweden) was used to measure 92 CVD-related circulating biomarkers. We used cluster analyses to investigate protein-based subphenotypes, and linear regression to examine associations of biomarkers with AAA and sac volume on CT scans. Results: Cluster analyses revealed two biomarker-based subgroups in both WW and EVAR patients, with higher levels of 76 and 74 proteins, respectively, in one subgroup versus the other. In WW patients, uPA showed a borderline significant association with AAA volume. Adjusting for clinical characteristics, there was a difference of −0.092 (−0.148, −0.036) loge mL in AAA volume per SD uPA. In EVAR patients, after multivariable adjustment, four biomarkers remained significantly associated with sac volume. The mean effects on sac volume per SD difference were: LDLR: −0.128 (−0.212, −0.044), TFPI: 0.139 (0.049, 0.229), TIMP4: 0.110 (0.023, 0.197), IGFBP-2: 0.103 (0.012, 0.194). Conclusion: LDLR, TFPI, TIMP4, and IGFBP-2 were independently associated with sac volume after EVAR. Subgroups of patients with high levels of the majority of CVD-related biomarkers emphasize the intertwined relationship between AAA and CVD. ClinicalTrials.gov Identifier: NCT03703947.

Biomarkers are expressed in Normalized Protein eXpression (NPX), which are relative units that result from the polymerase chain reaction and which are on the log2 scale.A high NPX value equals a high biomarker concentration and, because of the log2 scale, one unit difference in NPX represents a doubling of the protein concentration.All biomarkers are expressed as median (25 th -75 th percentile).
* For highly correlated biomarker pairs (Pearson's correlation coefficient >0.8), the biomarker with the highest mean correlation was excluded.Accordingly,11 biomarkers were excluded from the cluster analysis in the EVAR group: CASP-3, CPB1, EPHB4, GP6, JAM-A, LTBR, PAI, PECAM-1, SELP, TNF-R1, TNF-R2.Continuous variables with a normal distribution are presented as mean ± standard deviation (SD), or median (25 th -75 th percentile) in case of non-normality.Differences in baseline characteristics between patients in the two clusters were tested with Student t-tests or Mann Whitney U tests depending on their distributions.Categorical variables are presented as counts and percentages and differences between groups were tested using the chi-square test or Fisher's exact test, as appropriate.
*Coronary heart disease: history of myocardial infarction, and/or percutaneous coronary intervention and/or coronary artery bypass grafting.
AAA: abdominal aortic aneurysm..270The statistical analyses were repeated while excluding patients who initially underwent EVAR for ruptured AAA and patients with a re-intervention between the EVAR and current measurements.Linear regression analysis was applied to examine associations of individual biomarkers with sac volume.A) Biomarkers with an adjusted p-value <.05 in univariable linear regression were adjusted for age, sex and body surface area.B) Additionally, time between EVAR surgery and study visit was also added to the model.C) Next, additional pre-defined variables were added: history of coronary heart disease, hypertension, diabetes mellitus, smoking status, peripheral arterial occlusive disease, antiplatelet therapy, lipid lowering drug therapy and familial abdominal aortic aneurysm.D) Lastly, the four biomarkers of interest, together with the covariates from model C, were used to construct an adjusted multimarker model.
Results are presented as the mean effect with 95% confidence interval (CI) of a 1 standard deviation (SD) difference of the biomarker on sac volume (expressed as loge mL) .We corrected for multiple testing concluding significance with a Benjamini-Hochberg adjusted p-value <.05.
Abbreviations used: adj.p-value: adjusted p-value, β: regression coefficient, CI: confidence interval..485The statistical analyses were repeated using sac growth defined as absolute difference between current diameter and preoperative diameter.Linear regression analysis was applied to examine associations of individual biomarkers with sac growth.A) Biomarkers with an adjusted p-value <.05 in univariable linear regression were adjusted for age, sex and body surface area.Additionally, the biomarkers of interest from the sac volume model in the EVAR group are also shown, to allow comparison between the sac volume and sac growth results.B) Additionally, time between EVAR surgery and study visit was also added to the model.C) Next, additional pre-defined variables were added: history of coronary heart disease, hypertension, diabetes mellitus, smoking status, peripheral arterial occlusive disease, antiplatelet therapy, lipid lowering drug therapy and familial abdominal aortic aneurysm.
Results are presented as the mean effect with 95% confidence interval (CI) of a 1 standard deviation (SD) difference of the biomarker on sac growth (expressed as mm).As only one marker was selected by univariate linear regression and the other markers are merely illustratively portrayed, we did not correct the p-values for multiple testing.

Table S2 . Biomarker concentrations (in NPX value) in watchful waiting patients according to clusters
Biomarkers are expressed in Normalized Protein eXpression (NPX), which are relative units that result from the polymerase chain reaction and which are on the log2 scale.A high NPX value equals a high biomarker concentration and, because of the log2 scale, one unit difference in NPX represents a doubling of the protein concentration.All biomarkers are expressed as median (25 th -75th percentile).

Table S5 . Clinical characteristics of EVAR patients according to cluster membership
AAA: abdominal aortic aneurysm, ASA classification: Physical Status Classification System by the American Society of Anesthesiologists, EVAR: Endovascular Aneurysm Repair.