{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:47:59Z","timestamp":1760147279966,"version":"build-2065373602"},"reference-count":26,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2023,1,22]],"date-time":"2023-01-22T00:00:00Z","timestamp":1674345600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004587","name":"Instituto de Salud Carlos III","doi-asserted-by":"publisher","award":["PI1900075","PI-0324-2018","DOC_01421","847468","PE-COVID-0053-2020"],"award-info":[{"award-number":["PI1900075","PI-0324-2018","DOC_01421","847468","PE-COVID-0053-2020"]}],"id":[{"id":"10.13039\/501100004587","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Fundaci\u00f3n para la Investigaci\u00f3n Biom\u00e9dica de C\u00f3rdoba","award":["PI1900075","PI-0324-2018","DOC_01421","847468","PE-COVID-0053-2020"],"award-info":[{"award-number":["PI1900075","PI-0324-2018","DOC_01421","847468","PE-COVID-0053-2020"]}]},{"name":"Regional Ministry of Economic Transformation, Industry, Knowledge, and Universities of the Junta de Andaluc\u00eda, co-financed by the European Union through the European Social Fund \u201cThe ESF invests in your future\u201d","award":["PI1900075","PI-0324-2018","DOC_01421","847468","PE-COVID-0053-2020"],"award-info":[{"award-number":["PI1900075","PI-0324-2018","DOC_01421","847468","PE-COVID-0053-2020"]}]},{"name":"European Union\u2019s Horizon","award":["PI1900075","PI-0324-2018","DOC_01421","847468","PE-COVID-0053-2020"],"award-info":[{"award-number":["PI1900075","PI-0324-2018","DOC_01421","847468","PE-COVID-0053-2020"]}]},{"name":"Consejer\u00eda de salud y familias Junta de Andaluc\u00eda, Subvenciones para la financiaci\u00f3n de la I+D+i biom\u00e9dica y en ciencias de la salud en Andaluc\u00eda 2021, co-funded by European Union","award":["PI1900075","PI-0324-2018","DOC_01421","847468","PE-COVID-0053-2020"],"award-info":[{"award-number":["PI1900075","PI-0324-2018","DOC_01421","847468","PE-COVID-0053-2020"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Aortic stenosis (AS) is a frequent cardiac disease in old individuals, characterized by valvular calcification, fibrosis, and inflammation. Recent studies suggest that AS is an active inflammatory atherosclerotic-like process. Particularly, it has been suggested that several immune cell types, present in the valve infiltrate, contribute to its degeneration and to the progression toward stenosis. Furthermore, the infiltrating T cell subpopulations mainly consist of oligoclonal expansions, probably specific for persistent antigens. Thus, the characterization of the cells implicated in the aortic valve calcification and the analysis of the antigens to which those cells respond to is of utmost importance to develop new therapies alternative to the replacement of the valve itself. However, calcified aortic valves have been only studied so far by histological and immunohistochemical methods, unable to render an in-depth phenotypical and functional cell profiling. Here we present, for the first time, a simple and efficient cytometry-based protocol that allows the identification and quantification of infiltrating inflammatory leukocytes in aortic valve explants. Our cytometry protocol saves time and facilitates the simultaneous analysis of numerous surface and intracellular cell markers and may well be also applied to the study of other cardiac diseases with an inflammatory component.<\/jats:p>","DOI":"10.3390\/ijms24032194","type":"journal-article","created":{"date-parts":[[2023,1,23]],"date-time":"2023-01-23T02:27:45Z","timestamp":1674440865000},"page":"2194","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["A Straightforward Cytometry-Based Protocol for the Comprehensive Analysis of the Inflammatory Valve Infiltrate in Aortic Stenosis"],"prefix":"10.3390","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4153-2236","authenticated-orcid":false,"given":"Pablo","family":"\u00c1lvarez-Heredia","sequence":"first","affiliation":[{"name":"Immunology and Allergy Group (GC01), Maimonides Biomedical Research Institute of Cordoba (IMIBIC), University of Cordoba, Reina Sofia University Hospital, Avda Menedez Pidal s\/n, 14004 Cordoba, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9400-208X","authenticated-orcid":false,"given":"Jos\u00e9 Joaqu\u00edn","family":"Dom\u00ednguez-del-Castillo","sequence":"additional","affiliation":[{"name":"Cardiovascular Pathology Group (GA09), Maimonides Biomedical Research Institute, Reina Sofia University Hospital, University of Cordoba, Avda Menedez Pidal s\/n, 14004 Cordoba, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Irene","family":"Reina-Alfonso","sequence":"additional","affiliation":[{"name":"Immunology and Allergy Group (GC01), Maimonides Biomedical Research Institute of Cordoba (IMIBIC), University of Cordoba, Reina Sofia University Hospital, Avda Menedez Pidal s\/n, 14004 Cordoba, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Carmen","family":"Guti\u00e9rrez-Gonz\u00e1lez","sequence":"additional","affiliation":[{"name":"Immunology and Allergy Group (GC01), Maimonides Biomedical Research Institute of Cordoba (IMIBIC), University of Cordoba, Reina Sofia University Hospital, Avda Menedez Pidal s\/n, 14004 Cordoba, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1611-7089","authenticated-orcid":false,"given":"Fakhri","family":"Hassouneh","sequence":"additional","affiliation":[{"name":"Immunology and Allergy Group (GC01), Maimonides Biomedical Research Institute of Cordoba (IMIBIC), University of Cordoba, Reina Sofia University Hospital, Avda Menedez Pidal s\/n, 14004 Cordoba, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1875-0518","authenticated-orcid":false,"given":"Alexander","family":"Batista-Duharte","sequence":"additional","affiliation":[{"name":"Immunology and Allergy Group (GC01), Maimonides Biomedical Research Institute of Cordoba (IMIBIC), University of Cordoba, Reina Sofia University Hospital, Avda Menedez Pidal s\/n, 14004 Cordoba, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7773-004X","authenticated-orcid":false,"given":"Antonio","family":"Trujillo-Aguilera","sequence":"additional","affiliation":[{"name":"Immunology and Allergy Group (GC01), Maimonides Biomedical Research Institute of Cordoba (IMIBIC), University of Cordoba, Reina Sofia University Hospital, Avda Menedez Pidal s\/n, 14004 Cordoba, Spain"},{"name":"Immunology and Allergy Service, Reina Sofia University Hospital of Cordoba, Avda Menedez Pidal s\/n, 14004 Cordoba, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rosal\u00eda","family":"L\u00f3pez-Romero","sequence":"additional","affiliation":[{"name":"Cardiovascular Pathology Group (GA09), Maimonides Biomedical Research Institute, Reina Sofia University Hospital, University of Cordoba, Avda Menedez Pidal s\/n, 14004 Cordoba, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ignacio","family":"Mu\u00f1oz","sequence":"additional","affiliation":[{"name":"Cardiovascular Pathology Group (GA09), Maimonides Biomedical Research Institute, Reina Sofia University Hospital, University of Cordoba, Avda Menedez Pidal s\/n, 14004 Cordoba, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8985-0295","authenticated-orcid":false,"given":"Rafael","family":"Solana","sequence":"additional","affiliation":[{"name":"Immunology and Allergy Group (GC01), Maimonides Biomedical Research Institute of Cordoba (IMIBIC), University of Cordoba, Reina Sofia University Hospital, Avda Menedez Pidal s\/n, 14004 Cordoba, Spain"},{"name":"Immunology and Allergy Service, Reina Sofia University Hospital of Cordoba, Avda Menedez Pidal s\/n, 14004 Cordoba, Spain"},{"name":"Department of Cell Biology, Physiology and Immunology, University of Cordoba, Avda Menedez Pidal s\/n, 14004 Cordoba, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1169-8996","authenticated-orcid":false,"given":"Alejandra","family":"Pera","sequence":"additional","affiliation":[{"name":"Immunology and Allergy Group (GC01), Maimonides Biomedical Research Institute of Cordoba (IMIBIC), University of Cordoba, Reina Sofia University Hospital, Avda Menedez Pidal s\/n, 14004 Cordoba, Spain"},{"name":"Department of Cell Biology, Physiology and Immunology, University of Cordoba, Avda Menedez Pidal s\/n, 14004 Cordoba, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,1,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"765419","DOI":"10.3389\/fcvm.2021.765419","article-title":"Pathogenesis and Molecular Immune Mechanism of Calcified Aortic Valve Disease","volume":"8","author":"Bian","year":"2021","journal-title":"Front. Cardiovasc. Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1254\/jphs.13R05CR","article-title":"Recent Advances in Research on Human Aortic Valve Calcification","volume":"124","author":"Furukawa","year":"2014","journal-title":"J. Pharmacol. Sci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"503","DOI":"10.1056\/NEJMoa1109034","article-title":"Genetic Associations with Valvular Calcification and Aortic Stenosis","volume":"368","author":"Thanassoulis","year":"2013","journal-title":"N. Engl. J. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2262","DOI":"10.1161\/01.CIR.95.9.2262","article-title":"Prospective Study of Asymptomatic Valvular Aortic Stenosis. Clinical, echocardiographic, and exercise predictors of outcome","volume":"95","author":"Otto","year":"1997","journal-title":"Circulation"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1016\/j.atherosclerosis.2018.08.029","article-title":"Inflammatory and metabolic mechanisms underlying the calcific aortic valve disease","volume":"277","author":"Cho","year":"2018","journal-title":"Atherosclerosis"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"561","DOI":"10.1093\/eurheartj\/ehab395","article-title":"2021 ESC\/EACTS Guidelines for the management of valvular heart disease","volume":"43","author":"Vahanian","year":"2022","journal-title":"Eur. Heart J."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1395","DOI":"10.1056\/NEJMe0807001","article-title":"Calcific Aortic Stenosis\u2014Time to Look More Closely at the Valve","volume":"359","author":"Otto","year":"2008","journal-title":"N. Engl. J. Med."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Moln\u00e1r, A., P\u00e1sztor, D., and Merkely, B. (2022). Cellular Senescence, Aging and Non-Aging Processes in Calcified Aortic Valve Stenosis: From Bench-Side to Bedside. Cells, 11.","DOI":"10.3390\/cells11213389"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1159\/000354221","article-title":"Etiology of Valvular Heart Disease in the 21st Century","volume":"126","author":"Boudoulas","year":"2013","journal-title":"Cardiology"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1783","DOI":"10.1161\/CIRCULATIONAHA.110.006767","article-title":"Calcific aortic valve disease: Not simply a degenerative process: A review and agenda for research from the National Heart and Lung and Blood Institute Aortic Stenosis Working Group. Executive summary: Calcific aortic valve disease-2011 update","volume":"124","author":"Rajamannan","year":"2011","journal-title":"Circulation"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"68","DOI":"10.1016\/S0002-9149(00)00830-4","article-title":"Nonobstructive aortic valve calcium as a window to atherosclerosis of the aorta","volume":"86","author":"Adler","year":"2000","journal-title":"Am. J. Cardiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"683","DOI":"10.1093\/eurheartj\/ehab757","article-title":"Calcific aortic valve disease: From molecular and cellular mechanisms to medical therapy","volume":"43","author":"Kraler","year":"2021","journal-title":"Eur. Heart J."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2789","DOI":"10.1016\/j.jacc.2009.12.068","article-title":"Bicuspid Aortic Valve Disease","volume":"55","author":"Siu","year":"2010","journal-title":"J. Am. Coll. Cardiol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1016\/j.jjcc.2014.12.021","article-title":"The aortic stenosis complex: Aortic valve, atherosclerosis, aortopathy","volume":"65","author":"Boudoulas","year":"2015","journal-title":"J. Cardiol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2220","DOI":"10.1161\/CIRCULATIONAHA.111.088633","article-title":"Moderate aortic enlargement and bicuspid aortic valve are associated with aortic dissection in Turner syndrome: Report of the international turner syndrome aortic dissection registry","volume":"126","author":"Carlson","year":"2012","journal-title":"Circulation"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"630","DOI":"10.1016\/S0735-1097(96)00563-3","article-title":"Clinical Factors Associated with Calcific Aortic Valve Disease. Cardiovascular Health Study","volume":"29","author":"Stewart","year":"1997","journal-title":"J. Am. Coll. Cardiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"364","DOI":"10.1016\/j.ijcard.2016.11.180","article-title":"Understanding the structural features of symptomatic calcific aortic valve stenosis: A broad-spectrum clinico-pathologic study in 236 consecutive surgical cases","volume":"228","author":"Galli","year":"2017","journal-title":"Int. J. Cardiol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1016\/j.prp.2012.02.009","article-title":"Calcific aortic valve stenosis: Immunohistochemical analysis of inflammatory infiltrate","volume":"208","author":"Krbal","year":"2012","journal-title":"Pathol. Res. Pract."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"26","DOI":"10.4110\/in.2016.16.1.26","article-title":"Involvement of Immune Cell Network in Aortic Valve Stenosis: Communication between Valvular Interstitial Cells and Immune Cells","volume":"16","author":"Lee","year":"2016","journal-title":"Immune Netw."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1006","DOI":"10.4049\/jimmunol.1003521","article-title":"Circulating Activated and Effector Memory T Cells Are Associated with Calcification and Clonal Expansions in Bicuspid and Tricuspid Valves of Calcific Aortic Stenosis","volume":"187","author":"Winchester","year":"2011","journal-title":"J. Immunol."},{"key":"ref_21","first-page":"33","article-title":"Blood vessels and lymphatics in calcific aortic stenosis--in support of its inflammatory pathogenesis","volume":"46","author":"Steiner","year":"2010","journal-title":"Ceskoslovenska Patol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1318","DOI":"10.1016\/j.jtcvs.2017.01.052","article-title":"The shift of macrophages toward M1 phenotype promotes aortic valvular calcification","volume":"153","author":"Li","year":"2017","journal-title":"J. Thorac. Cardiovasc. Surg."},{"key":"ref_23","first-page":"319","article-title":"Calcific aortic valve disease and aortic atherosclerosis--two faces of the same disease?","volume":"47","author":"Florian","year":"2009","journal-title":"Rom. J. Intern. Med."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1007\/s00395-022-00935-6","article-title":"Innate immune cells in the pathophysiology of calcific aortic valve disease: Lessons to be learned from atherosclerotic cardiovascular disease?","volume":"117","author":"Broeders","year":"2022","journal-title":"Basic Res. Cardiol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"5329","DOI":"10.4049\/jimmunol.178.8.5329","article-title":"The Lymphocytic Infiltration in Calcific Aortic Stenosis Predominantly Consists of Clonally Expanded T Cells","volume":"178","author":"Wu","year":"2007","journal-title":"J. Immunol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1413","DOI":"10.1016\/j.ajpath.2017.02.012","article-title":"Interferon-\u03b3 Released by Activated CD8+ T Lymphocytes Impairs the Calcium Resorption Potential of Osteoclasts in Calcified Human Aortic Valves","volume":"187","author":"Nagy","year":"2017","journal-title":"Am. J. Pathol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/3\/2194\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T18:13:23Z","timestamp":1760120003000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/3\/2194"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,1,22]]},"references-count":26,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2023,2]]}},"alternative-id":["ijms24032194"],"URL":"https:\/\/doi.org\/10.3390\/ijms24032194","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2023,1,22]]}}}