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

Goren Saenz-Pipaon; Patxi San Martín; Núria Planell; Alberto Maillo; Susana Ravassa; Amaia Vilas-Zornoza; Esther Martinez-Aguilar; José Antonio Rodriguez; Daniel Alameda; David Lara-Astiaso; Felipe Prosper; José Antonio Paramo; Josune Orbe; David Gomez-Cabrero; Carmen Roncal

doi:10.1080/20013078.2020.1729646

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

Goren Saenz-Pipaon, Patxi San Martín, Núria Planell, Alberto Maillo, Susana Ravassa, Amaia Vilas-Zornoza, Esther Martinez-Aguilar, José Antonio Rodriguez, Daniel Alameda, David Lara-Astiaso, Felipe Prosper, José Antonio Paramo, Josune Orbe, David Gomez-Cabrero, Carmen Roncal^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

28 Scopus citations

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.

Original language	English (US)
Article number	1729646
Journal	Journal of Extracellular Vesicles
Volume	9
Issue number	1
DOIs	https://doi.org/10.1080/20013078.2020.1729646
State	Published - Jan 1 2020
Externally published	Yes

Bibliographical note

Funding Information:
The Foundation for Applied Medical Research, Universidad de Navarra (Spain); European Fund for Economic and Regional Development (FEDER) funds [PI18/01195], Ministry of Economy and Competitiveness, Institute of Health Carlos III [CB16/11/00371]. Patrimonio Praga (Mexico) and Virto Group (Spain). We particularly acknowledge the patients for their participation, the Biobank of the University of Navarra and Dr Juan Carlos Pastrana for their collaboration. We want to thank Lara Montori for her technical assistance (Laboratory of Atherothrombosis, CIMA) and David Gil-Carton for his assistance on the cryo-electron microscopy study (CIC bioGUNE). The analysis of EVs size distribution by NTA has been performed by José-Amable Bernabe at the ICTS “NANBIOSIS”, Unit 6 of the CIBER in Bioengineering, Biomaterials & Nanomedicne (CIBER-BBN) at the Barcelona Materials Science Institute.

Publisher Copyright:
© 2020, © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.

Keywords

Peripheral artery disease
RNA-Seq
extracellular vesicles
liquid-biopsy
thrombosis

ASJC Scopus subject areas

Histology
Cell Biology

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1080/20013078.2020.1729646

Cite this

Saenz-Pipaon, G., San Martín, P., Planell, N., Maillo, A., Ravassa, S., Vilas-Zornoza, A., Martinez-Aguilar, E., Rodriguez, J. A., Alameda, D., Lara-Astiaso, D., Prosper, F., Paramo, J. A., Orbe, J., Gomez-Cabrero, D., & Roncal, C. (2020). Functional and transcriptomic analysis of extracellular vesicles identifies calprotectin as a new prognostic marker in peripheral arterial disease (PAD). Journal of Extracellular Vesicles, 9(1), [1729646]. https://doi.org/10.1080/20013078.2020.1729646

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title = "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.",

keywords = "Peripheral artery disease, RNA-Seq, extracellular vesicles, liquid-biopsy, thrombosis",

author = "Goren Saenz-Pipaon and {San Mart{\'i}n}, Patxi and N{\'u}ria Planell and Alberto Maillo and Susana Ravassa and Amaia Vilas-Zornoza and Esther Martinez-Aguilar and Rodriguez, {Jos{\'e} Antonio} and Daniel Alameda and David Lara-Astiaso and Felipe Prosper and Paramo, {Jos{\'e} Antonio} and Josune Orbe and David Gomez-Cabrero and Carmen Roncal",

note = "Funding Information: The Foundation for Applied Medical Research, Universidad de Navarra (Spain); European Fund for Economic and Regional Development (FEDER) funds [PI18/01195], Ministry of Economy and Competitiveness, Institute of Health Carlos III [CB16/11/00371]. Patrimonio Praga (Mexico) and Virto Group (Spain). We particularly acknowledge the patients for their participation, the Biobank of the University of Navarra and Dr Juan Carlos Pastrana for their collaboration. We want to thank Lara Montori for her technical assistance (Laboratory of Atherothrombosis, CIMA) and David Gil-Carton for his assistance on the cryo-electron microscopy study (CIC bioGUNE). The analysis of EVs size distribution by NTA has been performed by Jos{\'e}-Amable Bernabe at the ICTS “NANBIOSIS”, Unit 6 of the CIBER in Bioengineering, Biomaterials & Nanomedicne (CIBER-BBN) at the Barcelona Materials Science Institute. Publisher Copyright: {\textcopyright} 2020, {\textcopyright} 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.",

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doi = "10.1080/20013078.2020.1729646",

language = "English (US)",

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Saenz-Pipaon, G, San Martín, P, Planell, N, Maillo, A, Ravassa, S, Vilas-Zornoza, A, Martinez-Aguilar, E, Rodriguez, JA, Alameda, D, Lara-Astiaso, D, Prosper, F, Paramo, JA, Orbe, J, Gomez-Cabrero, D & Roncal, C 2020, 'Functional and transcriptomic analysis of extracellular vesicles identifies calprotectin as a new prognostic marker in peripheral arterial disease (PAD)', Journal of Extracellular Vesicles, vol. 9, no. 1, 1729646. https://doi.org/10.1080/20013078.2020.1729646

TY - JOUR

T1 - Functional and transcriptomic analysis of extracellular vesicles identifies calprotectin as a new prognostic marker in peripheral arterial disease (PAD)

AU - Saenz-Pipaon, Goren

AU - San Martín, Patxi

AU - Planell, Núria

AU - Maillo, Alberto

AU - Ravassa, Susana

AU - Vilas-Zornoza, Amaia

AU - Martinez-Aguilar, Esther

AU - Rodriguez, José Antonio

AU - Alameda, Daniel

AU - Lara-Astiaso, David

AU - Prosper, Felipe

AU - Paramo, José Antonio

AU - Orbe, Josune

AU - Gomez-Cabrero, David

AU - Roncal, Carmen

N1 - Funding Information: The Foundation for Applied Medical Research, Universidad de Navarra (Spain); European Fund for Economic and Regional Development (FEDER) funds [PI18/01195], Ministry of Economy and Competitiveness, Institute of Health Carlos III [CB16/11/00371]. Patrimonio Praga (Mexico) and Virto Group (Spain). We particularly acknowledge the patients for their participation, the Biobank of the University of Navarra and Dr Juan Carlos Pastrana for their collaboration. We want to thank Lara Montori for her technical assistance (Laboratory of Atherothrombosis, CIMA) and David Gil-Carton for his assistance on the cryo-electron microscopy study (CIC bioGUNE). The analysis of EVs size distribution by NTA has been performed by José-Amable Bernabe at the ICTS “NANBIOSIS”, Unit 6 of the CIBER in Bioengineering, Biomaterials & Nanomedicne (CIBER-BBN) at the Barcelona Materials Science Institute. Publisher Copyright: © 2020, © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.

PY - 2020/1/1

Y1 - 2020/1/1

N2 - 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.

AB - 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.

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KW - RNA-Seq

KW - extracellular vesicles

KW - liquid-biopsy

KW - thrombosis

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

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

UN SDGs

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