Circulating CXCL9, monocyte percentage, albumin, and C-reactive protein as a potential, non-invasive, molecular signature of carotid artery disease in 65+ patients with multimorbidity: a pilot study in Age.It

Abstract

Background: Carotid endarterectomy (CEA) for the prevention of upcoming vascular and cerebral events is necessary in patients with high-grade stenosis (≥70%). In the framework of the Italian National project Age.It, a pilot study was proposed aiming at the discovery of a molecular signature with predictive potential of carotid stenosis comparing 65+ asymptomatic and symptomatic inpatients.

Methods: A total of 42 inpatients have been enrolled, including 26 men and 16 women, with a mean age of 74 ± 6 years. Sixteen symptomatic and 26 asymptomatic inpatients with ≥70% carotid stenosis underwent CEA, according to the recommendations of the European Society for Vascular Surgery and the Society for Vascular Surgeons. Plaque biopsies and peripheral blood samples from the same individuals were obtained. Hematobiochemical analyses were conducted on all inpatients, and plasma cytokines/molecules, such as microRNAs (miRs), IL-6, sIL-6Ralpha, sgp130, myostatin (GDF8), follistatin, activin A, CXCL9, FGF21, and fibronectin, were measured using the ELISA standard technique. MiR profiles were obtained in the discovery phase including four symptomatic and four asymptomatic inpatients (both plasma and plaque samples), testing 734 miRs. MiRs emerging from the profiling comparison were validated through RT-qPCR analysis in the total cohort.

Results and conclusion: The two groups of inpatients differ in the expression levels of blood c-miRs-126-5p and -1271-5p (but not in their plaques), which are more expressed in symptomatic subjects. Three cytokines were significant between the two groups: IL-6, GDF8, and CXCL9. Using receiver operating characteristic (ROC) analysis with a machine learning-based approach, the most significant blood molecular signature encompasses albumin, C-reactive protein (CRP), the percentage of monocytes, and CXCL9, allowing for the distinction of the two groups (AUC = 0.83, 95% c.i. [0.85, 0.81], p = 0.0028). The potential of the molecular signature will be tested in a second cohort of monitored patients, allowing the application of a predictive model and the final evaluation of cost/benefit for an assessable screening test.

Keywords: CXCL9; IL-6; asymptomatic patients; biomarkers; carotid artery disease; elders; microRNAs; symptomatic patients.

Figure 1

Venn diagram of common miRs between carotid plaque and plasma in the selected asymptomatic and symptomatic inpatients. Results of miR profiling in terms of shared miRs and FC (comparing asymptomatic and symptomatic inpatients, FC ≥ 2 and FC ≤ −2) in both carotid plaque and plasma. Data were validated in single RT-qPCR; only miR-126–5p and miR-1271–5p have been confirmed in plasma samples (see Supplementary Table S3 ; Table 2 ).

Figure 2

CXCL9, GDF8, and IL-6 differ between asymptomatic and symptomatic inpatients. Cytokine concentration was evaluated in plasma samples from asymptomatic inpatients (n = 26) and compared to symptomatic inpatients (n = 16). Data are reported as mean ± SD. Statistical analysis was performed with Student’s t-test. *p ≤ 0.05; **p ≤ 0.01.

Figure 3

ROC analyses with the model: CXCL9, % of monocytes, albumin, CRP, with and without miR-126–5p and miR-1271–5p. ROC analyses were obtained with a machine learning-based approach (see Methods). A 10× repetition of the analyses resulted in the SD calculation and p-values. The model with CXCL9 and without the identified miRs resulted in the most significant one (AUC = 0.83, 95% c.i. [0.85, 0.81], p = 0.0028).