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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 platelet-free 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 platelet-free 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.

SUBMITTER: Saenz-Pipaon G

PROVIDER: S-EPMC7048174 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Saenz-Pipaon Goren G San Martín Patxi P Planell Núria N Maillo Alberto A Ravassa Susana S Vilas-Zornoza Amaia A Martinez-Aguilar Esther E Rodriguez José Antonio JA Alameda Daniel D Lara-Astiaso David D Prosper Felipe F Paramo José Antonio JA Orbe Josune J Gomez-Cabrero David D Roncal Carmen C

Journal of extracellular vesicles 20200219 1

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 prospectiv ...[more]

PMID: 32158521

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Project description:Background and objectivesWe aimed to investigate the history of medical resource consumption and quality of life (QoL) in peripheral arterial disease (PAD) patients in Korea.MethodsThis was a prospective, multi-center (23 tertiary-hospitals, division of cardiology), non-interventional study. Adult patients (age ?20 years) suffering from PAD for the last 12-month were enrolled in the study if they met with any of following; 1) ankle-brachial index (ABI) ?0.9, 2) lower-extremity artery stenosis on computed tomography angiography ?50%, or 3) peak-systolic-velocity-ratio (PSVR) on ultrasound ?2.0. Medical chart review was used to assess patient characteristics/treatment patterns while the history of medical resource consumption and QoL data were collected using a patient survey. QoL was measured using EuroQoL-5-dimensions-3-level (EQ-5D-3L) score system, and the factors associated with QoL were analyzed using multiple linear regression analysis.ResultsThis study included 1,260 patients (age: 69.8 years, male: 77.0%). The most prevalent comorbidities were hypertension (74.8%), hyperlipidemia (51.0%) and diabetes-mellitus (50.2%). The 94.1% of the patients took pharmacotherapy including aspirin (76.2%), clopidogrel (53.3%), and cilostazol (33.6%). The 12.6% of the patients were receiving smoking cessation education/pharmacotherapy. A considerable number of patients (500 patients, 40.0%) had visit history to another hospital before diagnosis/treatment at the current hospital, with visits to orthopedic units (50.4%) being the most common. At the time, 29% (or higher) of the patients were already experiencing symptoms of critical limb ischemia. Baseline EQ-5D index and EQ VAS were 0.64±0.24 and 67.49±18.29. Factors significantly associated with QoL were pharmacotherapy (B=0.05053; p=0.044) compared to no pharmacotherapy, and Fontaine stage improvement/maintain stage I (B=0.04448; p<0.001) compared to deterioration/maintain stage II-IV.ConclusionsIncrease in disease awareness for earlier diagnosis and provision of adequate pharmacotherapy is essential to reduce disease burden and improve QoL of Korean PAD patients.

Project description:The prevalence of arterial stiffness and hypertension increases with age. This study investigates the effect of induced pluripotent mesenchymal stem cell-derived extracellular vesicles (EVs) on ageing-associated arterial stiffness and hypertension. EVs were collected and purified from induced pluripotent stem cell-derived mesenchymal stem cells (iPS-MSCs). Young and old male C57BL/6 mice were used. Mice in the EVs group were injected via tail vein once a week for four weeks (18 x 106 EVs/mouse/injection). Blood pressure (BP) was measured using the tail-cuff method and validated by direct cannulation. Pulse wave velocity (PWV) was measured using a Doppler workstation. PWV and BP were increased significantly in the old mice, indicating arterial stiffness and hypertension. Intravenous administration of EVs significantly attenuated ageing-related arterial stiffness and hypertension, while enhancing endothelium-dependent vascular relaxation and arterial compliance in the old EVs mice. Elastin degradation and collagen I deposition (fibrosis) were increased in aortas of the old mice, but EVs substantially improved ageing-associated structural remodelling. Mechanistically, EVs abolished downregulation of sirtuin type 1 (SIRT1), and endothelial nitric oxide synthase (eNOS) protein expression in aortas of the older mice. In cultured human aortic endothelial cells, EVs promoted the expression of SIRT1, AMP-activated protein kinase alpha (AMPKα), and eNOS. In conclusion, iPS-MSC-derived EVs attenuated ageing-associated vascular endothelial dysfunction, arterial stiffness, and hypertension, likely via activation of the SIRT1-AMPKα-eNOS pathway and inhibition of MMPs and elastase. Thus, EVs mitigate arterial ageing. This finding also sheds light into the therapeutic potential of EVs for ageing-related vascular diseases.AbbreviationsEV: Extracellular vesicles; iPS: induced pluripotent stem cell; MSC: mesenchymal stem cell; AMPKα: AMP activated protein kinase α; eNOS: endothelial nitric oxide synthase; Sirt1: sirtuin 1; JNC7: Seventh Report of the Joint National Committee; CVD: cardiovascular disease; PWV: pulse wave velocity; BP: blood pressure; SNP: sodium nitroprusside.

Project description:BackgroundClinical applications have shown extracellular vesicles (EVs) to be a major paracrine effector in therapeutic responses produced by human mesenchymal stromal/stem cells (hMSCs). As the regenerative capacity of EVs is mainly ascribed to the transfer of proteins and RNA composing its cargo, and to the activity attributed by the protein surface markers, we sought to profile the protein composition of small EVs released from hMSCs to identify hMSC-EV biomarkers with potential clinical relevance.MethodsSmall EVs were produced and qualified from five human bone marrow MSC donors at low passage following a 48-h culture in exosome-depleted medium further processed by steps of centrifugation, filtration, and precipitation. Quantitative proteomic analysis comparing the protein profile of the EVs released from hMSCs and their parental cell was conducted using tandem mass tag labeling combined to mass spectrometry (LC-MS/MS) to identify enriched EV protein markers.ResultsNanoparticle tracking analysis showed no differences in the EV concentration and size among the five hMSC donors (1.83 × 1010 ± 3.23 × 109/mL), with the mode particle size measuring at 109.3 ± 5.7 nm. Transmission electron microscopy confirmed the presence of nanovesicles with bilayer membranes. Flow cytometric analysis identified commonly found exosomal (CD63/CD81) and hMSC (CD105/CD44/CD146) markers from released EVs in addition to surface mediators of migration (CD29 and MCSP). Quantitative proteomic identified 270 proteins significantly enriched by at least twofold in EVs released from hMSCs as compared to parental hMSCs, where neuropilin 1 (NRP1) was identified among 21 membrane-bound proteins regulating the migration and invasion of cells, as well as chemotaxis and vasculogenesis. Validation by western blot of multiple batches of EVs confirmed consistent enrichment of NRP1 in the nanovesicles released from all five hMSC donors.ConclusionThe identification and verification of NRP1 as a novel enriched surface marker from multiple batches of EVs derived from multiple hMSC donors may serve as a biomarker for the assessment and measurement of EVs for therapeutic uses.

Dataset Information

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

Publications

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

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