Infectious seeds of valve calcification: Exploring the bacterial hypothesis in the pathogenesis of calcific aortic valve disease.

Antonella Forlino, Paola Giordani, Cristina Merla, Silvia Roda, Roberta Besio, Abeer Ahmed Qaed Ahmed, Angela Kuka, Irene Mileto, Greta Petazzoni, Marta Corbella, Patrizia Cambieri, Baldanti Fausto, Annalisa De Silvestri, Totaro Pasquale, Filippo Amoroso, Pelenghi Stefano, Maraschi Federica, Eloisa Arbustini, Viviana Vilardo, Alexandra Smirnova, Raffaele Bruno, Seminari Elena

Journal: European journal of clinical investigation 2026;56(3):e70188

PMID: 41795218

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Abstract

BACKGROUND

Calcific aortic valve disease (CAVD) is a multifactorial condition characterized by progressive leaflet calcification with a potential role for bacterial colonization in its pathogenesis. This study investigates clinical, microbiological and molecular features of calcified versus non-calcified aortic regurgitation (AR) valves.

METHODS

This is a prospective, observational study, whose primary objective was to compare the occurrence of bacterial detection between CAVD and AR. The secondary objectives included the evaluation of bone-related calcification markers in valves from CAVD and AR patients.

RESULTS

We analysed 31 CAVD and 8 AR valves, yielding 111 leaflets (84 calcified, 27 non-calcified). Light microscopy of CAVD leaflets revealed near-complete disruption of the three-layered valve architecture, with calcified masses extending through the leaflets, sparse cellularity and focal micro-angiogenesis; no bacteria were detected by GRAM, PAS or TEM. Enrichment culture detected low-virulence bacteria in 5.95% of CAVD and 4.16% of AR leaflets; 16S rRNA PCR was positive in 22.5% of CAVD and 12.5% of AR cases, with Staphylococcus and Streptococcus spp. predominating. Calcium content was significantly higher in CAVD leaflets (p = .001) and correlated with dyslipidemia (p = .02). Osterix expression was higher in valves with positive microbiological findings (p < .0001), while ALP was increased in CAVD and bicuspid valves regardless of microbial status. Valve interstitial cells from CAVD exhibited spontaneous in vitro calcification, unlike controls.

CONCLUSION

The early osteogenic marker osterix was found to be upregulated in patients whose valves tested positive for microbial DNA, suggesting a potential role for bacteria in driving cellular differentiation towards an osteoblastic phenotype in CAVD.

Address: Department of Molecular Medicine, Biochemistry Unit, University of Pavia, Pavia, Italy.; Biostatistics and Clinical Epidemiology, Fondazione IRCCS Policlinico san Matteo, Pavia, Italy.; Malattie Infettive, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy.; Microbiologia e Virologia, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy.; Infectious Diseases Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.; Department of Molecular Medicine, Biochemistry Unit, University of Pavia, Pavia, Italy.; Biostatistics and Clinical Epidemiology, Fondazione IRCCS Policlinico san Matteo, Pavia, Italy.; Cardiac Surgery Unit, Fondazione IRCCS Policlinico san Matteo, Pavia, Italy.; Department of Chemistry, University of Pavia, Pavia, Italy.; Department of Research, Centre for Inherited Cardiovascular Diseases, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy.; Malattie Infettive, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy.; Department of Medicine, Surgical, Diagnostic and Pediatric Science, University of Pavia, Pavia, Italy.

DOI: 10.1111/eci.70188

MeSH Terms: Humans, Calcinosis, Aortic Valve, Male, Female, Prospective Studies, Aged, Middle Aged, Aortic Valve Insufficiency, Aortic Valve Disease, RNA, Ribosomal, 16S, Calcium, Staphylococcus, Streptococcus, Sp7 Transcription Factor, Aortic Valve Stenosis