Multimodal Endothelial Phenotyping in Heritable Thoracic Aortic Diseases
M Tarnawska, L Wozniak-Mielczarek, R Knapczyk, A Parzuchowska, I Walczak, A Parteka, A Skoniecka, K Sledzinska, R Pawlaczyk, N Bednarz-Knoll, R Sabiniewicz, M Hellmann, B Kutryb-ZajacAbstract
Background
Hereditary Thoracic Aortic Diseases (HTADs) are associated with progressive aortic dilatation driven in part by dysregulated transforming growth factor beta signalling. While structural alterations of the aortic wall are well characterised, endothelial dysfunction (ED) and mitochondrial impairment may represent additional, insufficiently explored mechanisms contributing to disease progression.
Purpose
To establish a multimodal endothelial phenotyping strategy in HTAD integrating non-invasive microcirculatory assessment, circulating endothelial cell(CEC) quantification and functional characterisation of patient-derived aortic endothelial cells (AECs) (Figure 1).
Methods
The flow-mediated skin fluorescence (FMSF) to assessed vascular endothelial function was performed in 86 participants: 30 with MFS, 26 with LDS and 30 age-matched healthy controls. Among them, in three patients with genetically confirmed LDS undergoing aortic surgery were included in the translational arm: SMAD3 c.527_528del (p.Ile176ThrfsTer11), TGFBR1 c.1460G>A (p.Arg487Gln) and TGFBR2 c.1609C>T (p.Arg537Cys). CECs were isolated from peripheral blood using density gradient centrifugation followed by imaging cytometry with strict inclusion (CD31+, DAPI+) and exclusion markers (pan-keratins−, α-smooth muscle actin−, CD29−) and morphological verification (Figure 1). AECs were isolated from ascending aortic tissue by magnetic CD31-based selection (Figure 1). Cell purity was confirmed by flow cytometry (CD31+ 98–99%, CD14− <1%, CD309− <0.2%). Mitochondrial respiration was assessed using extracellular flux Seahorse analyser. Ex vivo modulation with empa- and dapagliflozin was performed for 24 hours (Figure 2).
Results
All patients demonstrated impaired microvascular function across multiple FMSF parameters, indicating nitric oxide-dependent dysfunction. CEC analysis revealed increased numbers of mature CD31+/DAPI+ events with preserved endothelial morphology, consistent with ongoing endothelial injury. Imaging flow cytometry enabled exclusion of non-endothelial artefacts and ensured high analytical specificity (Figure 1). Isolated AECs maintained a stable endothelial phenotype in culture. Bioenergetic analysis demonstrated reduced mitochondrial efficiency at baseline. Empagliflozin increased basal and adenosine triphosphate-linked respiration, indicating improved mitochondrial function in AECs (Figure 2).
Conclusion(s)
In this study, we applied a multidimensional approach to endothelial assessment in patients with HTAD, integrating non-invasive microvascular functional testing, circulating endothelial cell analysis and metabolic characterization of patient-derived aortic endothelial cells. Furthermore, we demonstrated the effect of SGLT2 inhibitors on endothelial and mitochondrial bioenergetics ex vivo. This integrated strategy aims to provide evidence on the involvement of ED in HTAD and to identify potential new diagnostic and therapeutic targets.Figure 1For image description, please refer to the figure legend and surrounding text.Figure 2For image description, please refer to the figure legend and surrounding text.