Functional and transcriptomic analysis of extracellular vesicles identifies calprotectin as a new prognostic marker in peripheral arterial disease (PAD)

Abstract

Peripheral arterial disease (PAD) is associated with a high risk of cardiovascular events and death and is postulated to be a critical socioeconomic cost in the future. Extracellular vesicles (EVs) have emerged as potential candidates for new biomarker discovery related to their protein and nucleic acid cargo. In search of new prognostic and therapeutic targets in PAD, we determined the prothrombotic activity, the cellular origin and the transcriptomic profile of circulating EVs. This prospective study included control and PAD patients. Coagulation time (Procoag-PPL kit), EVs cellular origin and phosphatidylserine exposure were determined by flow cytometry in plateletfree plasma (n = 45 PAD). Transcriptomic profiles of medium/large EVs were generated using the MARS-Seq RNA-Seq protocol (n = 12/group). The serum concentration of the differentially expressed gene S100A9, in serum calprotectin (S100A8/A9), was validated by ELISA in control (n = 100) and PAD patients (n = 317). S100A9 was also determined in EVs and tissues of human atherosclerotic plaques (n = 3). Circulating EVs of PAD patients were mainly of platelet origin, predominantly Annexin V positive and were associated with the procoagulant activity of plateletfree plasma. Transcriptomic analysis of EVs identified 15 differentially expressed genes. Among them, serum calprotectin was elevated in PAD patients (p < 0.05) and associated with increased amputation risk before and after covariate adjustment (mean follow-up 3.6 years, p < 0.01). The combination of calprotectin with hs-CRP in the multivariate analysis further improved risk stratification (p < 0.01). Furthermore, S100A9 was also expressed in femoral plaque derived EVs and tissues. In summary, we found that PAD patients release EVs, mainly of platelet origin, highly positive for AnnexinV and rich in transcripts related to platelet biology and immune responses. Amputation risk prediction improved with calprotectin and was significantly higher when combined with hs-CRP. Our results suggest that EVs can be a promising component of liquid biopsy to identify the molecular signature of PAD patients.

EV-origin: Enumerating the tissue-cellular origin of circulating extracellular vesicles using exLR profile

“…Recent evidence suggests that body fluid EVs are involved in numerous physiological processes and play essential roles in remodeling homeostasis of the body [6] . In particular, plasma EVs originate from host cells mediate their mutual regulation locally or remotely, these EVs have cell type-specific biomolecules and could be exploited as predictive biomarkers for disease treatment [7] , [8] , [9] , [10] , [11] , [12] , [13] , [14] . The brain cell originated EVs could also be detected in CSF reflect the physiological and pathological changes taking place in the originated brain tissue [15] , [16] , [17] .…”

Calprotectin predicts mortality after ischemic stroke and is present in the thrombus

“…Moreover, S100A9 gene knockout or neutralization reduces neutrophil recruitment and thrombotic effects by modulating platelet function without in uencing other haemostatic parameters [12]. S100A9 is expressed in carotid [28] and femoral atherosclerotic plaques [10], and in human coronary artery thrombus [13]. Our histological analysis showed that S100A9 is present in all ischemic stroke thrombi and correlated with in ammatory cells and platelets.…”

“…Stroke subtype classi cation was assessed by TOAST criteria and dichotomized etiological groups were created. NIHSS score was categorized ([0-7], [7][8][9][10][11][12][13][14] and [>14]) and analysis of variance and trend analysis were performed.…”

“…Studies focusing on coronary events have shown that plasma calprotectin was related to rst and future coronary events, independently of traditional cardiovascular risk factors and C-reactive protein [9]. Recently, circulating calprotectin was associated with increased peripheral artery disease risk prediction, further improved when combined with high sensitivity CRP [10]. Moreover, plasma calprotectin has been proposed as a diagnostic marker of AIS [11] and, inhibition of S100A9 has been reported to suppress thrombus formation in experimental models of stroke [12,13].…”

<p>Extracellular Vesicle-Related Thrombosis in Viral Infection</p>

“…[162][163][164][165] However, a similar role of exosomes and MVs is assumed in COVID-19 as other virus infectious diseases. [166][167][168][169][170] In particular, concerning thrombosis, there is no doubt that COVID-19 causes more symptoms in comparison with the past virus infectious disease. 171 Accumulation of important reports of EVs in conjunction with COVID-19 is expected in the future.…”