The first taxonomic and functional characterization of human CAVD-associated microbiota.

Introduction: Calcific aortic valve disease (CAVD) is the most common heart valve disorder, defined by a remodeling multistep process: namely, valve fibrosis with its area narrowing, impaired blood flow, and final calcification phase. Nowadays, the only treatment is the surgical valve replacement. As for other cardiovascular diseases, growing evidence suggest an active role of the immune system in the calcification process that could be modulated by the microbiota. To address this point, we aimed to investigate and characterize, for the first time, the presence of a valve microbiota and associated immune response in human CAVD. Method: Calcified aortic valve (CAV) samples from twenty patients (11 from Germany and 9 from Italy) with diagnosis of severe symptomatic CAVD were used to assess the presence of infiltrating T cells, by cloning approach, and to characterize the valve microbiota, by 16S rRNA gene sequencing (NGS). Results: We documented the presence of infiltrating T lymphocytes, especially the T helper subset, in CAV samples. Moreover, we found a tissue-associated microbiota in freshly collected CAV samples, which was significantly different in Italian and German patients, suggesting potential correlation with other cardiovascular risk factors. Conclusion: The presence of microbiota in inflamed CAV samples represents the right trigger point to explain the valve calcification process, encouraging further studies to explore the potential link between bacteria and adaptive immune response and to define the critical role of local microbiota-immunity axis on CAVD development.

Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat.

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high mortality, despite new therapeutic options such as transcatheter aortic valve replacement (TAVR). The complete pathways that start with valve calcification and lead to severe aortic stenosis remain only partly understood. By providing a close representation of the aortic valve cells in vivo, the assaying of T lymphocytes from stenotic valve tissue could be an efficient way to clarify their role in the development of calcification. After surgical excision, the fresh aortic valve sample is dissected in small pieces and the T lymphocytes are cultured, cloned then analyzed using fluorescence activated cell sorting (FACS). The staining procedure is simple and the stained tubes can also be fixed using 0.5% of paraformaldehyde and analyzed up to 15 days later. The results generated from the staining panel can be used to track changes in T cell concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific T cell subtypes of interest. In this study, we show the isolation of T cells, performed on fresh calcified aortic valve samples and the steps of analyzing T cell clones using flow cytometry to further understand the role of adaptive immunity in CAVD pathophysiology.

Calcific Aortic Valve Disease-Natural History and Future Therapeutic Strategies.

Calcific aortic valve disease (CAVD) is the most frequent heart valve disorder. It is characterized by an active remodeling process accompanied with valve mineralization, that results in a progressive aortic valve narrowing, significant restriction of the valvular area, and impairment of blood flow.The pathophysiology of CAVD is a multifaceted process, involving genetic factors, chronic inflammation, lipid deposition, and valve mineralization. Mineralization is strictly related to the inflammatory process in which both, innate, and adaptive immunity are involved. The underlying pathophysiological pathways that go from inflammation to calcification and, finally lead to severe stenosis, remain, however, incompletely understood. Histopathological studies are limited to patients with severe CAVD and no samples are available for longitudinal studies of disease progression. Therefore, alternative routes should be explored to investigate the pathogenesis and progression of CAVD.Recently, increasing evidence suggests that epigenetic markers such as non-coding RNAs are implicated in the landscape of phenotypical changes occurring in CAVD. Furthermore, the microbiome, an essential player in several diseases, including the cardiovascular ones, has recently been linked to the inflammation process occurring in CAVD. In the present review, we analyze and discuss the CAVD pathophysiology and future therapeutic strategies, focusing on the real and putative role of inflammation, calcification, and microbiome.