Substrate Stiffness Modulates Endothelial Inflammation and Infection Susceptibility in Cardiac Device Implantation

Introduction: : The vascular endothelium serves as a dynamic, mechanosensitive interface between blood and tissues, critically regulating thrombosis, vasomotor tone, and vascular homeostasis. Endothelial cells (ECs) rapidly respond to mechanical cues—such as shear stress and cyclic strain—and their dysfunction under aberrant biomechanical conditions contributes to pathologies ranging from atherosclerosis to cancer metastasis. In patients with implanted cardiac devices, altered hemodynamics and mechanical mismatch can further disrupt EC function. Recent studies have suggested that differences in host vascular response may play a role in susceptibility to the risk of cardiac device infection (CDI) in patients with Staphylococcus aureus bloodstream infection. CDI occurs in 1–3% of cardiac device recipients with mortality rates as high as 27.5% within three years. Notably, elevated levels of soluble EC markers in CDI patients suggest a pivotal role for endothelial dysfunction in CDI pathogenesis. However, the mechanistic links between device-related biomechanical perturbations and EC dysfunction remain poorly understood, partly due to the lack of physiologically relevant models. To address this, we developed a novel in vitro CDI model utilizing human-derived cells to investigate how substrate stiffness, a key mechanical variable altered by cardiac device implantation, modulate EC function and infection susceptibility.

Materials and

Methods: : We designed a tunable in vitro CDI model by incorporating cardiac device analogues within collagen gels of controlled mechanical properties. Human umbilical vein endothelial cells (HUVECs) were cultured on three substrate stiffness conditions: soft (~4 kPa, mimicking native cardiovascular tissue), intermediate stiffness (~20 kPa, simulating tissue-embedded devices), and high stiffness (~200 GPa, bare implant materials). Endothelial cell responses were comprehensively assessed via RT-qPCR analysis, focusing on three key categories: (1) mechanotransduction pathway components, (2) EC inflammation/activation markers, and (3) S. aureus adhesion/internalization mediators. Functional outcomes were evaluated by THP-1 monocyte adhesion assay under static conditions. To investigate the role of YAP-mediated mechanotransduction pathways, parallel experiments were conducted using verteporfin treatment, a selective YAP inhibitor, with subsequent comparison of gene expression patterns between treated and control cells. Additionally, to model bacterial challenge, HUVECs were treated with 1μg/ml LPS for 4h as a positive control for microbial stimulus.

Results, Conclusions, and Discussions:: ECs actively sensed and responded to substrates stiffness, as suggested by stiffness-dependent trends in mechanotransduction-related gene expression (Talin2, β-catenin), though the upregulation was not statistically significant. Interestingly, ECs grown on embedded device surfaces exhibited significant downregulation of IL8 and IL6 compared to soft and stiff substrates, suggesting a potentially impaired immune cell recruitment and weaken host defense against bacterial infection. This finding was further supported by reduced THP-1 monocyte adhesion to ECs on embedded devices. Following LPS stimulation, EC activation markers (VCAM-1, ICAM-1, CCL2, IL6, IL8) increased with substrate stiffness, indicating heightened inflammatory responses on rigid surfaces. Given these stiffness-dependent activation patterns, we hypothesize that substrate mechanics may influence S. aureus infection susceptibility in ECs. Future work with live bacteria and protein-level validation (e.g., Western blot) is needed. Despite these limitations, our model systematically uncovers how device-induced stiffness perturbations prime endothelial inflammation. The findings highlight substrate stiffness as a regulator of endothelial dysfunction in CDI.

Acknowledgements and/or References (Optional):: Supported by AHA grant (25PRE1365989)