P-116 - PATIENTS WITH SYMPTOMATIC CAROTID STENOSIS HAVE INCREASED CELL-FREE DNA LEVELS IN PLASMA INDEPENDENTLY OF PLASMA DNASEI ACTIVITY

TOPIC:
Vascular Biology
AUTHORS:
Emma P. (Haemostasis, Thrombosis, Arteriosclerosis and Vascular Biology Research Group. Medical Research Institute Hospital La Fe. ~ Valencia ~ Spain) , Julia O. (Haemostasis, Thrombosis, Arteriosclerosis and Vascular Biology Research Group. Medical Research Institute Hospital La Fe. ~ Valencia ~ Spain) , Raquel H. (Haemostasis, Thrombosis, Arteriosclerosis and Vascular Biology Research Group. Medical Research Institute Hospital La Fe. ~ Valencia ~ Spain) , Francesca Maria M. (Angiology and Vascular Surgery Service. University and Polytechnic Hospital La Fe ~ Valencia ~ Spain) , Noelia G. (Angiology and Vascular Surgery Service. University and Polytechnic Hospital La Fe ~ Valencia ~ Spain) , Fernando C. (Haemostasis, Thrombosis, Arteriosclerosis and Vascular Biology Research Group. Medical Research Institute Hospital La Fe. ~ Valencia ~ Spain) , Pilar M. (Haemostasis, Thrombosis, Arteriosclerosis and Vascular Biology Research Group. Medical Research Institute Hospital La Fe. ~ Valencia ~ Spain) , Manuel M. (Angiology and Vascular Surgery Service. University and Polytechnic Hospital La Fe ~ Valencia ~ Spain)
Introduction:
Plasma cell-free DNA (cfDNA) is increased in several disorders and it has previously been proposed as a biomarker for stroke (1). DNaseI activity in plasma may modulate cfDNA concentration. Our aim in the present study was to quantify cfDNA levels and DNaseI activity in plasma samples from patients with symptomatic and asymptomatic carotid stenosis to ascertain if they could be useful biomarkers of plaque vulnerability.
Methods:
Twenty-six patients with symptomatic carotid stenosis and 40 asymptomatic sex and age matched were recruited. Symptomatology was considered when stroke, transient ischaemic attack or amaurosis fugax occurred within <21 days before surgery. cfDNA was quantified using the Quant-iT™ PicoGreen™ dsDNA Assay kit (Invitrogen). Fluorescence emitted by the intercalating agent Picogreen in samples was interpolated in a standard curve prepared with known concentrations of λ DNA. DNaseI activity was measured with the single radial enzyme diffusion (SRED) assay in 1% agarose gels prepared in Tris-HCl 10mM, MgCl 5mM, CaCl2 1mM containing 22,5 µg/ml salmon testis DNA (Sigma) and Safeview (NBSbiological). DNaseI activity of 2 µl plasma was estimated as the halo area formed by DNA degradation during 17h at 37ºC in a humid chamber. Halo areas were measured with the ImageJ software. cfDNA concentration and DNaseI activity were compared between groups with a t- test and their correlation was estimated with a Pearson test (GraphPad_v8.0.1).
Results:
cfDNA concentration was higher in patients with symptomatic than asymptomatic carotid stenosis (1289 vs.1190 ng/ml; p= 0.047) (Figure 1A). No differences in DNaseI activity were observed between symptomatic and asymptomatic patients (Figure 1B). Moreover, plasma cfDNA concentration was not correlated with DNaseI activity in these patients.
Conclusion:
Patients with symptomatic carotid stenosis have an increased plasma cfDNA concentration, which may be caused by tissue damage and inflammation. This increase appears to be, at least in part, independent from plasma DNaseI activity. Additional studies would clarify the utility of cfDNA as a marker of plaque vulnerability and scoring of patient´s prognosis and diagnosis.
References:
1-Tieu et al. J Transl Genet Genom 2020;4: 133-143, DOI: 10.20517/jtgg.2020.18 ISCIII-FEDER (PI20/01171, PI20/00075, FI21/00171)
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