Mitochondria play a central role in vascular homeostasis, extending beyond ATP production to regulate redox signalling, calcium handling and cellular stress responses (Xu et al., 2025). In endothelial cells, mitochondria function primarily as signalling organelles rather than major energy producers, critically modulating vascular tone, inflammation and adaptation to metabolic stress (Xie et al., 2010; Groschner et al., 2012).

Accumulating evidence indicates that mitochondrial dysfunction contributes to the pathogenesis of cardiovascular and metabolic disorders. Impaired mitochondrial function leads to excessive reactive oxygen species (ROS) production, endothelial activation and reduced nitric oxide bioavailability, promoting vascular stiffness, inflammation and atherosclerosis (Walczak et al., 2026). These processes are particularly relevant in patients undergoing coronary artery bypass grafting (CABG), where the functional integrity of vascular grafts is essential for long-term outcomes (Emmert et al., 2024).

The left internal mammary artery (LIMA) is considered the gold standard conduit in CABG due to its superior long-term patency and resistance to atherosclerosis (Shadrin et al., 2023). Despite its clinical importance, the bioenergetic profile of LIMA remains poorly characterized, largely due to technical limitations in assessing mitochondrial function in intact human vascular tissue.

Recent advances in extracellular flux analysis, such as the Seahorse XF platform, have enabled real-time assessment of mitochondrial respiration in intact cells and isolated mitochondria (Brand and Nicholls, 2011; Divakaruni et al., 2014). However, their application to intact human vascular tissues remains limited.

In this study, we aimed to establish a novel ex vivo approach for real-time bioenergetic analysis of intact human vascular tissue using the Seahorse XF Flex 3D Analyzer. Using LIMA grafts obtained during CABG, we demonstrate the feasibility of measuring mitochondrial respiration in structurally preserved human arterial segments. This proof-of-concept study introduces, to our knowledge, the first application of Seahorse XF Flex 3D technology to intact human vascular tissue and provides a new platform for investigating vascular mitochondrial function in clinically relevant settings.

We successfully established an ex vivo protocol for measuring mitochondrial respiration in human LIMA tissue using the Seahorse XF Flex Analyzer. Histological analysis confirmed preservation of vascular structure in prepared LIMA segments (Figure 2).

Real-time OCR measurements demonstrated that mitochondrial respiration can be reliably assessed in intact human arterial tissue using the Seahorse XF Flex 3D platform (Figure 3). The applied protocol enabled reproducible measurement of key bioenergetic parameters, including basal respiration, ATP-linked respiration and maximal respiratory capacity.

In parallel, HPLC analysis confirmed the presence of measurable levels of nucleotides in LIMA tissue (Figure 4). Nucleotide profiling demonstrated preserved energy status of the analyzed samples, supporting the functional integrity of the vascular tissue under ex vivo conditions. Combined assessment of mitochondrial respiration and nucleotide concentrations provides complementary insight into vascular bioenergetics at both functional and metabolic levels.

In this study, we demonstrate the feasibility of real-time ex vivo assessment of mitochondrial respiration in human LIMA grafts using the Seahorse XF Flex 3D Analyzer. To our knowledge, this is the first application of the Seahorse XF Flex 3D platform for bioenergetic analysis in intact human vascular tissue.

To date, the assessment of mitochondrial function in the vascular system has been primarily performed in isolated endothelial or vascular smooth muscle cells as well as intact vessels from experimental animal models (Karaś et al., 2024; Yu et al., 2017; Fang et al., 2025). These approaches have provided important insights into vascular bioenergetics, but they do not fully capture the structural and cellular complexity of intact human vascular tissue.

The application of the Seahorse XF Flex 3D platform to structurally preserved human arterial segments therefore represents a methodological advancement, enabling bioenergetic measurements under conditions that more closely resemble the in vivo environment. The ability to measure oxygen consumption rate in real time in intact human vessels provides a novel tool for studying vascular bioenergetics in clinically relevant settings.

In this study, we complemented functional assessment of mitochondrial respiration with biochemical quantification of nucleotide levels using HPLC. While extracellular flux analysis provides dynamic information on mitochondrial activity, nucleotide profiling reflects the energetic state of the tissue. Importantly, the integration of these approaches allows simultaneous assessment of functional mitochondrial activity, cellular energy and redox status for a more comprehensive evaluation of vascular bioenergetics.

The left internal mammary artery, widely used in CABG due to its superior long-term patency, represents an ideal model for translational studies of vascular function (Karthi et al., 2006). Despite its clinical importance, its bioenergetic profile has been poorly characterized. The methodological approach presented here provides a new opportunity to investigate metabolic features of clinically relevant human vascular grafts. Thus, the integration of mitochondrial respiration measurements with nucleotide analysis may be particularly valuable in future studies aimed at identifying metabolic alterations associated with vascular dysfunction or patient comorbidities.

Importantly, the ability to assess mitochondrial respiration in human LIMA tissue may have potential clinical implications for evaluating graft quality. Although LIMA is widely regarded as the gold standard conduit in CABG, variability in endothelial function and metabolic status between patients may influence graft performance and long-term patency. Clinical studies have shown that both biochemical and patient-specific factors significantly affect short- and long-term outcomes following cardiac surgery (INFLACOR study) (Kowal et al., 2018). However, current intraoperative assessment of graft quality is largely limited to anatomical and flow-based measurements, which do not provide insight into the metabolic condition of the vessel (Arslanhan et al., 2026). Ex vivo bioenergetic profiling using this approach may therefore offer a complementary functional readout reflecting mitochondrial integrity and cellular viability within the vascular wall. Such an approach may help identify subtle alterations in vascular metabolism associated with patient comorbidities, such as diabetes or oxidative stress. In the future, this methodology could contribute to the development of biomarkers of graft quality and support personalized strategies in coronary artery bypass grafting.